rna-mediated virulence gene regulation in the human pathogen listeria...
TRANSCRIPT
RNA-mediated virulence gene regulation in the human pathogen
Listeria monocytogenes
Edmund Loh
Institutionen för Molekylärbiologi Laboratory for Molecular Infection Medicine Sweden (MIMS)
Umeå Universitet Umeå 2010
2
Edmund Loh Institutionen för Molekylärbiologi Laboratory for Molecular Infection Medicine Sweden (MIMS) Umeå Universitet S-901 87, Umeå. Sweden
Copyright © Edmund Loh ISSN: 0346-6612 New series nr: 1337 ISBN: 978-91-7264-994-1 Printed by: Print & Media Umeå, Sweden 2010
3
To my mother…...
(Kepada Ibuku)
Holy mother of Jesus!
&
Now you must work like a dog!
(Jörgen Johansson)
4
5
Table of contents
ABSTRACT .................................................................................................................................................. 7
SAMMANFATTNING PÅ SVENSKA....................................................................................................... 8
PAPERS IN THIS THESIS ......................................................................................................................... 9
INTRODUCTION...................................................................................................................................... 10
1. LISTERIA MONOCYTOGENES................................................................................................................. 102. PATHOPHYSIOLOGY OF LISTERIA INFECTION........................................................................................ 10
2.1 Crossing the intestinal barrier ...................................................................................................... 112.2 Multiplications within the liver and spleen................................................................................... 122.3 Bacteraemia .................................................................................................................................. 132.4 Crossing the placental barrier ...................................................................................................... 132.4.1 Neonatal infection...................................................................................................................... 132.5 Crossing the blood-brain barrier.................................................................................................. 142.6 Other complications of an L. monocytogenes infection ................................................................ 142.7 Summary ....................................................................................................................................... 15
3. INTRACELLULAR LIFE CYCLE OF LISTERIA MONOCYTOGENES.............................................................. 153.1 Internalisation of L. monocytogenes ............................................................................................. 163.2 Intracellular growth and spread ................................................................................................... 163.3 Virulence factors involved in the intracellular growth cycle of L. monocytogenes ...................... 173.3.1 Internalins.................................................................................................................................. 173.3.1.1 InlA.......................................................................................................................................... 173.3.1.2 InlB.......................................................................................................................................... 183.3.1.3 Other internalins..................................................................................................................... 193.3.2 Other virulence factors .............................................................................................................. 203.3.2.1 Vip........................................................................................................................................... 203.3.2.2 p60 .......................................................................................................................................... 203.3.2.3 Ami and Auto........................................................................................................................... 203.3.2.4 Sortases................................................................................................................................... 213.3.2.5 Bsh and BilE ........................................................................................................................... 213.3.2.6 Listeriolysin O......................................................................................................................... 223.3.2.7 Phospholipases ....................................................................................................................... 233.3.2.8 Hpt .......................................................................................................................................... 243.3.2.9 ActA......................................................................................................................................... 24
4. REGULATION OF LISTERIA MONOCYTOGENES VIRULENCE GENES......................................................... 254.1 Sigma Factor B (σB) ...................................................................................................................... 264.2 PrfA............................................................................................................................................... 274.2.1 Transcriptional regulation of prfA expression........................................................................... 284.2.2 Translational regulation of prfA expression .............................................................................. 294.2.3 Post-translational regulation of PrfA ........................................................................................ 30
5. RNA REGULATION................................................................................................................................ 315.1 CIS-ACTING RNAS.............................................................................................................................. 32
5.1.1 Nutritional depravation.............................................................................................................. 325.1.2 pH .............................................................................................................................................. 345.1.3 Thermosensors ........................................................................................................................... 345.1.4 Riboswitches .............................................................................................................................. 355.1.4.1 Molecular recognition of SAM................................................................................................ 375.1.4.2 Structure of a SAM riboswitch ................................................................................................ 385.1.4.3 SAM riboswitch-mediated gene regulation ............................................................................. 385.1.4.4 Other types of riboswitches..................................................................................................... 39
5.2 TRANS-ACTING RNAS......................................................................................................................... 405.2.1 ncRNAs that sequester proteins ................................................................................................. 40
6
5.2.1.1 CsrB and CsrC........................................................................................................................ 405.2.1.2 6S RNA.................................................................................................................................... 415.2.2 ncRNAs that bind to target mRNAs............................................................................................ 425.2.3 DsrA........................................................................................................................................... 435.2.4 RyhB........................................................................................................................................... 455.3 Protein factors important for the function of trans-encoded ncRNAs........................................... 475.3.1 Hfq ............................................................................................................................................. 475.3.2 RNase E and RNase III .............................................................................................................. 48
5.4 RNA-MEDIATED GENE REGULATION IN PATHOGENS........................................................................... 495.4.1 RNAIII in Staphylococcus aureus .............................................................................................. 505.4.2 VrrA in Vibrio cholerae ............................................................................................................. 515.4.3 LhrA-C in Listeria monocytogenes ............................................................................................ 52
AIMS OF THIS THESIS ........................................................................................................................... 53
RESULTS AND DISCUSSION................................................................................................................. 54
6.0 PRFA .................................................................................................................................................. 546.1 Novel regulation of PrfA............................................................................................................... 546.1.1 Length of the prfA coding sequence correlates to its expression level....................................... 546.1.2 Possible mechanisms?................................................................................................................ 556.2 Another factor involved in the regulation of PrfA?....................................................................... 576.2.1 S-box riboswitches in L. monocytogenes.................................................................................... 576.2.2 A direct interaction between SreA and the 5´-UTR of prfA........................................................ 586.2.3 Temperature involvement?......................................................................................................... 596.2.4 In vitro studies............................................................................................................................ 596.2.5 Novel function of S-box riboswitches regulating PrfA translation............................................. 596.3 Characterisation of 5´-UTRs in L. monocytogenes....................................................................... 606.3.1 Possible mechanisms for 5´-UTR-mediated regulation............................................................. 61
7. SWITCHING FROM SAPROPHYTISM TO VIRULENCE................................................................................ 627.1 Novel ncRNAs in L. monocytogenes.............................................................................................. 627.2 Novel cis-regulatory RNA elements in L. monocytogenes ............................................................ 637.3 Other discoveries .......................................................................................................................... 647.3.1 Overlapping antisense UTRs and normal antisense RNAs ........................................................ 647.3.2 Antisense regulation of flagellar biosynthesis ........................................................................... 647.4 Transcriptional changes from saprophytism to virulence............................................................. 64
CONCLUDING REMARKS..................................................................................................................... 66
ACKNOWLEDGEMENTS....................................................................................................................... 67
REFERENCES ........................................................................................................................................... 71
APPENDIX: PAPER I-IV ......................................................................................................................... 90
7
Abstract
RNA-mediated virulence gene regulation in the human pathogen Listeria monocytogenes
The Gram-positive human pathogen Listeria monocytogenes uses a wide range of virulence factors for its pathogenesis. The majority of its virulence genes are encoded on a 9-kb pathogenicity island and are controlled by the transcriptional activator PrfA. Expression of these genes is maximal at 37°C and minimal at 30°C in a mechanism involving an RNA thermosensor. This thesis brings up different aspects of RNA-mediated regulation, including regulatory RNA structures within coding mRNA controlling expression to 5-untranslated RNA (5´-UTR) that controls downstream genes (cis-acting) as well as small non-coding RNAs (ncRNAs) that bind other target RNA (trans-acting).
We investigated the importance of the coding region of the prfA-mRNA for its expression. Various lengths of prfA-mRNA were fused with reporter genes. Our finding suggested that the first 20 codons of prfA-mRNA were essential for efficient translation in Listeria monocytogenes. Translation of the shorter constructs was shown to be reduced. The expression level showed an inverse correlation with the RNA secondary structure stability in the beginning of the coding region. Riboswitches have previously been known to control expression of their downstream mRNA in a cis-acting manner. A trans-acting S-adenosylmethionine-binding riboswitch termed SreA was identified in Listeria monocytogenes. It was found to control the expression of the virulence regulator PrfA, by binding to the prfA-UTR and thereby affecting its translation. We examined the RNA locus encoding different virulence factors in Listeria monocytogenes. Several of them were preceded by 5´-UTRs of various lengths. We speculate that these 5´-UTRs could control expression of the downstream mRNA, provided they are of sufficient length. These findings prompted us to examine where and when Listeria monocytogenesswitches on gene expression. Tiling array was used to compare RNAs isolated from wild-type and mutant bacteria grown at different growth conditions. Antisense RNAs covering parts of or whole open-reading frames as well as 29 new ncRNAs were identified. Several novel riboswitches possibly functioning as upstream terminators were also found. My thesis work compiles together a variety of novel RNA-mediated gene regulatory entities. A first coordinated transcriptional map of Listeria monocytogenes has been set up. My work has also revealed that the expression of the virulence regulator PrfA is controlled at several levels, indicating the importance of both the 5´-UTR and the coding RNA for regulated expression.
8
Sammanfattning på Svenska
RNA-medierad virulensgenreglering i den humana patogenen Listeria monocytogenes
Den Gram-positiva humana patogenen Listeria monocytogenes använder en mängd olika virulensfaktorer för sin patogenes. Majoriteten av dess virulensgener är kodade på en 9-kb patogenicitetsö och styrs av den transkriptionella aktivatorn PrfA. Uttrycket av dessa gener är maximalt vid 37°C och minmalt vid 30°C vilket regleras av en RNA-termostat. Denna avhandling behandlar olika aspekter av RNA-medierad reglering, inklusive RNA-strukturer inom kodande mRNA som kan kontrollera uttryck, 5´-icke-translaterade regioner (5´-UTR) som styr nedströmsgener (cis-agerande) såväl som små icke-kodande RNA (ncRNA) som binder andra mål-RNA (trans-agerande).
Vi undersökte betydelsen av den kodade regionen i prfA-mRNA för dess uttryck. Olika längder av prfA-mRNA placerades framför reportergener. Våra upptäckter tyder på att de 20 första kodonerna av prfA-mRNA är nödvändiga för effektiv translation i Listeria monocytogenes. Translationen av kortare konstruktioner visade sig vara reducerad. Det är sedan tidigare känt att riboswitchar styr uttrycket av sina nedströms mRNA genom att binda olika metaboliter. En trans-agerande S-adenosylmethionine-bindande riboswitch, kallad SreA, identifierades i Listeria monocytogenes. Det konstaterades att den styrde uttrycket av virulensregleraren PrfA genom att binda till prfA-UTR och därmed påverka dess translation. Vi undersökte mRNA-kodande för olika virulensfaktorer. Ett flertal av dessa föregicks av 5´-UTR:er av olika längd. Vi tror att dessa 5´-UTR:er kan styra uttryck av nedströms mRNA, om de är av tillräcklig längd. Dessa upptäckter gjorde att vi undersökte var och när Listeria monocytogenes slår på sina gener. Tiling-array användes för att jämföra RNA isolerade från vildtyps- och mutantbakterier som odlats vid olika växtbetingelser. Antisense-RNA som täcker delar av eller hela gener, såväl som 29 nya ncRNA identifierades. Flera nya riboswitchar, som kan slå på och av gener, hittades också.
Mitt avhandlingssarbete sammanställer en mängd nya RNA-baserade regulatoriska enheter. En första koordinerad transkriptionell karta över Listeria monocytogenes har satts upp. Mitt arbete har också uppenbarat att uttrycket av virulensregleraren PrfA styrs genom flera mechanismer, vilket indikerar betydelsen av både 5´-UTR och det kodande RNA för reglerat uttryck.
9
Papers in this thesis The Results and Discussion of this thesis are based on the following papers and
manuscript labelled with Roman numerals (I-IV).
Paper I
Loh, E., Memarpour F., Johansson, J. and Sondén, B. The first 20 codons of the prfA-
mRNA are required for efficient translation in Listeria monocytogenes. Manuscript
Paper II
Loh, E*., Dussurget, O*., Gripenland, J*., Vaitkevicius, K., Tiensuu, T., Mandin, P. Repoila, F., Buchrieser, C., Cossart, P. and Johansson, J. A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes. Cell, 2009, 139:770-779.
Paper III
Loh, E*., Gripenland, J*. and Johansson J. Control of Listeria monocytogenes virulence by 5´-untranslated RNA. Trends in Microbiology, 2006 (Original article, Genome analysis section) 14:294-298.
Paper IV
Toledo-Arana, A., Dussurget, O., Nikitas, G., Sesto, N., Guet-Revillet, H., Loh, E., Gripenland, J., Tiensuu, T., Vaitkevicius, K., Barthelemy, M., Vergassola, M., Nahori, M-A., Soubigou, G., Regnault, B., Coppee, J-Y, Lecuit, M., Johansson J. and Cossart P. The transcriptional landscape of Listeria monocytogenes: Switch from saprophytism to virulence. Nature, 2009, 459:950-956.
* Authors have contributed equally.
Paper II, III and IV are reproduced with the permissions from Cell, Trends in Microbiology and Nature respectively.
10
Introduction
1. Listeria monocytogenes Listeria monocytogenes is a Gram-positive bacterium discovered in 1926. The genus
Listeria consists of six species: L. monocytogenes, L. invanoii, L. innocua, L. welshimeri,
L. seeligeri and L. grayi. Among these six species, only L. monocytogenes is a true
human pathogen with L. ivanoii being an opportunistic human pathogen. L. seeligeri
possesses many of the virulence factors found in L. monocytogenes but is non-pathogenic
(Hain et al., 2006; Steinweg et al., 2010).
This facultative anaerobe is 0.4 to 1.5 µm long, rod-shaped, does not form spores, is non-
capsulated and is motile at temperatures between 10 to 30°C. It is commonly found in
decaying soil, plants, sewage and in food. In humans, L. monocytogenes is responsible
for causing meningitis, meningo-encephalitis, foetal infection, neonatal abortion and
febrile gastroenteritis (Vazquez-Boland et al., 2001). The disease termed listeriosis is
common among weakened immune system individuals, pregnant women and elderly.
The foods frequently causing listeriosis are usually unpasteurised soft cheeses, dairy
products, sausages, salami, smoked fish and “ready-to-eat” frozen food. The bacterium is
a serious threat to food safety as it can tolerate high concentrations of salt and acidic
conditions and is able to multiply at refrigeration temperature (Vazquez-Boland et al.,
2001). Beside humans, a wide range of vertebrates such as birds and mammals also
succumb to listeriosis. The transmission of Listeria in animals usually occurs via
consumption of contaminated fodder from the silo, which leads to a herd outbreak.
L. monocytogenes possesses the ability to cross three barriers: the intestinal, the foetal-
placental and the blood-brain barrier (BBB). The animal model for studying listeriosis is
the mouse model which therefore has provided us with most of our knowledge of the
Listeria infection process.
2. Pathophysiology of Listeria infectionListeriosis is usually a very severe disease with a mortality rate in humans of around 30%
or even higher despite early antimicrobial therapies. Human listeriosis is usually acquired
11
via ingestion of contaminated food, which is the major cause of epidemics and sporadic
incidents (Farber and Peterkin, 1991; Pinner et al., 1992). The primary entry site of L.
monocytogenes into the host is the gastrointestinal tract (GIT), with relatively high initial
dosage of bacteria (105) needed, at least in the murine infection model. Lower dosages
however, are capable of causing infection in immunosuppressed individuals, the elderly
and pregnant women. Dosages as low as 102 bacteria per gram of food have been
reportedly linked to listeriosis (Farber and Peterkin, 1991; McLauchlin et al., 1991). The
infectious incidence also varies depending upon the pathogenicity and virulence of the L.
monocytogenes strain ingested in addition to host risk factors.
Clinical manifestations usually appear 20 hours after the ingestion of contaminated food,
with symptoms ranging from the typical fever, myalgia and nausea to diarrhoea.
Fig. 1. Schematic representation of the pathophysiology of Listeria infection.
2.1 Crossing the intestinal barrier Once inside the human GIT, L. monocytogenes must withstand the harsh conditions of
the stomach before reaching the small intestine. The acidic conditions in the stomach
destroy a significant number of Listeria organisms. Those that survive and reach the
small intestine induces their entry into the epithelial cell. Initially, the bacteria are
detected at the absorptive epithelial cells of the apical area of the villi. Later, most of
them are detected inside the macrophages of the stroma of the villi. This indicates that L.
12
monocytogenes penetrates the intestinal barrier by invading the intestinal epithelium
(Racz et al., 1972). Once inside the enterocytes, it passes directly from cell to cell
utilising host cell actin polymerisation. This invasion of neighbouring enterocytes by
basolateral spreading causes enteritis. From animal model studies, the internalisation and
translocation into deep organs occurs very rapidly (within minutes), suggesting that
crossing the intestinal barrier does not require intracellular growth prior to translocation
to other organs (Pron et al., 1998).
2.2 Multiplications within the liver and spleen L. monocytogenes that have crossed the intestinal barrier are carried by the blood and/or
lymphatic system to the mesenteric lymph nodes, liver and the spleen (Cousens and Wing,
2000; Mackaness, 1962; Marco et al., 1992; Pron et al., 1998). Animal models showed
that once L. monocytogenes is intravenous, they are rapidly cleared from the bloodstream
by macrophages in the liver and spleen (North et al., 1991). Almost 90% of the bacteria
accumulate in the liver, as they are captured by the Kupffer cells lining the sinusoid. The
bacteria enter the liver hepatocytes through either the Kupffer cells, cell-to-cell spread or
by direct invasion (Dramsi et al., 1993; Wood et al., 1993).Within the first 6 hours of
accumulation, most of the bacteria are cleared by resident macrophages resulting in a
decrease of the bacterial population. In a normal healthy person, the hepatocytes will
respond to Listeria infection by releasing neutrophil chemo-attractants and exhibiting an
increase in adhesion to neutrophils, resulting in microabscess formation (Conlan and
North, 1991). However, not all the bacteria are destroyed, the surviving L.
monocytogenes will start to reproduce and increase in numbers. This process usually
takes around 2 to 5 days and neutrophils are replaced by blood-derived mononuclear cells
together with lymphocytes to form granulomas (Heymer et al., 1988). The hepatocytes in
the liver are the primary site for L. monocytogenes replication (Cousens and Wing, 2000;
Gaillard et al., 1987). In an immunocompetent human, the infection is cleared usually
within 5 to 7 days. This clearing process is mediated once L. monocytogenes is degraded
within the cytosol, resulting in the presentation of Listerial peptides (such as LLO and
Mpl) on the cell surfaces to CD8 T-cells. The clearance process is also a result of γ-
interferon-mediated macrophage activation. However, if the infection is not controlled
13
by adequate immune response in the liver and spleen, it might result in the release of
bacteria into the circulation due to the proliferation process of unchallenged bacteria.
2.3 Bacteraemia Bacteraemia is one of the most common manifestations of listeriosis. Clinical symptoms
usually include fever and myalgia. As L. monocytogenes is a multisystemic pathogen
capable of infecting various cells, it is capable of causing septicaemia involving multiple
organs.
2.4 Crossing the placental barrier During gestation, pregnant women usually develop a slight impairment in cell-mediated
immunity and therefore are at higher risk of developing listerial bacteraemia (17-fold
higher than non-pregnant women) (Mylonakis et al., 2002; Weinberg, 1984). Abnormally
high oestrogenic hormones that occur during late pregnancy could also lead to disruption
of the T-cell-mediated resistance to infection (Pung et al., 1985). Bacteraemia may be
prolonged, causing secondary infections specifically at the placenta. L. monocytogenes
can proliferate in the placenta where the usual defence mechanisms are not reachable by
haematogenous penetration due to the placental barrier. This translocation process across
the placental-endothelial barrier enables the bacteria to gain access to the foetal
bloodstream. Untreated bacteraemia is usually self-limiting, however maternal fever may
persist, subsequently leading to neonatal abortion. 22% of perinatal infections result in
stillbirth or neonatal death; premature labour is also common (Vazquez-Boland et al.,
2001).
2.4.1 Neonatal infection From animal model experiments, it is known that orally administrated L. monocytogenes
causes stillbirth and the bacteria can be isolated from placenta and foetal tissues (Smith et
al., 2003). When utero infection arises, spontaneous abortion can be expected. The foetus
may be stillborn or die within hours when granulomatosis infantiseptica occurs. During
the early onset of infection, bacteria can be isolated from the eye (conjunctivae), external
14
ear, nose, amniotic fluid, placenta, blood and occasionally the cerebrospinal fluid (CSF).
At later stages, the highest concentrations of bacteria could be found in the lung and gut
of the neonates. This indicates that the acquisition of Listeria is not via the
haematogenous route but through infected amniotic fluid instead (Becroft et al., 1971).
2.5 Crossing the blood-brain barrier The mechanism behind central nervous system (CNS) infection by L. monocytogenes is
still largely unknown. However, it has been shown that Listeria exhibits tropism towards
the brain; it can infect axons and spread along the nerve cell body (Dons et al., 1999). A
L. monocytogenes CNS infection is primarily manifested as meningitis. Listeria
monocytogenes was ranked as the fifth most common pathogen to cause bacterial
meningitis but first when it comes to mortality rate (22%) from a survey by the “Centers
for Disease Control and Prevention” (CDC) (Wenger et al., 1990). L. monocytogenes
crosses the blood-brain barrier (BBB) predominantly via the choroid plexus, the site
where CSF is produced. Alternatively, direct uptake by the endothelial cells could be
another way for free circulating L. monocytogenes in the blood to cross the BBB (Kirk,
1993). However, this haematogenous route of infection requires a large dosage of
bacteria. 30% of the total L. monocytogenes circulating in the bloodstream after
intravenous inoculation are engulfed by phagocytes (Drevets, 1999). These entrapped
bacteria spread to the endothelial cells and across the BBB in vitro although it is
unknown whether this occurs during Listeria infection in vivo. Macroscopic brain
abscesses account for approximately 10% of the CNS listerial infections. Subcortical
abscesses in thalamus, pons and medulla are typical for L. monocytogenes but not for
other pathogens. Mortality is very high and patients who survive the infection usually
have serious sequelae (Cone et al., 2003).
2.6 Other complications of an L. monocytogenes infection Approximately 8% of patients with listerial infection may develop endocarditis. Patients
with a previous history of heart-valve problems are at higher risk as they could develop
complicated sepsis with a mortality rate of 48% (Carvajal and Frederiksen, 1988). For the
15
past three decades, many case studies of non-lethal localised listerial infection have been
published. These infection sites range from the skin to the conjunctivae of the eye (Cain
and McCann, 1986; Schwartz et al., 1989). L. monocytogenes could also cause febrile
gastroenteritis, although these cases are quite rare. The primary targets are usually young
children. Symptoms such as fever, watery diarrhoea, nausea and malaise could emerge as
fast as 24 hours after ingestion of a large inoculum of bacteria.
2.7 Summary Listeria monocytogenes is taken up via the GIT through a of contaminated food source.
There are three variables that might affect the outcome of the infection: the inoculum of
bacteria in the food ingested, the pathogenic properties of the strain ingested, and the
immune-status of the host. In a healthy individual, L. monocytogenes does not pose much
threat, as it is self-limiting. However, this is not the case for immunocompromised
individuals. The failure to completely clear L. monocytogenes might result in an
increased amount of bacteria in the liver and subsequent release into the bloodstream.
These individuals may develop septicaemia due to prolonged bacteraemia. Other
complications such as meningoencephalitis could occur, and if the host is pregnant, it
might lead to neonatal abortion.
3. Intracellular life cycle of Listeria monocytogenes Being a facultative intracellular bacterium, L. monocytogenes is capable of invading,
surviving and growing within its host cell. As mentioned in the previous chapter, L.
monocytogenes can invade and reproduce inside macrophages, epithelial cells,
hepatocytes, endothelial cells and nerve cells. Its intracellular life cycle is shown in Fig. 2.
16
Fig. 2. Intracellular life cycle of Listeria monocytogenes. Proteins important for the different steps are shown within parenthesis. See text for further details.
3.1 Internalisation of L. monocytogenesL. monocytogenes begins by adhering to the surface of the eukaryotic cell. It recognises a
large number of different eukaryotic receptors. These receptors range from the globular
domain of the complement factor Clq (Braun et al., 2000), the Met receptor for
hepatocyte growth factor (HGF) (Shen et al., 2000), components of extracellular matrix
(ECM) such as heparin sulphate proteoglycans (HSPG) (Alvarez-Dominguez et al., 1997)
and fibronectin (Gilot et al., 1999) to the most prominently studied receptor the
transmembrane glycoprotein E-cadherin (Mengaud et al., 1996). These bacterial ligands
identified are all surface proteins, such as internalins InlA and InlB, InlJ, p60 and Vip.
InlA recognises and interacts with the adhesion molecule E-cadherin, whereas InlB
interacts with the Clq receptor, the c-Met receptor and glycosaminoglycans (including
heparin sulphate).
3.2 Intracellular growth and spread During the process of internalisation, L. monocytogenes is engulfed within a phagocytic
vacuole (Gaillard et al., 1987). The Listeria-containing vacuole becomes acidic by the
increased influx of hydrogen ions (H+) from the cytoplasm (Beauregard et al., 1997), and
the phagolysosomal formation is prevented (Alvarez-Dominguez et al., 1997). After 30
minutes, the bacteria begin to disrupt the phagosome membrane (Gaillard et al., 1987).
Two hours later, approximately 50% of the bacteria have been released into the
cytoplasm (Tilney and Portnoy, 1989). This process is mediated by the haemolysin
17
together with 2 phospholipases and is the initial but essential step for L. monocytogenes
intracellular survival and growth. Once inside the cytosol, it replicates with a doubling
time of 1 hour (Gaillard et al., 1987; Portnoy et al., 1988). It exploits hexose phosphate
from the host cell cytoplasm for intracellular growth (Ripio et al., 1997). The cytoplasmic
bacteria are immediately surrounded by a cloud of fibrillar material made up of actin
filaments. Around 2 hours post-infection, these filaments rearrange to form an actin tail at
one of the poles of the bacterium. This actin tail is made up of 2 populations of cross-
linked actin filaments: one formed by long, axially arranged actin bundles, while the
other is made of short and randomly arranged filaments. The unique actin tail formation
allows the bacterium to be propelled in the cytoplasm at an average speed of 0.3 µm s-1.
This propulsion movement is random and eventually the bacteria will reach the cell
periphery, thereby pushing its membrane outwards to form a finger-like protrusion. These
pseudopods will penetrate their neighbouring cells, forming a double-membrane
phagosome. The degradation of the double-membrane vacuole is rapid (around 5
minutes) and once the bacterium is released into the cytosol, a new round of intracellular
infection cycle will occur (Dabiri et al., 1990; Mounier et al., 1990; Robbins et al., 1999).
3.3 Virulence factors involved in the intracellular growth cycle of L. monocytogenes
3.3.1 Internalins The Internalin family in L. monocytogenes is comprised of 24 members. These internalins
are characterised by the presence of amino-terminal regions with tandemly arranged
leucine-rich repeats (LRRs) (Fig. 3) (Pizarro-Cerda and Cossart, 2006).
3.3.1.1 InlA InlA is an 800-amino-acid long protein, initially identified in a transposon mutagenesis
screen while searching for non-invasive L. monocytogenes mutants. The absence of InlA
severely impaired bacterial entry into human epithelial Caco-2 cells (Gaillard et al.,
1991). From a deletion mutational study, it was shown that the LRR and inter repeat (IR)
regions were sufficient to induce entry into target cells (Lecuit et al., 1997). E-cadherin
was identified to be the receptor for InlA (Mengaud et al., 1996). E-cadherin belongs to
18
the cadherin superfamily of calcium-dependent cell adhesion molecules. This
transmembrane glycoprotein is located within adherence junctions and mediates cell-cell
adhesion.
Fig. 3 Schematic representation of InlA and InlB. The internalin InlA contains a LPXTG motif at its carboxy-terminus that is recognised by Sortase A, allowing covalent binding to the cell wall peptidoglycan (Bierne et al., 2002). The other internalin, InlB, has a glycine/tryptophan-rich (GW) module at its C-terminus that can interact electrostatically with lipoteichoic acid enabling itself to loosely attach to the bacteria cell wall (Jonquieres et al., 1999).
Bacterial InlA interaction with human E-cadherin is initially mediated by the presence of
LRRs and the extracellular domain of E-cadherin. This interaction is species-specific as
the presence of proline at position 16 on E-cadherin (naturally in humans and guinea
pigs) is essential for InlA binding. A mutation from proline to glutamic acid at position
16 (naturally in mice and rats) inhibits adhesion of L. monocytogenes to E-cadherin
leading to a failure to invade the host cell (Lecuit et al., 1999). Transgenic mice
expressing human E-cadherin in the intestine showed that the InlA/E-cadherin interaction
is essential for bacterial translocation across the intestinal barrier (Lecuit et al., 2001). In
humans, this InlA/E-cadherin interaction is also required for the crossing of the maternal-
foetal barrier by L. monocytogenes (Lecuit et al., 2004). The presence of E-cadherin on
the epithelial cells where it is in contact with the encephalorachidean fluid could be a site
where InlA interacts with and translocates the bacterium across the blood-brain barrier.
3.3.1.2 InlB InlB is a 630-amino-acid long protein from the internalin family that is encoded on the
same operon as inlA (Gaillard et al., 1991). Similar to InlA, InlB is required for invasion
19
of the human epithelial cell; however, InlB enters through a larger group of target cells
than InlA (Braun et al., 1998; Dramsi et al., 1995). InlB is detachable from the bacterial
cell wall, and the GW domain at the C-terminal can interact with cellular targets
(Jonquieres et al., 2001). A crystal structure study reveals that InlB adopts an elongated
curve formation, suggesting that it can accommodate several ligands (Marino et al.,
1999). The hepatocyte growth factor receptor (HGF), Met, is the main receptor of InlB
(Shen et al., 2000) and it belongs to a family of receptor tyrosine kinases (RTK)
(Hubbard and Till, 2000). Upon binding to its ligand, RTKs transduce extracellular
signals into the cytoplasm by autophosphorylation: these phosphorylated molecules
activate several pathways involved in cell proliferation, migration and differentiation.
Ubiquitin ligase Cbl is recruited to Met upon cellular stimulation by InlB (Ireton et al.,
1999). During this stimulation, Cbl ubiquitinates Met and triggers the activation of the
endocytosis machinery leading to clathrin-dependent internalisation of Met and L.
monocytogenes (Li et al., 2005; Veiga and Cossart, 2005). The receptor for the globular
domain of the complement component Clq (known as Gc1q-R) has also been identified
as a receptor for InlB (Braun et al., 2000).
3.3.1.3 Other internalins Besides InlA and InlB, there are 22 additional internalins identified in the Listeria
monocytogenes genome. InlC, for example, is a secreted protein, homologous to InlA and
InlB at the cap region. Regions of homology are the signal peptide, LRRs and B repeats,
although InlC lacks a cell wall anchoring motif at the C-terminus (Domann et al., 1997).
An inlC deletion mutant had reduced virulence in a mouse model but could not replicate
intracellularly in cell lines (Engelbrecht et al., 1996). The other internalins share the same
features as InlC, possessing the cap region but lacking the cell-wall anchoring motif and
are therefore thought to be released into the extracellular medium (Dramsi et al., 1997;
Engelbrecht et al., 1996; Kuhn and Goebel, 2000; Raffelsbauer et al., 1998). Recently, it
has been shown that the InlH internalin assists L. monocytogenes survival into host cells
by tempering the inflammatory response (Personnic et al., 2010). The level of InlA is
increased but not its transcript level in a ∆inlH mutant, suggesting the involvement of
inlH expression in inlA post-transcriptional regulation.
20
3.3.2 Other virulence factors
3.3.2.1 Vip Vip is an LPXTG-anchored cell wall protein identified from comparison of genomes
between pathogenic and non-pathogenic Listeria spp. This protein is required for
invasion of Caco-2 and L2071 cell lines (Cabanes et al., 2005). The cellular receptor for
Vip is Gp96, which is a chaperone resident in the endoplasmatic reticulum and also
present at the plasma membrane (Cabanes et al., 2005). Gp96 modulates the immune
response by affecting the cellular trafficking of several molecules such as Toll-like
receptors (Tsan and Gao, 2004). Therefore, besides being involved as a receptor for Vip,
Gp96 can be sequestered and thereby reducing the immunological response in the host
during infection (Cabanes et al., 2005).
3.3.2.2 p60 p60 is an 60-kDa extracellular protein encoded by the iap (invasion-associated protein)
gene. It encodes a 484-amino-acid long protein containing a central repeat region of Thr-
Asn units (Köhler et al., 1990). This protein is found both in the supernatant (Kuhn and
Goebel, 1989) and associated with the cell wall (Ruhland et al., 1993). When added
exogenously in a cell culture condition, it restores the invasiveness of L. monocytogenes
to wild-type levels in an iap mutant background (Kuhn and Goebel, 1989). ActA is
unevenly distributed in a ∆iap strain, leading to malformation of the actin tail and
reduction in intracellular movement (Pilgrim et al., 2003).
3.3.2.3 Ami and Auto Ami is a 917-amino-acid long protein with an N-terminal domain that has similarities to
the amidase domain of Staphylococcus aureus Atl autolysin. Its C-terminal domain has 8
GW domains which anchor to the bacterial cell wall (Milohanic et al., 2001). ami
mutants are attenuated in a mouse model and Ami mediates bacterial adhesion to target
cells in a ∆inlAB background (Milohanic et al., 2001). These experiments indicate the
importance of Ami during L. monocytogenes virulence.
21
Auto is another GW-anchored autolysin that is absent in the avirulent strain Listeria
innocua (Cabanes et al., 2004). A ∆auto strain showed a reduced invasiveness in
nonprofessional phagocytes, however when Auto was expressed into L. innocua, it could
not induce an invasive phenotype (Cabanes et al., 2004). Fine-tuned regulation of Auto is
essential in L. monocytogenes as overexpression of Auto also impairs invasion of host
cells (Cabanes et al., 2004).
3.3.2.4 Sortases Sortases are transpeptidases involved in the cell wall anchoring of surface proteins and
virulence. Surface proteins displaying C-terminal LPXTG motifs are covalently bound to
the bacteria cell wall peptidoglycan by sortases. The analysis of the L. monocytogenes
genome sequence revealed two genes encoding sortases, srtA and srtB (Bierne et al.,
2002). Sortase A, encoded by srtA is responsible for anchoring InlA and several other
LPXTG proteins to the peptidoglycan (Bierne et al., 2002; Garandeau et al., 2002;
Pucciarelli et al., 2005). This protein is essential for the efficient entry of L.
monocytogenes into human enterocytic and hepatocytic cells, as well as for colonising the
liver and spleen of mice (Bierne et al., 2002; Garandeau et al., 2002).
A second sortase, sortase B encoded by srtB anchors a small group of proteins and
recognises two different sorting motifs: NXZTN and NPKXZ (Pucciarelli et al., 2005).
One of these proteins is SvpA, a surface protein reported to promote Listeria escape from
the phagosome of macrophages that has been shown to be involved in virulence (Bierne
et al., 2004; Borezee et al., 2001). Sortase B is required for efficient colonisation of the
liver, spleen and intestine of the mice by the oral route (Newton et al., 2005).
3.3.2.5 Bsh and BilE Bile is a dark green, bitter-tasting fluid produced by the liver in most vertebrates. It aids
the process of lipid digestion in the small intestine. In humans, bile is stored in the
gallbladder and is discharged into the duodenum upon eating. Bile is alkaline and has an
important function: acting as bactericide. However, Listeria monocytogenes is able to
22
tolerate high concentrations of bile (Begley et al., 2002; Begley et al., 2003; Begley et al.,
2005; Dussurget et al., 2002; Sleator et al., 2005).
Bsh (bile salt hydrolase) is present in L. monocytogenes but not in the non-pathogenic
species, such as L. innocua (Dussurget et al., 2002). Bsh is usually produced by
commensal enteric and lactic bacteria as a protective mechanism against bile toxicity,
working by deconjugating the conjugated bile salts. The deletion of bsh, results in
decreased survival of L. monocytogenes in the liver of mice after intravenous injection
and reduced bacterial faecal carriage after oral infection of guinea pigs (Dussurget et al.,
2002). bsh is controlled by σB (Sue et al., 2003) and positively regulated by PrfA (see
below) (Dussurget et al., 2002), with σB playing a more important role (Begley et al.,
2005; Toledo-Arana et al., 2009).
BilE (bile exclusion), has a role in listerial bile tolerance and is essential for intestinal
colonisation and virulence in L. monocytogenes (Sleator et al., 2005). BilE is encoded by
a two-gene operon, bilEA-bilEB. bile expression is up-regulated in the presence of PrfA,
and is also controlled by σB.
3.3.2.6 Listeriolysin O Listeriolysin O (LLO, also called haemolysin) is one of the major virulence determinants
of L. monocytogenes. It is a member of the pore-forming, cholesterol-dependent cytolysin
toxin family (CDC). The hly gene encoding LLO was the first virulence gene to be
identified in Listeria and is a key virulence factor essential for pathogenesis. The main
function of LLO is its role in the escape of L. monocytogenes from primary and
secondary vacuoles (Gedde et al., 2000; Portnoy et al., 1988). The activity of LLO is pH
dependent; optimal at acidic pH (inside the phagosome) and less active at neutral pH
(inside the cytoplasm). This mechanism prevents excessive damage to the newly invaded
cell. LLO binds to the phagosomal membrane as a monomer and oligomerises into large
complexes that finally penetrate the membrane by forming pores. This enables the
bacteria to escape via the pores into the cytosol where it then replicates. L.
monocytogenes mutants lacking LLO fail to survive and proliferate within macrophages
and non-professional phagocytes (Cossart et al., 1989; Gaillard et al., 1986; Kathariou et
al., 1987; Portnoy et al., 1988). LLO has also been shown to be involved in the
23
internalisation of L. monocytogenes into cells, although the full mechanism is yet to be
elucidated (Dramsi and Cossart, 2003).
3.3.2.7 Phospholipases The observation that L. monocytogenes is still capable of escaping into the cytosol and
proliferating in the absence of LLO suggests that other membrane-active products may be
involved in phagosome disruption (Portnoy et al., 1988). Pathogenic Listeria spp.
produce three different enzymes with phospholipase C (PLC) activity that are involved in
virulence. L. monocytogenes secretes two of them, PlcA and PlcB whereas L. ivanovii
secretes SmcL. These phopholipases are responsible for the lysis of intracellular vacuoles.
PlcA, or phosphatidylinositol-specific PLC (PI-PLC) enzyme is encoded by the plcA
gene and is highly specific for the glycosyl-PI (GPI)-anchored eukaryotic membrane
proteins (Goldfine and Knob, 1992; Leimeister-Wachter et al., 1991; Mengaud et al.,
1991).
PlcB, or phosphatidylcholine PLC (PC-PLC) enzyme is encoded by the plcB gene and is
a broad substrate range phospholipase. PlcB hydrolyses phosphatidylcholine (PC),
phosphatidylethanolamine (PE), phosphatidylserine (PS) and sphingomyelin (SM)
(Geoffroy et al., 1991; Goldfine et al., 1993; Vazquez-Boland et al., 1992). PlcB is
synthesised as a proenzyme and requires zinc metalloprotease encoded by the gene mpl
for maturation (Domann et al., 1991; Raveneau et al., 1992). Both PlcA and PlcB act
synergistically with LLO to lyse the primary and secondary vacuoles (Camilli et al.,
1991; Grundling et al., 2003; Smith et al., 1995). However, PlcB can also promote lysis
of the primary vacuole in human epithelial cell lines in the absence of LLO (Grundling et
al., 2003; Marquis et al., 1995).
Both phospholipases are required for virulence in mice and are essential for evasion of
cell autophagy (Birmingham et al., 2007; Camilli et al., 1991; Camilli et al., 1993; Py et
al., 2007; Raveneau et al., 1992; Schluter et al., 1998; Smith et al., 1995). Autophagy is a
catabolic process involving the degradation of a cell’s own components through the
lysosomal machinery. Expression of LLO is essential for the induction of an autophagic
reaction at early infection, suggesting a role for permeabilisation of the vacuole in the
induction of the degradative pathway. PlcA and PlcB expression are required for bacterial
24
escape from autophagic degradation in nonprofessional phagocytic cells and
macrophages (Birmingham et al., 2007; Py et al., 2007). Phospholipases mediate L.
monocytogenes escape from the double membrane autophagosome by a mechanism
preventing autophagic killing
3.3.2.8 Hpt Hpt was the first virulence factor to be identified as specifically involved in the
replication of a facultative intracellular pathogen and it is encoded by the uhpT gene.
Once inside the host cell, L. monocytogenes expresses specific determinants to obtain
nutrients necessary for its intracellular multiplication. It exploits the hexose phosphates
from the host as a source of carbon and energy to fuel its intracellular growth. Hpt, being
a hexose phosphate transporter, mediates hexose phosphate uptake allowing L.
monocytogenes to grow intracellularly and is required for Listeria proliferation in mouse
organs (Chico-Calero et al., 2002). The expression is controlled by the L. monocytogenes
transcriptional activator, PrfA.
3.3.2.9 ActA After escaping from the vacuole and intracellular proliferating, L. monocytogenes induces
polymerisation of actin filaments to move through the cytoplasm and to spread from cell
to cell. ActA, encoded by the actA gene, is responsible for this actin-based motility in
Listeria (Kocks et al., 1992) (Fig 4). L. monocytogenes move from one infected cell to an
uninfected neighbouring cell, favouring bacteria tissue spreading without being exposed
to the extracellular environment (Gouin et al., 2005; Kocks et al., 1992). The central
domain of the ActA, is composed of four proline-rich repeats that bind to
enabled/vasodilator-stimulated phosphor (Ena/VASP) family proteins and modulate
bacterial speed and directional movement (Auerbuch et al., 2003; Geese et al., 2002;
Laurent et al., 1999). The N-terminal region alone is sufficient to induce bacterial
movement (Lasa et al., 1997), since it binds to and actives Arp2/3, a seven-protein host
complex that induces actin polymerisation (Boujemaa-Paterski et al., 2001; Skoble et al.,
2001; Skoble et al., 2000).
25
Fig. 4. Fluorescence micrograph of HeLa cells infected with L. monocytogenes EGDe. Comet-like tail of actin filaments (green) and bacteria (red). Regions of overlap of red and green fluorescence appear yellow. (Picture taken by Tiago Costa)
The intracellular movement force is provided by the continuous deposition of actin
monomers at the interface between the bacterial cell surface and the growing tail of actin
filaments in the cytosol that serves as support for propulsion. The motility system only
functions at one of the poles, ensuring that the bacterium moves unidirectionally. This is
because ActA accumulates at one of the bacterial poles and is absent from the other pole
(Kocks et al., 1993).
Besides its intracellular role in cell to cell spreading, ActA has also been implicated in L.
monocytogenes invasion. The N-terminus of ActA presents several clusters of positively
charged amino acids that could be involved in cell attachment and entry by recognition of
heparan sulphate (Alvarez-Dominguez et al., 1997). Inactivation of ActA impairs L.
monocytogenes invasion of macrophages and epithelial cells (Alvarez-Dominguez et al.,
1997; Suarez et al., 2001). Expression of ActA in L. innocua is sufficient to confer the
ability to enter epithelial cells (Suarez et al., 2001), indicating that ActA alone is
sufficient for uptake.
4. Regulation of Listeria monocytogenes virulence genes As mentioned in the previous chapters, L. monocytogenes can respond rapidly to
changing environmental conditions. This ranges from the saprophytic state when it is in
the environment to the pathogenic state when it is inside the host. Regulated expression
26
of virulence genes is vital in a pathogen such as L. monocytogenes as it faces multiple
environments during its lifecycle. These important mechanisms for regulating the
expression of appropriate virulence genes under rapidly changing environmental
conditions are controlled mainly by σB and PrfA.
4.1 Sigma Factor B (σB) Under different environmental stress conditions, different alternative sigma (σ) factors
are associated with a core RNA polymerase. The association of different σ factors with
the core RNA polymerase is dissociable. The σ factor is responsible for enzyme
recognition of specific DNA sequences and promoter regions. Association of the core
polymerase to different σ factors enables the RNA polymerase to target specific promoter
regions leading to expression of a different set of target genes at certain/varying
environmental conditions. The L. monocytogenes genome encodes five σ factors: one
housekeeping σ factor (σA) and four alternative σ factors (σB, σC, σH, σL) (Glaser et al.,
2001).
Some alternative σ factors such as σB are normally in an inactive form through direct
interaction with anti-σ-factor proteins until the bacterium encounters conditions that
require the expression of genes regulated by that σ factor. σB is encoded by sigB in L.
monocytogenes and is recognised as a general stress-responsive sigma factor. This
alternative sigma factor has only been identified in the low G-C-containing, Gram-
positive genera Bacillus, Listeria and Staphylococcus (Binnie et al., 1986; Brody and
Price, 1998; Fouet et al., 2000; Knobloch et al., 2001; Wu et al., 1996). The regulation of
σB in L. monocytogenes is not as well studied as in Bacillus subtilis but a comparative
genomic study showed that the sigB operon is well conserved in L. monocytogenes, with
similar gene organisation (Ferreira et al., 2004). Although the sigB operon structures are
identical, the signal transduction pathway differs between Listeria and Bacillus. In L.
monocytogenes, this pathway is regulated by regulatory proteins (anti-sigma factors and
anti-anti-sigma factors) encoded in the sigB operon (RsbT, RsbU, RsbV and RsbW).
RsbW (anti-sigma) binds to and inactivate σB and under certain stress conditions, RsbV
(anti-anti-sigma) is produced and binds to RsbW, releasing σB. σB in L. monocytogenes
regulates expression of virulence and virulence-associated genes, such as inlA, inlB, bsh,
27
gadB, opuCA and the RNA-chaperone protein, Hfq (see below) (Christiansen et al., 2004;
Kazmierczak et al., 2003; McGann et al., 2007; Nadon et al., 2002; Sue et al., 2003).
σB in L. monocytogenes also controls expression of the transcriptional activator, PrfA. Of
the two promoters directly upstream of the gene encoding for PrfA, P1prfA is σB dependent
(Fig. 6) (Nadon et al., 2002). Loss of σB reduces its invasiveness in human intestinal
epithelial cells through down-regulation of PrfA and InlA (Kim et al., 2004).
4.2 PrfA Rapid transcriptional regulation of gene expression in L. monocytogenes is an essential
mechanism for adaptation in new environments. Positive regulatory factor A (PrfA ) was
among the 201 putative transcriptional activators identified in L. monocytogenes after a
genome sequence study (Glaser et al., 2001). PrfA is a 233-amino-acid long protein that
binds to a 14-bp palindromic sequence in the -41 region of genes in the PrfA regulon,
thereby activating their transcription (Mengaud et al., 1989; Sheehan et al., 1996). It was
first identified as a regulatory factor for hly transcription (Leimeister-Wachter et al.,
1990) with the numbers of newly identified virulence genes controlled by PrfA increasing
to date. PrfA is the master regulator of virulence gene expression in L. monocytogenes. It
is a protein member of the cAMP receptor protein (Crp)/catabolite gene activator protein-
fumarate nitrate reductase regulatory (Cap-Fnr) family of bacterial transcription factors
(Lampidis et al., 1994).
PrfA regulates most of the known L. monocytogenes virulence genes (i.e. prfA, plcA, hly,
mpl, actA, plcB, inlA, inlB, inlC, uhpt, bsh, bilE and vip) (Dussurget et al., 2004). Six of
these are well established factors in the successive steps of intracellular growth of L.
monocytogenes, prfA, plcA, hly, mpl, actA and plcB and are physically linked in a 9-kb
chromosomal island termed Listeria pathogenicity island 1 (LIPI-1) (Fig. 5). PrfA is vital
for the transcription of these virulence genes and it is therefore not unexpected that the
PrfA itself is also tightly controlled by multiple mechanisms: 1) transcriptional, 2)
translational and 3) post-translational.
28
Fig. 5. Structure and transcriptional organisation of the PrfA-dependent virulence gene cluster (LIPI-1) and other PrfA-dependent virulence genes in L. monocytogenes. prfA: positive regulator factor A; plcA: phosphatidylinositol-specific phospholipase C; hly: listeriolysin O; mpl: metalloprotease; actA: actin polymerisation protein; plcB: broad range phospholipase C (lecithinase); inlA, inlB: cell wall bound internalins A and B; inlC: small secreted internalin C; uhpT: hexose phosphate transporter; bsh: bile salt hydrolase; bilE: bile exclusion gene; vip: LPXTG-anchored cell wall protein. Red arrows indicate induction while the dotted red arrow indicates repression of expression by PrfA. Green arrows indicate the direction and size of the different transcripts.
4.2.1 Transcriptional regulation of prfA expression The transcriptional regulation of the prfA gene is mediated by three promoters (Fig. 6).
Two promoters, P1prfA and P2prfA are located upstream of the monocistronic prfA
transcript. Another promoter, PplcA leads to the production of a bicistronic plcA-prfA
transcript (Camilli et al., 1993; Freitag and Portnoy, 1994).
The transcripts generated by the P1 and P2 promoters are vital to achieve basal PrfA
expression. Further expression of prfA is governed by the PrfA-dependent plcA promoter.
Fig. 6. Transcriptional regulation of prfA. Three promoters that contribute to prfA expression; PplcA, P1prfA, P2prfA.
29
Both P1 and P2 promoters of prfA are required by L. monocytogenes to escape from the
vacuole after internalisation (Freitag and Portnoy, 1994), and the PlcA-mediated prfA
expression of the plcA-prfA transcript for cell-to-cell spread (Camilli et al., 1993). Both
P1 and P2 promoters of prfA are transcribed by RNA polymerase with the housekeeping
Sigma factor, σA. However, the P2 promoter has also been shown to be transcribed by the
stress response sigma factor σB with overlapping σA and σB binding consensus sites
(Schwab et al., 2005). Expression of the bicistronic transcript requires PrfA whereas both
the P1 and P2 promoters of prfA are negatively controlled by PrfA (Freitag et al., 1993)
(Fig. 6).
4.2.2 Translational regulation of prfA expression
Fig. 7. Translational regulation of prfA. Transcript originating from P1prfA promoter has a region that forms a closed stem loop structure masking the Shine-Dalgarno (SD) at temperatures below 30°C. At 37°C, the closed stem loop structure is unstable allowing the ribosome to bind to the SD and initialise translation.
Temperature regulated expression of virulence genes in several bacteria has been known
since a decade ago (Konkel and Tilly, 2000). Temperature plays a vital role in the
expression of L. monocytogenes virulence genes. The mechanism of thermosensing was
not known until 2002, when Johansson and colleagues demonstrated that the 5´-
untranslated RNA upstream of the coding mRNA forms a secondary structure at lower
temperatures (such as 30°C) masking the Shine-Dalgarno (SD) site and inhibiting
translation (Fig. 7) (Johansson et al., 2002).
This temperature regulated region, termed “thermosensor”, forms a more flexible
secondary structure at higher temperatures (such as 37°C), with the SD region exposed
30
allowing the ribosome to bind the SD and initialising translation. The thermosensor
mechanism has so far only been found at the P1prfA, and not P2prfA or PplcA, suggesting that
these promoters could have alternative roles to play in the regulation of PrfA at lower
temperatures. In vitro and in vivo studies in cells of Drosophila melanogaster at 25°C and
30°C showed that at lower temperatures, L. monocytogenes is still capable of expressing
some virulence genes (Cheng and Portnoy, 2003; Mansfield et al., 2003) (Loh et al.
unpublished data). It could be speculated that at lower temperatures, other yet
undiscovered mechanisms, could be regulating the virulence genes when expression of
PrfA is low.
4.2.3 Post-translational regulation of PrfA To date, although some information is known, the full mechanism of post-translational
regulation of PrfA in L. monocytogenes is yet to be elucidated. Medium composition has
been shown to determine expression of PrfA-dependent genes. Starvation conditions
(minimal essential (MEM) medium) (Bohne et al., 1994) or nutrient rich conditions
(brain-heart infusion (BHI) medium) supplemented with active charcoal (Ripio et al.,
1996) induce the PrfA regulon. PrfA switching between a transcriptionally active to an
inactive form upon binding/releasing a hypothetical factor has been proposed (Renzoni et
al., 1997; Ripio et al., 1996; Vega et al., 1998). The true identity of a hypothetical PrfA
co-factor is still yet to be discovered, although observations from different experiments
have linked PrfA activity to the availability of nutrients. One such system is the
phosphoenolpyruvate phosphotransferase system (PTS) where the uptake of non-
phosphorylated sugars is controlled via this system. It has been shown that PrfA-
dependent gene expression is directly influenced by the phosphorylation status of the PTS
permeases (Stoll et al., 2008). Upon uptake of these unphosphorylated carbohydrates,
they receive phosphate groups from enzyme II (EII) domain A. This will in turn
accumulate the amount of non-phosphorylated EIIA that has been postulated to sequester
PrfA activity. When L. monocytogenes is within its host, it utilises a non-PTS-dependent
transport system, such as the Hpt transporter (Freitag et al., 2009) for the acquisition of
carbohydrate. In this case, EIIA remains phosphorylated, alleviating repression of PrfA.
31
From structural and functional studies of PrfA mutants, it was found that PrfA requires
conformational changes in order to bind and regulate PrfA-dependent genes (Eiting et al.,
2005; Vega et al., 2004). This suggests that binding to a co-factor within the binding
pocket of PrfA is vital for its activity (Eiting et al., 2005; Velge et al., 2007). It has been
proposed that the PTS system might affect the synthesis of this co-factor in L.
monocytogenes, analogous to the CRP system binding its co-factor, cAMP in Escherichia
coli (E. coli) (Gorke and Vogel, 2008).
5. RNA regulation As mentioned above, bacteria such as L. monocytogenes possess many different survival
strategies, ranging from sensing nutrients and proliferating in different environments to
evading host immune defences. They can adapt to harsh conditions by modifying their
gene expression. Gene regulation in bacteria has long been studied; however the main
focus has been placed on elucidating how proteins act as regulators responding to
environmental stimuli.
It has become evident that RNAs have much more diverse functions than previously
anticipated. Previously, it was thought that RNA only come in three categories: tRNA,
rRNA and mRNA, and that RNA do not play a large role other than assisting in the
process of protein synthesis from DNA. Today, there is another category of RNA termed
non-coding RNAs (ncRNAs). These ncRNAs are found in higher organisms such as
humans, worms, plants to the lower organisms such as bacteria. These RNA molecules
that can function as regulators were first identified in bacteria long before the first
microRNAs (miRNAs) and short interfering RNAs (siRNAs) were discovered in
eukaryotes.
ncRNAs in bacteria are categorised into two major classes: 5´-UTR regions of mRNA
that control downstream genes (cis-acting) and ncRNAs that bind protein or control other
distally located target RNA (trans-acting). 5´-UTRs affect cis gene expression by
adopting different conformational changes in response to cellular and/or environmental
signals such as nutritional depravation (stalling ribosomes, uncharged tRNA),
temperature (thermosensors), pH and small molecule ligands (riboswitches). Trans-acting
RNAs are classified into two major categories that work by sequestration of proteins or
32
by controlling target mRNA expression by affecting translation and/or target mRNA
stability. There are also a number of ncRNAs that carry out specific housekeeping
functions, such as: 4.5S RNA of the signal recognition particle, RNase P for processing
tRNAs, and tmRNA which is important to resolve stalled ribosomes.
5.1 cis-acting RNAs Numerous ncRNAs are embedded in the 5´-UTRs in front of protein coding mRNA.
These 5´-UTRs affect their downstream mRNA expression in various ways:
1) Nutritional depravation (stalling ribosomes, uncharged tRNA)
2) Temperature (thermosensors)
3) pH
4) Small molecule ligands (riboswitches)
5.1.1 Nutritional depravation The first attenuation mechanism was shown to control E. coli tryptophan synthesis
(Yanofsky, 1981). The set of genes responsible for the production of tryptophan is known
as the trp operon, which control the biosynthesis of tryptophan in the cell from the initial
precursor, chorismic acid. This operon is a repressible system that consists of a repressor,
a promoter, an operator and structural genes. There are two mechanisms of negative
feedback in the trp operon. The repressor for the trp operon is produced upstream by the
trpR gene which is consecutively expressed at a low level. The tetramers of the TrpR
protein are inactive and free within the cell. When tryptophan is present, it binds to the
tryptophan repressor causing a change in conformation, which allows the repressor to
bind the operator. This prevents RNA polymerase from binding to and transcribing the
operon. When tryptophan is not present, the repressor is in its native conformation and
cannot bind the operator region, allowing expression of the trp-operon.
33
Fig. 8. Attenuation of the trp operon. The 5´of trpE gene contains 4 domains. When the cellular levels of tryptophan are low, translation of a leader peptide is slow, allowing domain 2 and 3 to bind with each others thereby continuing transcription. When cellular levels of tryptophan are high, there are higher levels of tryptophan tRNA thereby increasing the translation process. This quicker process allows domain 3 to bind with domain 4 forming a stem-loop structure terminating transcription.
The trp operon has a leader peptide sequence in the 5´-UTR of the trpE gene containing
several trp-codons. This 5´-UTR also contains 4 domains (Fig. 8). Domain three
(nucleotide 108-121) of the mRNA can base pair with either domain two (nucleotide 74-
94) or domain four (nucleotide 126-134). If domain three pairs with domain four, a stem
loop structure is formed on the mRNA, terminating transcription. If domain three pairs
with domain two, no stem loop structure will be formed, thus allowing transcription of
the downstream mRNA. When the cellular level of tryptophan is high, the level of
transfer RNA (tRNA) charged with tryptophan is also high, preventing the ribosome from
stalling at the trp-operon located at the 5´ end of the transcript. The quick translation
process enables the ribosome complex to bind to domain two, allowing domain three and
four to form the stem loop structure thus terminating transcription. If the ribosome
attempts to translate this peptide while tryptophan levels in the cell are low, it will stall at
either domain one or two, which contain several trp codons. While it is stalled, the
ribosome physically shields domain one of the transcript allowing domain two to pair
with domain three, as the ribosome complex is not associated with domain two. This
structure allows transcription to continue and when trpE-A genes are translated, the
biosynthesis of tryptophan occurs.
34
5.1.2 pH Recently, a new class of cis-acting RNAs, the pH-responsive riboregulator, has joined the
ever growing ncRNA family (Nechooshtan et al., 2009). The alx gene in E. coli encodes
a putative transporter and its expression is induced at high alkaline conditions. The RNA
region preceding the alx open reading frame (ORF) has been identified as a pH-
responsive element sensing different pH conditions. Under normal growth conditions (pH
7.0), the 5´-UTR of alx forms a translationally inactive structure, whereas under high
alkaline conditions, an alternative structure that is translationally active is formed. The
“active structure” formation occurs when transcription is in progress during alkaline
condition. At high pH condition, the RNA polymerase stalls at two different sites,
interferes with the formation of the “inactive structure”, thereby promoting the active
structure formation.
5.1.3 Thermosensors Temperature is an important parameter to monitor for the bacteria. This sensing
mechanism is vital especially for pathogens to fine-tune gene expression in the
environment and in the host. As mentioned in Chapter 4.2.2, L. monocytogenes possesses
a “thermosensor”. The P1prfA UTR in front of the mRNA of prfA forms a secondary
structure at lower temperature, masking the Shine-Dalgarno (SD) site and inhibiting
translation. It forms an alternative secondary structure at higher temperature (such as
37°C), with the SD region exposed allowing translation of PrfA (Johansson et al., 2002).
The control of gene expression by an RNA thermosensor is not just restricted to L.
monocytogenes. Thermosensors have been identified in different bacteria such as E. coli
(Morita et al., 1999a; Morita et al., 1999b), the plant symbiont Bradyrhizobium
japanicum (Narberhaus et al., 1996), Salmonella (Waldminghaus et al., 2005) and
Yersinia (Hoe et al., 1992). The expression of many genes essential for growth at higher
temperatures is dependent on the heat-shock factor σH, encoded by rpoH in E. coli. The
translation of rpoH is controlled by a thermosensor mechanism. Two regions in the rpoH
mRNA can interact to form a secondary structure preventing translation at low
temperature (Morita et al., 1999a; Morita et al., 1999b). At increased temperatures, this
35
structure is weakened and the sequence downstream of the AUG start codon is accessible
to the ribosome, allowing translation initiation.
Recently, a thermosensor regulating E. coli cold-shock protein (CspA) has been
identified (Giuliodori et al., 2010). At 37°C, the 5´-UTR and the nucleotides of cspA
coding region form a secondary structure sequestering both the SD site and the AUG start
codon. At lower temperatures (10°C), the 5´-UTR and the cspA coding region form a
totally different secondary structure where the SD site is presented at the tip of a stem
loop. The new structure possesses a pseudoknot and its formation has been speculated to
be an important component of this RNA thermosensor. Besides the 5´-UTR and the
beginning of the codon region of cspA mRNA, its 3´-end also undergoes structural
modification during temperature switching and has recently been found to bind to Hfq in
vitro (Hankins et al., 2010).
Besides the well known thermosensor regulation of prfA, there are least three PrfA
regulated virulence genes in L. monocytogenes that require an intact 5´-UTR for maximal
expression; inlA, actA and hly (Shen and Higgins, 2005; Stritzker et al., 2005; Wong et
al., 2004). Truncated mutations within the 5´-UTRs decrease the expression of these gene
products, indicating the importance of their respective UTRs. The increased level of InlA
but not its transcript level in a ∆inlH mutant has linked inlH expression to inlA post-
transcriptional regulation (Personnic et al., 2010). The 5´-UTR of the hly mRNA has
been shown to be important especially during infection, suggesting that it might be used
for sensing its environment (Shen and Higgins, 2005). A similar mechanism could be
applied to actA, as the expression level of ActA is high within its host (Moors et al.,
1999). In the case of actA, when various mutations are introduced, the 5´-UTR structure
is severely disrupted, leading to a decreased expression of ActA. This indicates a role of
the structure for adequate ActA expression (Wong et al., 2004).
5.1.4 Riboswitches Some 5´-UTRs can bind small molecules directly, which induce structural changes in the
RNA (Mandal and Breaker, 2004; Montange and Batey, 2008; Nudler and Mironov,
2004). Generally, these 5´-UTRs termed riboswitches directly regulate the genes
encoding proteins involved in the uptake and usage/synthesis of the metabolite. The
36
number and variety of riboswitches identified in bacteria are increasing. Riboswitches
have also been identified in fungi, green algae and plants (Bocobza and Aharoni, 2008).
In Bacillus subtilis, as many as 2% of the genes are regulated by different classes of
riboswitches. Generally, riboswitches consist of two domains: the aptamer region that
binds the ligand and the expression platform which regulates gene expression by
controlling either transcription or translation through RNA structural alterations (Mandal
and Breaker, 2004; Montange and Batey, 2008; Nudler and Mironov, 2004). There are
over 10 distinct classes of riboswitches identified to date as well as a putative riboswitch
that binds molybdenum cofactor “Moco RNA motif” (Regulski et al., 2008; Weinberg et
al., 2007).
The first riboswitch to be identified in Bacillus subtilis was the S-
adenosylmethionine (SAM) riboswitch (SAM-I). An S-box riboswitch binds its effector
molecule SAM to form a terminator structure that interrupts transcription and inhibits
synthesis of the downstream mRNA. In the absence of SAM, the S-box riboswitch forms
an anti-terminator structure, allowing continuous transcription. (Fig. 9) (McDaniel et al.,
2003; Whitford et al., 2009; Winkler et al., 2003). S-box riboswitches were identified and
characterised using genetic, biochemical and bioinformatic methods. With genetic and
mutational analysis, it was proven that conserved S-box riboswitch structures function in
a transcription termination manner (Auger et al., 2002; Grundy and Henkin, 1998).
Eventually, it was demonstrated that SAM was the effector molecule binding directly to
the aptamer region of the S-box riboswitch (Winkler et al., 2003). Four additional RNA-
structures binding SAM were subsequently discovered and termed: SAM-II (Corbino et
al., 2005), SAM-III (Fuchs et al., 2006), SAM-IV (Weinberg et al., 2008) and SAM-V
(Poiata et al., 2009). These five classes of S-box riboswitches demonstrate that it is
possible for the bacteria to sense the same effector molecule, i.e., SAM, to control
expression of a variety of genes. SAM-I and SAM-II riboswitch have not been found in
the same species (Barrick and Breaker, 2007). Thus, despite sharing the same effector
molecule, S-box riboswitches appear to have evolved independently through evolution.
37
Fig. 9. Mode of action of an S-box riboswitch. During high SAM conditions, an S-box riboswitch binds its effector molecule SAM (Green circle), thereby forming a terminator structure that interrupts transcription and inhibits synthesis of the downstream mRNA. In the absence of SAM, the S-box riboswitch forms an antiterminator structure allowing continuous transcription and subsequently translation of methionine biosynthesis protein.
5.1.4.1 Molecular recognition of SAM The S-box aptamer was first examined using RNA in-line probing in the presence of
SAM and other SAM analogues (Corbino et al., 2005; Lim et al., 2006; Soukup and
Breaker, 1999; Winkler et al., 2003). The dissociation constant (Kd) values of the ligands
were obtained by observing the structural changes of the riboswitch when these ligands
bind (Soukup et al., 2001). The Kd value is a measurement used to describe the affinity
between two molecules i.e. how tightly a ligand binds to a particular RNA. A low
dissociation constant indicates that the ligand binds the RNA with high affinity. The
minimal aptamer domain of the SAM-I riboswitch in the yitJ mRNA of B. subtilis has a
Kd value of ~4 nM for SAM, whereas the SAM-II aptamer of Agrobacterium tumefaciens
has a Kd value of ~1 µM (Corbino et al., 2005). Binding of SAM to the S-box riboswitch
is extremely specific. Both SAM-I and SAM-II bind to SAM and discriminate against S-
adenosylhomocysteine (SAH) with a Kd value difference of ~100- and ~1000-fold,
respectively (Corbino et al., 2005; Winkler et al., 2003). Studies have shown that by
modifying the amino acid, sugar and nucleobase moieties of SAM, the binding affinity to
SAM-I and SAM-II is significantly reduced (Corbino et al., 2005; Lim et al., 2006). This
38
suggests that all of these functional groups on the ligand are essential for the molecular
recognition and binding by the S-box riboswitch.
5.1.4.2 Structure of a SAM riboswitch In 2006, the structure of a SAM-I aptamer from Thermoanaerobacter tengcongensis in
complex with SAM at 2.0-Å resolution was published (Montange and Batey, 2006). This
aptamer contains all conserved nucleotides and secondary structure features which are
common to all SAM-I riboswitches. The SAM-I aptamer structure contains two pairs of
coaxially stacked helices (P1 with P4 and P2 with P3) and they are packed together at an
angle of ~ 70 degrees. There is a kink-turn motif within the P2 loop that redirects the
orientation of this P2 loop by roughly 100 degrees to form a pseudoknot between the
terminal loop of P2 and the junction connecting P3 and P4 (J3/J4) (Klein et al., 2001;
McDaniel et al., 2005; Winkler et al., 2001). With the help of the kink-turn and the
pseudoknot formation, the highly conserved P1 and the lower part of P3 loop are brought
together, forming a space in which SAM can nicely fit. Briefly, the RNA aptamer forms a
pocket interacting with SAM by utilising van der Waals forces. As mentioned above, it
was shown that all the functional groups of SAM are required for proper binding with the
RNA aptamer (Lim et al., 2006; Winkler et al., 2003).
5.1.4.3 SAM riboswitch-mediated gene regulation Aptamers from the same riboswitch can be combined with different expression platforms.
In Bacillus subtilis, there are eleven SAM-I riboswitches and they all form transcriptional
terminators in the presence of SAM (McDaniel et al., 2003; Tomsic et al., 2008; Winkler
et al., 2003). Many of these expression platforms can also form the anti-terminator
structure. In the case of SAM-I riboswitches, absence of SAM allows the more stable
anti-terminator structure to form and transcription to proceed. The terminators of SAM-I
riboswitches are strong stem-loops usually followed by six or more U residues, which
abort transcription in the 5´-UTR. This mechanism allows the riboswitch to repress gene
expression by transcriptional termination before the coding region of mRNA to save
“energy”. However, the SAM-I riboswitches of bacteria from other species have been
39
predicted to regulate gene expression by control of translation initiation (Barrick and
Breaker, 2007; Rodionov et al., 2004).
One SAM-I riboswitch in Clostridium acetobutylicum resides directly downstream and
on the opposite DNA strand of genes coding for proteins converting methionine to
cysteine. In this regulation, the riboswitch controls transcription of a transcript that is
believed to interfere with the coding transcript through antisense RNA (Barrick and
Breaker, 2007; Rodionov et al., 2004). In the absence of SAM, the read-through form of
the riboswitch is thought to be formed by an antisense interaction that represses
expression of the gene involved in the conversion of methionine to cysteine. When SAM
is abundant, transcription could terminate and the antisense arrangement would be
inverted, allowing the gene for converting methionine to be expressed.
In general, the aptamer region of a riboswitch binds its respective metabolite and the
structure of the regulatory domain changes. This structural alteration can form or disrupt
transcriptional terminators, or block or expose the SD region (Fig. 10).
Fig. 10. Typical gene arrangement of riboswitches. Upon binding to its ligand, structural conformation changes of the riboswitches lead to; A) Stem-loop formation terminating transcription, B) Anti-terminator formation allowing continuous transcription, C) SD site inaccessible, thereby terminating translation, D) SD site is exposed, initiating translation.
5.1.4.4 Other types of riboswitches Besides classical riboswitches, there are riboswitches with the same aptamer domain that
mediate different regulatory mechanisms (i.e the cobolamin riboswitch). Cobalamin is the
40
coenzyme form of vitamin B12. Binding of cobolamin to the riboswitch forces the
formation of a terminator, stopping transcription of the btuB gene in Gram-positive
bacteria whereas binding controls translation initiation of the cob operon in Gram-
negative bacteria (Barrick and Breaker, 2007; Franklund and Kadner, 1997; Nahvi et al.,
2002; Vitreschak et al., 2003).
In addition, there is a riboswitch that acts as a ribozyme to catalyse self cleavage. This
glucosamine-6-phosphate (glmS) riboswitch cleaves itself upon binding to the effector
molecule. This inactivates expression of the downstream mRNA that codes for proteins
required for the synthesis of glucosamine-6-phosphate. Hence, the glmS ribozyme
generates a negative feedback loop by sensing glucosamine-6-phosphate (Collins et al.,
2007).
5.2 trans-acting RNAs As mentioned in the beginning of Chapter 5, trans-acting RNAs are classified into two
major categories that work by either sequestration of protein or by controlling target
mRNA expression by modifying translation and/or target mRNA stability.
5.2.1 ncRNAs that sequester proteins Some regulatory RNAs act by binding to proteins to modify their activity. Three protein-
binding ncRNAs (CrsB and 6S RNA) act by binding to a protein and sequestering its
activity.
5.2.1.1 CsrB and CsrC Carbon storage regulator, B and C (CsrB and CsrC) are ncRNAs that bind to and regulate
the carbon storage protein CsrA in E. coli. The CsrA protein binds to a GGA motif in the
5´-UTR of target mRNAs, affecting their translation and/or stability. CsrB and CsrC each
contain 22 and 13 multiple GGA-binding sites for CsrA respectively (Babitzke and
Romeo, 2007). When the level of CsrB and CsrC increases, they bind to the CsrA protein
and sequester it from its target mRNA. The level of CsrA has been suggested to exist in
equilibrium with CsrB ncRNA or CsrA target mRNA (Romeo, 1998). CsrB could be a
41
regulator for CsrA protein activity but also a “carrier”, as the CsrA protein aggregates
without CsrB ncRNA. CsrA also positively regulates csrB transcription, thereby forming
an autoregulatory circuit (Gudapaty et al., 2001). The stability of these ncRNAs, CsrB
and CsrC, are regulated by the CsrD protein, a cyclic di-GMP-binding protein that
recruits RNase E to degrade the ncRNAs (Fig. 11) (Suzuki et al., 2006).
Fig. 11. Mode of action of CsrB and CsrC. A) When levels of CsrB and CsrC are low, CsrA binds to its target mRNAs affecting their translation/stability. B) When levels of CsrB and CsrC are high, CsrA binds to them instead of its target mRNA and thereby allowing target mRNAs to be translated. Unbound CsrB and CsrC are degraded by CsrD protein and RNase E.
5.2.1.2 6S RNA 6S RNA was originally identified as a transcript transcribed at stationary phase (Hsu et al.
1985). The 6S RNA forms a tertiary structure resembling a sigma 70 (σ70) open complex
promoter. Therefore, it can associate with the σ70-containing RNA polymerase and
initiate transcription (Trotochaud and Wassarman, 2004; Wassarman and Saecker, 2006;
Wassarman and Storz, 2000). When the 6S RNA level is high, i.e., stationary phase, it
binds to σ70 but not to σS-containing RNAP holoenzymes (Trotochaud and Wassarman,
2005). The interaction between 6S RNA and σ70 allows transcription of σS -dependent
genes essential for survival at stationary phase. This is the only ncRNA that acts
transcriptionally rather than post-transcriptionally.
42
Two 6S RNA have been identified in B. subtilis, although their functions are yet to be
elucidated. One of the 6S RNA homologues shows maximal expression at stationary
phase (similar to E. coli) and the other at mid-logarithmic phase.
5.2.2 ncRNAs that bind to target mRNAs In contrast to the few protein-sequestering ncRNAs, most of the ncRNAs identified to
date act as antisense regulators by base pairing to their target mRNAs and affecting their
stability or translational efficiency. Base pairing can either be exerted by, cis- or trans-
encoded ncRNAs.
I) cis-encoded ncRNAs are encoded on the DNA strand opposite the target mRNAs and
are therefore fully complementary to their target, usually 75 nucleotides or more (Brantl,
2007; Wagner et al., 2002). Despite being encoded in the same region, these two
transcripts are uniquely transcribed from their own promoters and function in trans as
diffusible molecules. Most of the known cis-encoded ncRNAs are found in
bacteriophages, plasmids, transposons and toxin/antitoxin genes (Brantl, 2007; Wagner et
al., 2002). One example is the OhsC and IstR ncRNAs mechanism discovered in E. coli
(Fozo et al., 2008; Gerdes and Wagner, 2007). Although not bona fide antisense RNAs,
these two ncRNAs are encoded directly adjacent to genes encoding for toxic proteins.
They are constitutively expressed and have a rather short complementary sequence (19
and 23 nucleotides) respective to the toxin mRNAs. These ncRNAs can bind to their
target mRNAs promoting degradation and/or repressing translation and thereby
regulating the level of toxins. Fine-tuning of this toxin-antitoxin mechanism is required
as moderate levels of toxin slow the growth of bacteria. The slower growth process may
be essential for the repair and survival of the bacteria in a new environment (Kawano et
al., 2007; Unoson and Wagner, 2008). It has been proposed that the antitoxin is essential
for bacterial defence against foreign plasmids encoding a toxin.
Other cis-encoded ncRNAs range from the stationary phase-induced GadY in E. coli to
the virulence plasmid pJM1 of Vibrio anguillarum. GadY functions by base pairing with
gadXW mRNA in an antisense manner. This leads to cleavage of the RNA:RNA complex
between the gadX and the gadW gene, increasing the level of gadX transcript (Opdyke et
43
al., 2004; Tramonti et al., 2008). The RNAβ ncRNA from the pJM1 is expressed on the
other strand of the fatDCBAangRT operon. Expression of RNAβ terminates the fatA gene
by blocking transcription thereby reducing expression of the downstream angRT mRNA
(Stork et al., 2007).
II) trans-encoded ncRNAs are generally found at loci far from their target mRNAs and
hence share only limited complementarity with them. Most of the known trans-encoded
ncRNAs function negatively (Aiba, 2007; Gottesman, 2005). The base pairing process
between a trans-encoded ncRNA and its target mRNA usually induces mRNA
degradation and/or disrupts protein production by translational inhibition. In many cases,
these ncRNAs interact with both their target mRNA as well as the RNA-chaperone Hfq
(see below). In this chapter, two of the best known ncRNAs will be briefly described;
DsrA and RyhB.
5.2.3 DsrA One well known trans-encoded ncRNA is DsrA. It is 87 nucleotides long, expressed from
a single promoter ending with a Rho-independent terminator. DsrA was originally shown
to regulate colonic acid capsule synthesis (Sledjeski and Gottesman, 1995). It was later
shown to negatively regulate expression of the global transcriptional silencer H-NS and
positively regulate translation of the stationary phase sigma factor, σS, by directly base
pairing to the hns and rpoS target mRNAs (Lease et al., 1998; Majdalani et al., 1998;
Sledjeski et al., 1996).
Fig. 13. Sequence and proposed secondary structure of DsrA RNA
44
Temperature is vital as it also determines the rate of transcription and stability of the
DsrA transcript (Repoila and Gottesman, 2001). In the absence of DsrA, the rpoS 5´-UTR
forms a repressive structure, closing the rpoS SD site thereby blocking translation.
Binding of DsrA to the rpoS upstream region opens up the SD site, allowing binding of
the ribosome and subsequently translation initiation (Majdalani et al., 1998) (Fig 14). The
DsrA:rpoS interaction requires the RNA chaperone protein Hfq, which binds to two
different interaction surfaces (Mikulecky et al., 2004) as Hfq promotes the opening of the
rpoS closed 5´-UTR, allowing DsrA to bind to the anti-SD site (Arluison et al., 2007;
Soper and Woodson, 2008). Hfq is displaced rapidly by conformational changes in DsrA,
leaving DsrA on the rpoS mRNA to perform its regulatory effects (Lease and Woodson,
2004). RNase III (see below), which is the major double-strand specific nuclease, has
been proposed to have dual functions in this regulation (Resch et al., 2008). It processes
rpoS-mRNA in the absence of DsrA and it also cleaves the DsrA-rpoS duplex.
Fig. 14. Dual-actions of DsrA. Left: In the absence of DsrA, rpoS-UTR forms a structure masking the SD site thereby blocking translation. In the presence of DsrA, the first loop of DsrA binds to the rpoS-UTR opening its SD thereby initiating translation. Right: In the absence of DsrA, the SD site on hns-mRNA is uninterrupted thereby translation can progress. In the presence of DsrA, the binding of the second loop of DsrA to hns-mRNA occludes the SD site, terminating translation. The second loop on the DsrA RNA regulates translation of the global transcriptional
silencer H-NS. DsrA RNA has a rather mild repressing effect on H-NS translation by
binding to the complementary region in the hns mRNA including the SD site. This
interaction blocks the accessibility of the ribosome to the SD site thus blocking
translation (Fig. 14) (Lease and Belfort, 2000).
45
Genes encoding stress response proteins and virulence factors in pathogenic E. coli have
been known to be regulated by RpoS and H-NS. One study has shown that dsrA mutants
seem to be acid resistant (Lease et al., 2004). This suggests that DsrA might be involved
in virulence of E. coli although the direct interaction between DsrA and its targets
involved in the virulence process are yet to be elucidated.
5.2.4 RyhB Iron is an essential element for all living organisms ranging from humans to bacteria.
Although iron is abundant in the environment, it is usually inaccessible due to its
chemical properties rendering it poorly soluble under aerobic conditions at neutral pH.
Iron in the form of ferric ion (Fe2+) is highly sought after by microorganisms. Bacterial
environmental sensing typically includes mechanisms for iron acquisition and regulation.
The fine-tuning of iron concentrations is vital, since Fe2+ is highly reactive and too high
concentrations cause damage to many components in the bacteria, leading to death. The
intracellular concentration of Fe2+ is regulated by the ferric uptake regulator (Fur) protein,
a DNA-binding protein that represses transcription of genes involved in the acquisition of
iron when iron is abundant (Hantke, 2001). Besides regulating genes involved in iron
uptake, Fur also represses the transcription of the RyhB ncRNA (Masse et al., 2007). The
discovery that RyhB possesses a Fur consensus binding site overlapping its -10 box in the
putative promoter suggests that RyhB is involved in regulating iron homeostasis in E. coli.
RyhB was originally identified in a genome search for novel ncRNAs in E. coli
(Wassarman et al., 2001). RyhB is involved in iron metabolism in E. coli by
downregulating genes encoding for non-essential Fe2+-utilising proteins (Masse and
Gottesman, 2002). The ryhB gene lies in the intergenic region between two genes; yhhX
and yhhY (Argaman et al., 2001; Wassarman et al., 2001). RyhB is expressed at late
stationary phase as well as in minimal medium and its regulation is Hfq dependent
(Masse et al., 2003). In the absence of Hfq, RyhB RNA is unstable and subjected to
degradation by RNase E. RyhB lowers the level of six transcripts which previously were
discovered to be positively regulated by Fur (Fig.15) (Masse and Gottesman, 2002;
Masse et al., 2005). Due to its complementary sequence, RyhB exerts it action on each of
these six transcripts by direct base pairing. When iron is scarce, Fur is depleted of Fe2+
46
and is inactivated. This allows RyhB to be transcribed and interact with its target mRNAs,
exemplified by the sodB mRNA (Vecerek et al., 2003). The RNA chaperone Hfq
regulates the interaction by binding to the sodB mRNA, opening a loop on the transcript
thereby allowing binding of RyhB (Geissmann and Touati, 2004). After base pairing,
both RyhB and sodB mRNA are immediately subjected to degradation by RNase E
(Masse et al., 2003). This enables a very rapid down-regulation of these six genes coding
for non-essential Fe2+-utilising proteins when the bacterium is in an iron-depleted
environment. In contrast, when the iron concentration is high, this regulation is reversed
rapidly.
Fig. 15. Mode of action of RyhB. When iron (Fe2+) is abundant, it binds and activates FUR thereby repressing rhyB and allowing translation of target mRNAs. When Fe2+ is limited, FUR is inactive therefore allowing transcription of ryhB. RyhB binds to its target mRNAs with the assistant of Hfq leading to degration by RNAse E.
Homologues of RyhB have been identified in several pathogens: Vibrio cholerae (Mey et
al., 2005), Salmonella (Masse and Gottesman, 2002) and Shigella (Oglesby et al., 2005).
In Pseudomonas aeruginosa, two functional homologues of RyhB were identified; PrrF1
and PrrF2 (Wilderman et al., 2004). These two ncRNAs have been found to function
similarly to RyhB, by base pairing to their target mRNA leading to degradation. The
involvement of Hfq in the interaction of PrrF1 and PrrF2 with their target mRNA remains
to be elucidated.
47
5.3 Protein factors important for the function of trans-encoded ncRNAs
5.3.1 Hfq In many cases, the RNA-binding chaperone Hfq is required to mediate the interactions
between the ncRNA and its target mRNA. Hfq is necessary to facilitate the RNA-RNA
interaction despite poor complementarity between the ncRNA and the mRNA (Aiba,
2007; Brennan and Link, 2007; Chao and Vogel, 2010; Valentin-Hansen et al., 2004).
Hfq was originally discovered as a host factor needed for replication of the Qβ RNA
bacteriophage (Franze de Fernandez et al., 1968). In Escherichia coli, the hfq gene is part
of a complex superoperon (amiB-mutL-miaA-hfq-hflX-hflK-hflC). It contains 3 σ32-
dependent heat shock promoters and 4 σ70-dependent promoters (Tsui and Winkler, 1994;
Valentin-Hansen et al., 2004). In E. coli, the Hfq protein is 11.2 kDa, heat-stable and
present in 30,000-60,000 molecules per exponentially growing cell (Kajitani and
Ishihama, 1991). The level of Hfq peaks at early exponential growth phase and is
correlated to its growth rate (Ali Azam et al., 1999; Kajitani et al., 1994). Besides
binding to RNA, Hfq has also been found to bind to DNA (Azam and Ishihama, 1999),
70S ribosomes through the 30S ribosome and RNA polymerase (DuBow et al., 1977;
Sukhodolets and Garges, 2003).
The structure of Hfq was first described in Staphylococcus aureus (Schumacher et al.,
2002) and later confirmed with a truncated version of Hfq in E. coli (Sauter et al., 2003)
and full-length Hfq in Pseudomonas aeruginosa (Nikulin et al., 2005). The protein forms
a doughnut-shaped structure consisting of two homo-hexameric rings. Hfq is homologous
to Sm and Sm-like proteins (Moller et al., 2002; Zhang et al., 2002), which are involved
in splicing and mRNA decay in eukaryotes. The crystal structure of Hfq bound to RNA
showed the preference of single-stranded A/U-rich sequences for the interaction
(Schumacher et al., 2002). The main function of Hfq is believed to be a platform that
facilitate binding of ncRNAs to their target mRNAs as well as increasing the local
concentrations of ncRNAs and mRNAs (Aiba, 2007; Brennan and Link, 2007; Chao and
Vogel, 2010; Valentin-Hansen et al., 2004).
Most of the ncRNAs are less stable in the absence of Hfq as it seems to protect ncRNAs
from degradation in the absence of base pairing with their target mRNAs. Upon base
48
pairing, Hfq may also function to induce RNA degradation by recruiting RNase E or
other components of the degradosome.
Hfq in L. monocytogenes is an 8.7-kDa protein encoded on a region similar to that of E.
coli, but only containing the miaA and hflX genes. Transcription of hfq is induced under
various conditions; osmotic stress, ethanol stress and early stationary phase (Christiansen
et al., 2004). σB is directly involved in the regulation of hfq transcription, indicating its
involvement in Listeria survival under stress conditions. The Hfq proteins of E. coli and
L. monocytogenes have recently been shown to have the same basic properties. When
Listeria Hfq was introduced into an ∆hfq background in E. coli, it could restore the
effects of two ncRNAs, DsrA and RyhB (Nielsen et al., 2010).
Not all ncRNAs seem to require extra assistance from Hfq. It has been shown that an
ncRNA found in Vibrio cholerae, termed VrrA, that represses the expression of outer
membrane protein A (OmpA) still can function in the absence of Hfq (Song et al., 2008).
The interaction is, however, slightly weaker in a ∆hfq mutant strain than in the wild-type
strain. Hfq homologues are found in almost half of all eubacterial species, many of which
are pathogens (Chao and Vogel, 2010)
5.3.2 RNase E and RNase III Ribonuclease E (RNase E) is a large, 1061-aminoacid multidomain protein. It is an
endonuclease recognising monophosphate transcripts. RNase E can be divided into two
sections, the N-terminus with a catalytic domain and the C-terminus with a long
unstructured domain of no known function. The catalytic site of RNase E is structurally
related to DNase I. X-ray crystallography revealed that the RNase E structure is a
homotetramer in the form of a dimer of dimers (Callaghan et al., 2005). RNase E is the
backbone of a protein complex called the degradosome and is the principal ribonuclease
in E. coli. This ribonuclease plays an important role in the process of post-transcriptional
regulation of gene expression by ncRNAs (Gottesman, 2004; Wagner et al., 2002).
Briefly, RNase E inactivates polyribosomal mRNA by cleaving within the translational
initiation site or within the inter-cistronic regions of polycistronic mRNAs. Once
translation of the truncated mRNA is finished, RNase E further cleaves the ribosome-free
mRNA. One of the very well known ncRNAs, RyhB, utilises Hfq to protect itself from
49
being degraded by RNase E. Upon binding to its target mRNA, sodB, translation is
terminated and both RyhB and sodB are later degraded in a coordinated manner mediated
by RNase E (Geissmann and Touati, 2004). Possibly, the base pairing allows RNase E or
other degradosome components to get access to sites that otherwise are shielded (Masse
et al., 2003; Morita et al., 2005). sodB mRNA itself is known to be degraded by RNase E.
It is difficult to determine whether RyhB-stimulated degradation is secondary to blocking
translation or a direct consequence of the pairing.
Endonuclease III (RNase III) belongs to the family of dsRNA-specific
endoribonucleases and is active as a dimer of two identical 25.4-kDa polypeptides.
RNase III in E. coli is Mg2+-dependent and cleaves phosphodiester bonds creating 5´-
phosphate and 3´-hydroxyl termini with an overhang of 2 nucleotides (Court, 1993).
ncRNA-mediated gene-regulation has been shown to be dependent on RNase III activity
in many cases, such as with the DsrA ncRNA and its target rpoS-mRNA. In the absence
of DsrA, rpoS-mRNA itself forms a complex secondary structure, masking its own SD
site. RNase III cleaves within the double-stranded segment of this secondary structure,
destabilising the rpoS transcript. In the presence of DsrA, this ncRNA base pairs with the
complementary region of the rpoS SD region, disrupting its original secondary structure
and opening the SD site for translation of RpoS (Repoila et al., 2003). Subsequently,
RNase III can cleave within the DsrA/rpoS duplex but leaves the rpoS mRNA intact as it
is covered by ribosomes.
5.4 RNA-mediated gene regulation in pathogens Gene regulation in pathogenic bacteria has been studied for a long time. However, the
main focus has been to elucidate how proteins regulate virulence and stress-tolerance
genes responding to environmental stimuli. RNA involvement has been underestimated
but is gradually emerging as a major theme in the field of gene regulation. It is not
surprising that ncRNAs are involved in the regulation of virulence genes, as they are
small, cheap, “fast” to produce and could have pleiotropic effects.
50
5.4.1 RNAIII in Staphylococcus aureus Staphylococcus aureus is a Gram-positive human pathogen capable of causing serious
infections, such as septicaemia, endocarditis, osteitis and toxic shock syndrome. One of
its virulence loci is controlled by a “two-component” quorum sensing system encoded by
the agr locus (Novick and Geisinger, 2008). Similar two-component systems have also
been discovered in Streptoccoccus pyogenes and Clostridium perfringens (Kreikemeyer
et al., 2001; Shimizu et al., 2002; Steiner and Malke, 2002). The agr locus in S. aureus
consists of two transcriptional units that are transcribed from divergent promoters, P2 and
P3. The P3 promoter is responsible for the transcription of a 512 nt-long transcript,
RNAIII. RNAIII also encodes a δ-haemolysin protein (encoded by hld) at its 5´-end
(Janzon et al., 1989), making RNAIII a bi-functional molecule.
Fig. 16. Mode of actions of RNAIII. Upper panel: The agr operon. The transcription of RNAIII and its 5´-encoded δ-haemolysin is activated by the response regulator AgrA upon phosphorylation of the signal sensor AgrC. Lower panel: In the absence of RNAIII, hla-mRNA forms a secondary structure masking the SD site thereby blocking translation. The binding of RNAIII to hla-mRNA opens the SD site and allows initiation of translation.
The P2 promoter is responsible for transcribing four genes: agrB, D, C, A(Fig. 16). agrA
and agrC encode the classical two-component signal transduction system, whereas agrB
encodes a transporter and agrD encodes the auto-inducing peptide (AIP). Expression of
RNAIII peaks at late logarithmic and stationary phase. At these phases, there are
sufficient levels of AIP that can bind to the signal sensor AgrC. Upon binding, AgrC
phosphorylates the response regulator AgrA, leading to activation of RNAIII expression.
51
It has been suggested that RNAIII is able to form 14-stem loop structures (Benito et al.,
2000). Specific domains of RNAIII are capable of base pairing with complementary
RNA sequences within their targets (Novick et al., 1993). The 5´-end of RNAIII base
pairs upstream of the SD site of one its target mRNAs, hla (encoding α-haemolysin)
(Morfeldt et al., 1995; Novick et al., 1993). Upon base pairing, the SD site of the hla
transcript becomes accessible to the ribosome, allowing translation of the haemolysin
protein (Fig 16). RNAIII is also known to regulate another target mRNA, spa (encoding
the IgG-binding protein, protein A). RNAIII mediates inhibition of translation upon base
pairing, which induces degradation of the spa mRNA by RNase III (Huntzinger et al.,
2005). These studies have revealed that RNAIII binds to its target through loop-loop
interactions (kissing-complex) in an interaction not requiring Hfq.
5.4.2 VrrA in Vibrio cholerae Vibrio cholerae is a Gram-negative human pathogen causing acute diarrhoeal disease. It
remains a major public health problem as cholera affects five million people and causes
two hundred thousand deaths per year. The main virulence factor in V. cholerae is the
cholera toxin (CT), encoded by the ctxAB operon. There are 36 ncRNAs predicted in V.
cholerae as identified in a bioinformatics study (Livny et al., 2008). 9 have been
investigated and they were found to be involved in quorum sensing and biofilm formation
(Hammer and Bassler, 2003; Kovacikova and Skorupski, 2002; Zhu et al., 2002). Hfq has
also been found to be required for quorum sensing repression and involved in the
regulation of virulence gene expression (Lenz et al., 2004). The 140-nt long ncRNA
termed VrrA (Vibrio regulatory RNA of OmpA), was recently identified and found to be
conserved among Vibrio species (Song et al., 2008). VrrA acts as a repressor of the
synthesis of an outer membrane porin, OmpA. The expression of VrrA is activated by the
alternative stress sigma factor, σE. During outer membrane stress conditions, VrrA limits
the synthesis of the abundant OmpA protein by base pairing with the ompA mRNA,
blocking the SD site. As mentioned previously, the interaction between VrrA and ompA
is not stringently Hfq dependent (Song et al., 2008). VrrA is the first ncRNA known to be
involved in the production of outer membrane vesicles (OMVs).
52
It was shown that a vrrA mutant was able to colonise the intestine of infant mice 5-fold
better than the wild-type strain (Song et al., 2008). Toxin coregulated pilus subunit
(TcpA) level was significantly reduced when VrrA was overexpressed (Song et al., 2008).
The combined results suggest that VrrA is involved in the survival of bacteria in different
environments and that it also modify host infection.
5.4.3 LhrA-C in Listeria monocytogenesAs mentioned in the first chapter, L. monocytogenes is a Gram-positive human pathogen
capable of causing several potentially fatal diseases. In the beginning of 2009, 21
ncRNAs had been found in L. monocytogenes (Christiansen et al., 2004; Mandin et al.,
2007). Three of these ncRNAs termed LhrA, B and C, were originally discovered by co-
immunoprecipitation with the Hfq protein (Christiansen et al., 2006). Among the three
ncRNAs, only LhrA acts like a classical Hfq-binding ncRNA. LhrA was recently further
characterised. It is now known that LhrA regulates one target mRNA, lmo0850, encoding
a hypothetical protein, by base pairing (Nielsen et al., 2010). LhrA binds to the upstream
region of lmo0850, masking its SD site thus inhibiting translation. Hfq seems to be
essential for the LhrA-mediated lmo0850 regulation by stabilising the LhrA transcript. In
vitro experiments showed that Hfq stimulates the formation of a duplex between LhrA
and lmo0850 mRNA. This is the first time Hfq has been found to be directly involved in
mediating an ncRNA interaction in a Gram-positive bacterium. However, the function of
Hfq in Gram-positive bacteria is still very limited. In contrast to Gram-negative bacteria,
the deletion of Hfq in S. aureus did not show any apparent phenotypes (Bohn et al.,
2007). A similar effect has been seen in other Gram-positive bacteria such as B. subtilis
(Heidrich et al., 2006; Preis et al., 2009; Silvaggi et al., 2006). Some bacteria such as the
Gram-positive lactic acid bacteria belonging to the genus Lactobacillus, lack hfq or hfq-
homologues completely (Chao and Vogel, 2010).
53
Aims of this thesis
The main aims of my thesis work were to elucidate the functions of novel ncRNAs and RNA-mediated virulence gene regulation in L. monocytogenes.
Specific aims:
• To establish the involvement of the first 20-codons of prfA-mRNA for translation (Paper I).
• To investigate whether an S-box riboswitch could function in trans as an ncRNA (Paper II) .
• To elucidate whether 5´-UTRs of virulence and virulence-associated mRNA in L. monocytogenes are subjected to post-transcriptional regulation (Paper III) .
• To set up a coordinated transcriptional map of L. monocytogenes grown under different conditions (i.e. from saprophytism to virulence) (Paper IV).
• To perhaps identify novel ncRNAs or RNA-related elements involved in L. monocytogenes virulence. (Paper IV).
54
Results and Discussion
In this section, I will present and discuss the main results obtained during my thesis work.
Data represented in the papers of the thesis are referred to by their Roman numerals (I-
IV) and corresponding figure numbers.
6.0 PrfA
6.1 Novel regulation of PrfA As mentioned in the previous chapter, PrfA is the master regulator of virulence gene
expression in L. monocytogenes and its regulation is tightly controlled by multiple
mechanisms acting: 1) transcriptionally, 2) translationally and 3) post-translationally.
6.1.1 Length of the prfA coding sequence correlates to its expression levelIt was shown that 6 codons (18 bases) of the prfA mRNA was sufficient for the
thermosensor to function, when fused upstream of gfp (Johansson et al., 2002). With this
knowledge, we decided to construct fusions containing different lengths of the prfA
mRNA to study its effect on thermosensing (originally designed for developing a thermo-
inducible translational system for Mycobacteria spp). Four different codon lengths; 1
codon (3 bases), 4 codons (12 bases), 9 codons (27 bases) and 20 codons (60 bases) were
fused upstream of a lacZ reporter gene. Beta-galactosidase (β-gal) assay and Western
blotting were performed with results indicating a direct correlation between β-gal
expression levels with the length of the prfA-coding sequences (Fig. 1A, Paper I).
Hypothetically, the lacZ-mRNA could interfere with the prfA-coding region causing the
differences observed. 1 or 20 prfA-codons were inserted upstream of the gfp reporter gene.
Similar differences in expression between the prfA1-gfp and prfA20-gfp compared to the
prfA1-lacZ and prfA20-lacZ were observed by both Western blotting and fluorescence
plate experiments, arguing against an effect by the reporter gene. The expression
difference between the 1 and 20 codon constructs could theoretically be linked to the
respective transcripts stability, with the short transcript being less stable compared to the
55
long transcript, leading to an altered amount of translational product. The RNA
expression of the fusions was tested by rifampicin experiments to detect their half-lives.
We could not detect any differences in the stability between the prfA1-gfp and prfA20-gfp
transcripts, with both having the same half-life of approximately 8 minutes (Fig 3, Paper
I) . All of the effects observed so far were in E. coli. Therefore, the prfA1-gfp and the
prfA20-gfp fusions were introduced into their native host L. monocytogenes. The amount
of PrfA20-GFP was higher than the amount of PrfA1-GFP, in a similar fashion to the
difference observed in E. coli (Compare Fig. 2B. and 4, Paper I). It could be speculated
that the 19 additional codons in the prfA20 construct somehow stabilise the protein. The
stability of the prfA1-lacZ and prfA20-lacZ was investigated by protein stability
experiments. The results indicated that the PrfA20-LacZ had the same half-life as the
PrfA1-LacZ. Using in vitro studies, we examined if factors within the transcript itself
were responsible for the expression differences. A similar method was used by us in
another study (Paper II) . In brief, prfA1-gfp and the prfA20-gfp plasmid were in vitro
transcribed and translated before quantification by Western blotting. PrfA20-GFP showed
a much higher level compared with PrfA1-GFP (Fig. 6, Paper I), similar to prfA1-gfp and
prfA20-gfp in both E. coli and L. monocytogenes. β-lactamase was used as a control as it is
encoded on the same plasmid as prfA-gfp. The expression of β-lactamase did not differ
between the samples (Fig. 6, Paper I), indicating that the increased expression of PrfA20-
GFP was not due to a general up-regulation of the plasmid.
6.1.2 Possible mechanisms? Compiling our above data, we know that the difference in expression observed between
the 1 codon and 20 codon constructs was not due to altered transcription or the stability
of prfA mRNA nor the stability of the PrfA protein. Also, a similar expression pattern
was observed in L. monocytogenes as well as in in vitro experiments. These results
strongly argue that the mechanism giving a different expression of PrfA1 or PrfA20-codon
constructs resides within the transcript itself.
In other cases where the 5´-part of the coding region has proven important for the
translation, three different mechanisms have been suggested. These are: 1) downstream
box, 2) specific bases/codons and 3) RNA secondary structures.
56
The initiation of protein synthesis in E. coli requires a range of features of the mRNA to
form the ribosomal binding site (RBS) and to direct ribosomes to the correct translational
start site (Dreyfus, 1988; Gold, 1988; McCarthy and Brimacombe, 1994). The efficiency
of the RBS in some cases has been suggested to be dependent on the nucleotide +15 to
+26 of the coding region, termed “downstream box” (DB) (Sprengart et al., 1990). The
DB is suggested to base pair with the 16S rRNA, although this hypothesis is still
controversial. We investigated the different prfA mRNA constructs, but failed to identify
sequences complementary to the 16S rRNA, ruling out the involvement of a DB.
Codon usage bias is due to the difference in the frequency of occurrence of synonymous
codons in the bacterium’s genomic DNA. Fast-growing bacteria tend to use optimal
codons to achieve faster translation rate with high accuracy. There are many
mathematical methods proposed to measure codon usage bias and one of them is “codon
adaptation index” (CAI). We used the CAI measurement to investigate whether the
shorter construct with less translational product harbours an excess of rare codons.
Although the CAI values were slightly lower for the shorter constructs, all constructs had
CAI values higher than 0.5. We also failed to observe a correlation between expression
and polyadenines (Brock et al., 2007), CA repeats (Martin-Farmer and Janssen, 1999) or
specific codons. Our results indicate that the difference in expression observed between
the constructs is not due to the presence of rare/certain codons or stretches of bases.
It has also been suggested that a strong secondary structure at the 5´-end of the coding
sequence will have a negative effect on translation due to a difficulty for the ribosome to
bind the SD site (Kudla et al., 2009). Therefore, the strength of the secondary structures
in our constructs was measured using the RNA Vienna software. Our results showed an
inverse relationship between the expression of the PrfA-fusion with the stability of their
RNA secondary structure (Fig. 7, Paper I).
We are still unclear on why such mechanism exists. The secondary mRNA structures of
virulence genes and virulence-associated genes were studied and compared. Despite
being less stable, the RNA secondary structures of virulence mRNAs were not
significantly lower than control mRNAs. For prfA, the first 12 codons appear to have
evolved to maintain a loose RNA secondary structure, harbouring an excess of As and Us
(Fig. 8, Paper I). This was also observed for RNA thermosensors in other bacterial
57
species. We believe that such a mechanism ensures maximal translation as soon as the
appropriate temperature is reached.
6.2 Another factor involved in the regulation of PrfA? Besides the well known translational regulation of prfA mRNA by temperature
(thermosensor) (Johansson et al., 2002), we also discovered a novel S-box riboswitch that
functions as an ncRNA and controls expression of PrfA in L. monocytogenes (Paper II)
(Loh et al., 2009).
6.2.1 S-box riboswitches in L. monocytogenesL. monocytogenes harbours seven putative SAM-I riboswitches that were identified by a
tiling array study (Paper IV). These seven SAM-I riboswitches were designated SreA-G
(SAM riboswitch element). SreA-G are situated upstream of genes encoding proteins
involved in methionine and cysteine transport/metabolism. Due to their abundance and
length, we were interested to know whether a terminated S-box riboswitch (upon binding
to SAM) could still have an extra function by acting as an ncRNA. Briefly, we
constructed a sreA mutant and conducted a transcriptome analysis comparing the sreA
mutant with the wild-type strain (Table S2, Paper II). Among the genes that showed
differences in expression level, two of them were selected for further studies: lmo2230-
encoding a protein homologous to bacterial arsenate reductase and lmo0049 encoding the
listerial homologue of the S. aureus autoinducing peptide, AIP. We were able to
complement the expression level of these two genes with Northern blots and quantitative
PCRs (qPCR) by expressing SreA on a plasmid in the ∆sreA strain (Fig. 1, Paper II).
Due to high identity among SAM-I riboswitches sequences, SreB was also tested and
shown to regulate expression of lmo2230, but not lmo0049. (Fig. 1, Paper II). It could be
speculated that there are more unbound SAM in the ∆sreA strain, thereby causing the
effect observed. A riboswitch element, which is unable to bind SAM but still able to form
a terminator stem loop was constructed, (Fig. 2A, Paper II). The strain was able to
restore the expression of lmo2230 to the wild-type levels (Fig. 2B, Paper II), indicating
that the sequences and structures of SreA and not the level of SAM that caused the effect
58
observed on lmo2230 expression. Regulation of lmo2230 is known to be controlled by
two regulators, σB and PrfA. We observed a two-fold up-regulation of PrfA in the ∆sreA
strain. Expression of PrfA was restored to wild-type levels in the complemented strain
(Fig. 3A, Paper II). A similar pattern was also observed when expression of a PrfA-
regulated mRNA, hly was tested in these strains (Fig. 3B, Paper II). Putative PrfA-
binding sites were observed upstream of both SreA and SreB and expression of both
SreA and SreB was reduced in a∆prfA strain (Fig. 4A, Paper II). These findings
generated a complex regulatory cycle where a high level of PrfA activates SreA
transcription, which in turn down-regulates prfA transcription (Fig. 3 and 4, Paper II).
The expression of PrfA and PrfA-regulation genes is higher once L. monocytogenes has
adhered to and/or invaded into its host cell (Moors et al., 1999; Renzoni et al., 1999;
Scortti et al., 2007; Shetron-Rama et al., 2002). In line with this, expression of SreA was
seven-fold higher when L. monocytogenes was intracellular compared to extracellular
(Fig 4B, Paper II). It is hard not to speculate that while L. monocytogenes is inside its
host, the upregulation of SreA could serve as a component to fine-tune the expression
PrfA.
6.2.2 A direct interaction between SreA and the 5´-UTR of prfADirect base pairing between ncRNAs and their target mRNA, have been shown in many
different bacterial species (see introduction). We continued our investigation on how
SreA controls prfA expression by analysing the SreA sequence and the 5´-UTR region of
prfA. To our delight, the searches revealed a high degree of complementary between the
loop-3 of SreA and the 5´-distal side of the prfA-UTR (Fig. 5A, Paper II). An ectopic
system (E. coli) was selected to investigate this interaction as E. coli does not harbour
any SAM-I riboswitches or prfA. A plasmid expressing a prfAWT-gfp fusion (Johansson et
al., 2002) and a plasmid harbouring SreA, psreAWT were used to monitor a putative
interaction. GFP expression was measured by direct fluorescence measurement on agar
plates and Western blots with antibodies raised against GFP. In the presence of psreAWT,
the fluorescence intensity and GFP-expression was greatly reduced (Fig. 5B, C Paper II).
In addition, SreB was also able to suppress the expression of PrfA (Fig. 5D, Paper II).
To identify the interaction site between SreA and prfA-UTR, base substitution mutations
59
were introduced (Fig. 5A, Paper II). Such base substitutions should theoretically weaken
the repressive interaction. Indeed, the result showed that the repressive effect of psreAWT
was almost abolished when the SreAM1 was expressed in the presence of prfAWT-gfp (Fig.
5C, Paper II).
6.2.3 Temperature involvement? Expression of PrfA is greatly dependent on temperature (Johansson et al., 2002). We
therefore investigated whether temperature would affect the repressive effect of SreA on
PrfA expression. SreA could not repress PrfA expression at 30°C, in contrast to 37°C
(Fig. 5E, Paper II).
6.2.4 In vitro studies The interaction between SreA and prfA-UTR was further investigated by in vitro
experiment gel-shifts and in vitro transcription/translation assays. By gel-shift
experiments, it was observed that SreAWT was able to cause the prfAwt-UTR fragment to
shift, whereas no shift was detected when SreAWT was added to prfAM1*-UTR. The shift
could be restored when both SreAM1 and prfAM1*-UTR were added together (Fig. 6A,
Paper II) . This suggests that SreA and prfA-UTR interact directly and the region M1 and
M1* is essential for the interaction. In addition, the expression of PrfA-GFP was shown
to be reduced in the presence of SreA in an in vitro transcription/translation assay (Fig.
6B, Paper II).
6.2.5 Novel function of S-box riboswitches regulating PrfA translation Our in vivo and in vitro results indicate that SreA can function as an ncRNA and regulate
its target mRNA (prfA-UTR) by a direct RNA:RNA interaction. This is supported by the
findings that SreA show a high complementary to the 5´-UTR of the prfA mRNA, the
ability of SreA to interact with and repress expression of prfA in E. coli and by in vitro
experiments. Although SAM does not seem to be involved in the interaction between
SreA and prfA, we cannot rule out that SAM is part of the SreA:prfA complex. Our
finding has unravelled a novel regulatory mechanism linking cis- and trans-acting
60
ncRNAs together. Many pathogenic bacteria possess riboswitches and we propose that
these riboswitches should be studied further and be considered as potential trans-acting
RNAs. It would not be surprising if other pathogens also possess such a mechanism in
regulating their virulence genes.
Fig. 17. Schematic summary of Paper I, II and III.
6.3 Characterisation of 5´-UTRs in L. monocytogenesL. monocytogenes uses a wide range of virulence factors for its pathogenesis. It is known
from previous studies that at least three Listeria virulence genes (inlA, hly and actA) are
regulated by their 5´-UTRs (Shen and Higgins, 2005; Stritzker et al., 2005; Wong et al.,
2004). These findings together with the identification of the prfA-UTR thermosensor,
prompted us to investigate the presence of long 5´-UTRs upstream of L. monocytogenes
virulence and virulence-associated genes (surface proteins), as whether they potentially
could be post-transcriptionally regulated (Paper III) (Loh et al., 2006). Besides the
housekeeping sigma factor σA, searches were undertaken to identify σ
B consensus site
61
(see Introduction) within the promoter regions of the virulence-associated mRNAs. Using
a computational method, many of the virulence and virulence-associated genes were
suggested to contain σB consensus binding sites (Table 2, Paper III). A σB binding site
together with a σA binding site would allow the bacteria to switch transcriptional start
sites depending on the conditions, generating different transcript lengths. Our work
identified 8 of the virulence factors to harbour a 5´-UTR of sufficient length for
regulation.
6.3.1 Possible mechanisms for 5´-UTR-mediated regulation We suggest that Gram-positive bacteria such as L. monocytogenes could utilise two
pathways for its 5´-UTR mediated regulation: Stability of messenger or initiation of
translation.
I) Stability of messenger: 5´-UTRs could stabilise their messengers by protecting them
from degradation. The process could be mediated via alternative secondary structures,
binding to target proteins or sequestering the ribosome (Condon, 2003; Mathy et al.,
2007). Sequestration of ribosomes is dependent on stability-SD (STAB-SD), which
function as false SD-site. When the ribosome binds to the 5´-end of the UTR, it blocks
ribonuclease accessibility, thereby stabilising the transcript. One example is the presence
of a STAB-SD site at the 5´-UTR of inlAB mRNA, which is suggested to be important for
transcript stability (Agaisse and Lereclus, 1996). Our in silico study revealed that some L.
monocytogenes virulence genes; inlA, uhpT, inlC together with a virulence-associated
gene; lmo0842 harbour the STAB-SD sequence (Fig. S1, Paper III).
II) Initiation of translation: Metabolites binding 5´-UTRs (riboswitches, depending on
temperature as well as pH) (see introduction) can alter the UTR secondary structure,
stimulating or obstructing the binding of the ribosome to the SD. We hypothesised that
upon uptake of Listeria into its host cells, there could be putative host factors or bacterial
factors induced by the host, capable of binding to the UTR region and controlling
expression of the downstream virulence genes. For actA, it is known that mutations
within the 5´-UTR of actA greatly affect its secondary structure and disrupt ActA
62
production (Wong et al., 2004). Our study suggests that bsh forms alternative structures,
one with the SD site opened and one closed (Fig. S2, Paper III). This implies that the
expression of bsh is controlled at the level of translation initiation. It is also known that
the UTR of iap (70 nucleotides) forms a structure suggested to mask the SD site (Köhler
et al., 1991) hence it was used as the “minimal size” in our study (Paper III) .
In summary, we have analysed 5´-UTR regions located in front of virulence and
virulence-associated genes (surface proteins) of L. monocytogenes. Our findings suggest
that many of the 5´-UTRs are possibly controlled post-transcriptionally, via interaction
with host or bacterial factors.
7. Switching from saprophytism to virulence Until now, I have only discussed Papers I, II and III, all regarding L. monocytogenes
virulence and virulence-associated mechanisms. Much is known about how L.
monocytogenes regulates its virulence genes during pathogenesis, but not much effort has
been placed into elucidating how the bacterium switches its gene expression from
saprophytism to virulence. From now on, I will be discussing my fourth publication
(Paper IV), in which we performed a transcriptional tiling array to investigate RNAs
isolated from L. monocytogenes grown under various conditions. The tiling microarrays
were made of 25-base oligonucleotides with a 9-nucleotide overlap, covering both strands
of the L. monocytogenes chromosome. From the study, we discovered that L.
monocytogenes expresses more than 98% of its open reading frames (ORFs) under any
conditions with 517 polycistronic operons coding for 1719 genes.
7.1 Novel ncRNAs in L. monocytogenes Prior to the publication of Paper IV, 21 ncRNAs in L. monocytogenes had been
identified (Fig. 1B, C, Paper IV), where 5 were absent from L. innocua and 3 acted as
antisense RNAs (Christiansen et al., 2006; Mandin et al., 2007). 29 novel ncRNAs were
identified from our study, with sizes ranging from 77 to 534 nucleotides. Among these 29
ncRNAs, 15 were absent in L. innocua (possibly involved in virulence) and 5 were
predicted to contain small ORFs with appropriate ribosome-binding sites. PrfA and Hfq
63
do not seem to be involved in the regulation of these ncRNAs, whereas σB was important
for the expression of some (Fig. 1B, Paper IV). One of these newly identified ncRNA
was Rli38. It is absent in L. innocua and had approximately a 25-fold increased
expression in blood (Fig 1C, 2A, Paper IV). Rli38 was also induced when bacteria were
grown in the presence of hydrogen peroxide, H2O2 (Fig. 18).
Fig. 18. Expression of Rli38 during various stress conditions. Cells were grown in BHI before the onset of each stress-condition (NaCl (0.5 M), H2O2 (0.15%), Triton X-100 (2%), EtOH (5%), 10°C and pH 2.5).
In this study, we generated a ∆rli38 mutant and orally fed it to humanised E-cadherin
knock-in mice. The ∆rli38 mutant strain was attenuated, suggesting a role of Rli38 in
virulence. Various organs were harvested and the results indicated a general trend of
lower bacterial count for the ∆rli38 strain compared with the EGDe strain in all of the
organs tested (Fig. 2 right panel, Paper IV), again emphasising the involvement of
Rli38 in L. monocytogenes virulence.
7.2 Novel cis-regulatory RNA elements in L. monocytogenesIn addition to the discovery of new ncRNAs, we also identified 13 new long 5´-UTRs.
These cis-regulatory RNA elements can be long 5´-UTRs (paper III) . One newly
identified 5´-UTR was located in front of mpl, which is located upstream of actA. A short
transcript of the mpl was identified (starting from mpl transcription start site and stopping
just upstream of mpl RBS), supporting our previous work (Paper III) as being involved in
post-transcriptional regulation. 42 riboswitches were annotated in L. monocytogenes prior
to this study, but none of them have been experimentally tested except SreA (Paper II) .
In this study, we identified 40 riboswitches and studied their on and off states under
different growth conditions (Supplementary Table 2, Paper IV). We chose to study the
lysine riboswitch (LysRs) in more detail. Briefly, besides functioning as a “classical
64
riboswitch” regulating lysine, LysR was found to also act as a transcriptional terminator
of an upstream gene (Fig. 2C, D, Paper IV).
7.3 Other discoveries
7.3.1 Overlapping antisense UTRs and normal antisense RNAs We also identified 9 long overlapping antisense lying 3´-UTRs of genes on the opposite
strand. Transcription of these overlapping antisense 3´-UTRs end either inside or after the
coding operon located on the opposite strand. There are 4 antisense RNAs with long
overlapping 5´-UTRs, where transcription starts from promoters that are located within
the ORF of the opposite strand (Fig. 3, Paper IV).
7.3.2 Antisense regulation of flagellar biosynthesis MogR is a transcriptional repressor of the temperature-regulated flagellum genes in L.
monocytogenes. In this study, we identified 2 promoters for mogR: P1 and P2 (Fig. 4A, B,
Paper IV). P1 contained a σB consensus binding box and generated a long 5´-UTR that
lies antisense of three genes required for the synthesis of the Listeria flagellum (Fig. 4A
and B, Paper IV). P2 was located just upstream of the ATG of mogR, and was
constitutively expressed. Both promoters share a common Rho-independent
transcriptional terminator (Fig 4B, Paper IV). Transcription from P1 was not affected by
temperature, although it was overexpressed at stationary phase and not expressed in a
∆sigB mutant (Fig. 4D, Paper IV). A ∆sigB strain was previously shown to be more
motile at lower temperature, in support of our finding. We suggest that the increased
motility of a ∆sigB strain is due to a lower P1-mediated transcription, generating less
antisense RNA and mogR repressor transcript.
7.4 Transcriptional changes from saprophytism to virulence In this paper, we also analysed protein coding gene expression using the gene-expression
array present on the same chip. Venn diagrams were used to analyse differentially
expressed genes under five conditions (Fig 5, Paper IV). Briefly, we discovered that
PrfA and its virulence gene regulon seem to be involved in L. monocytogenes survival
65
and replication within the blood. Several ncRNAs present only in L. monocytogenes
exhibit the same expression pattern as the virulence genes, suggesting a role for them in
virulence. SigB was found to be more important for its adaptation within the intestinal
surroundings, also controlling expression of several virulence genes. Finally, this study
has revealed and categorised the coordinated global transcriptional process of L.
monocytogenes from the environment to the infectious site.
66
Concluding remarks
• The first 20 codons of prfA-mRNA were shown to be essential for
efficient translation in L. monocytogenes.
• An S-adenosylmethionine binding riboswitch (SreA) was shown to be
able to act in trans, controlling the expression of the virulence gene
transcriptional activator PrfA in L. monocytogenes.
• SreA acts by binding to the prfA-UTR, thereby repressing its
translation.
• Virulence- and virulence-associated mRNAs preceded by 5´-UTRs of
sufficient length in L. monocytogenes could control expression their
downstream mRNA post-transcriptionally.
• For the first time, tiling arrays were used to investigate RNAs isolated
from L. monocytogenes strains grown under various conditions; in
vitro, in vivo and ex vivo.
• The Tiling arrays revealed: 29 novel ncRNAs, several putative
riboswitches, riboswitches that are able to act as terminators for
upstream genes and antisense RNAs lying on the other strand of open-
reading frames.
67
Acknowledgements Holy mother of Jesus!!!!!
Believe it or not, I actually did not write this section first as many people would have done..... but then again, Edmund is not really a ”normal” guy, is he? ☺ What can I say here......hmmmmmmm...... I am soon done!!!!!!!!! (If you are reading this after the 12th
of May 2010, then I AM DONE!!!!!!)
The first person I would like to thank who had ”contributed” to my PhD study here in Umeå is Daniel Arvidsson (NOT YOU JÖRGEN!!!!! ☺). It seems like just yesterday when we first knew each other…….. 1998 wasn’t it? Travelling to England to visit me and I finally came to Umeå for holiday in the summer of 2000. I fell in love with this very charming little town. People are so friendly, beautiful lakes (fishing and BBQing) and most important of all ....Sunlight!!!!!!!!! (It is not surprising when you have lived in England for some time and you start craving for some glimpse of sunlight). Meeting new friends and their families here in Umeå made me want to stay even more. Since 2000, I have been visiting Umeå at least twice a year, (Summer for fishing, Winter for rolling down the snowy hill aka ”skiing”) until august 2003 when I finally moved to Umeå to start my Biomedicine Research school. (Thank you Vicky and Dan H. for picking me to fill 1 of the 12 places among the 60 to 70 odd applicants) I met a lot of new friends when I was studying in the research school and would like to thank those who I still have contacts with; Jim Silver, Anders Persson and Mia Sundström. You guys were fantastic and thank you for making that one year so wonderful for me. We had to do three different research rotations in three different groups during that one year of research school. For me: (Grp Ivor Tittawella, Grp Jörgen Johansson and Grp Tommy Olsson). I would like to thank Ivor for helping and guiding me in the lab and thank you for telling Jörgen that I was a super good student when he came to you and spied on me (Ivor told me….. Jörgen…... I have a lot more spies in the department!!! So, be careful!! ☺) . My third rotation was in Grp. Tommy Olsson, there I met Sven-Anders Bergström (Svenne). We had a wonderful 3 months together in the lab (and out of the lab), coming back during weekends to work and found fun in the lab (making cacao cream ice-cream with liquid nitrogen, bringing antibiotics to try to save you and Sofia’s gold fish), getting drunk with you at your student dorm and going to Fysikgränd during brännbollsyran (climbing over police fences, meant to block out minors and fell down……. the police were just laughing at me.. and didn’t even asked for my ID afterwards… ). HAAHHAHHAHA ……………. Now, it’s your turn Jörgen… ☺ Recently, it has become evident………..I did my second rotation of my research school in Jörgen Johansson’s group (Dec-2003 to Feb-2004) and later started my PhD studies till now (May, 2010). It was originally Anna Arnqvist who recommended me to Jörgen and I owe her a lot now!!! Jörgen is one of the most wonderful people you can ever meet… He just came back from Pasteur in Paris when I started my research project with him…. He had a bruise on his face, and he told me he got it while fighting someone in France and I actually BELIEVED him that time.. HAHAHAHAH ☺ Jörgen is my mentor but most important of all…. he is also a friend to me who I can go to and talk anything with…. ranging from bullshit to science… There are times when things were really tough
68
during my PhD time, he was the pillar of the lab…. pushing everything back up in a straight line again and cheered me up when I needed it the most…Usually PhD students are a bit reluctant to give their mobile numbers to their supervisors as they might be called back during weekends for extra work and so on.. I think Jörgen might have regretted for exchanging his phone number with me.. I think I called him more than he ever called me… (Jörgen!!!! I have results!!!!!!!!!!! or I have some questions, or I am stucked!!!) … Well, he had his revenges as well, calling me when I was travelling (03:00… when I was “zzzzzzzzzz” in Chicago, and… talked for 30 mins about work), (04:30 in Kuala lumpur when I was “zzzzzzzz” to tell me that he and the group members were drunk…… celebrating our Cell paper……..), (called me to help him with his snowplough and later setting up Berit ’s green house) (In this case I was rewarded with hamburgers☺)(You still owe me a lot of Viktors!!!!) (Thanks to Berit also for supplying the cookies and biscuits stored in your office).. I need to thank Simon and Alexandra for picking up the phone and giving it to your pappa (E: Hej, Simon/Alex… hur e det? S/A:PAPPA!!!! DET ÄR EDMUND!!!!) People always tell me that I am so lucky to have Jörgen as my supervisor and I totally agree with them!!!
What about the people within the group J.J. then? Jonas G. (my partner in crime in the lab)……. What to say about you……. Well, I need to thank you a lot for the enjoyable time we spent in and out of the lab together…. We have travelled so much together, had so much wonderful times during parties, fishing, bastu”ing” and much more…. I need to thank your fiancée Malin as well… for the wonderful dinners, BBQs and parties we all had together. Karolis - thank you so much for our fishing trips together, listening to me in the lab and discussions about science and other gossip (Do not discuss privatisation of healthcare system with him, unless you want to see him raise his voice☺). I need to thank Rima as well as baby Greta for the great time we have spent together. Teresa T.-she is almost like my “personality -twin” in the group, we can discuss from everything to nothing (when either one of us is hungry/grumpy) ☺ Thank you for all the time we have had in the lab and parties (I still do not remember you winning over me at Karaoke). Sakura N.- Thank you for being a good friend in the lab, we can also talk about science to discussions about tea from China or Ceylon…. And also the parties and dinners we have had together. Christopher A./Grasshopper- thank you for the time we spent playing squash and in the lab discussing science and politics ☺ Past and present members of the group Micke, Jessica, Nina, Aoife and Johnny. Big thanx!!! Our Journal club!!!! I really need to thank Bernt Eric for all the help during our JC and wed. seminars. You always managed to ask important questions that I had overseen. Sun- for talks about our S.E. Asian food + cultures. Monica for always helping me whenever I needed to “borrow” anything from Grp BEU. Thanks to Barbro L . for all the time we spent together, Hjortron and mushroom-picking trips…. (she cooks excellent Pea-soup!!!!!!). (Morning fika gang- Stina, Connie, Annika S, Hyun-Sook, TianYan, Ryoma, occasionally Ting and Anna Å)- Thank you for the time we all spent together fika”ing” and also for those wonderful cookies and cakes). Jurate!!! Thank you for keeping me updated with all the latest juicy gossips and most of all …the time you and Darius spent with us fishing and also dinner/wines/snacks from Lithuania. Patricia—thank you for the lunches and jokes we had together. Ex-member such as Claudia aka Dr Müller - thank
69
you for all the time we spent together, singing, going to IKEA and most of all your “feuerzangbole”. ☺ Carlos-for singing with your deep voice and your jokes! Micke S.- Really thank you for being my friend, I really enjoyed our time together.. I think you are the only one out from our Listeria group who knows the most about my project. I really enjoyed our discussions together, also thank you for all the ice-fishing, normal fishing trips!!!!! (I am still waiting for my dinner for hooking you up with Sandra ☺). P.S. remember our promise! We will try to see each other in Umeå within a few years! you know what I mean ;) Maria L. aka my personal trainer- Thank you for the time we spent together at IKSU, my place and your place (Wine and food but mostly wine! ☺) Group SvB- Thank you Sven B. for being there whenever I needed something signed (Quickly with no questions asked ☺)Past and present members of the group (Betty- for all the time we spent together, Marie A ., Pär o Anna, Jenny o Christer (for making our daily lives so interesting), Ingela, Mari , Elin for the lunches and talks we had at the lunch room. Marija P. for the Latvian Vodka!! ☺ Patrik - for being positive all the time. Ignas B. for being charming always, calm and cool to chat with!! ☺ Group UvPR- Ulrich -for the old times we had back in the old lab. Jenny- thank you for the time we spent together in the old office and when will you get your driving licence? (I WILL “ SOON” - ☺). People from the department (present or past); such as Tiago (thanks for the picture of Listeria), Stefanie, Stefan “North” , Maria N . (For allowing me to use your office for the thesis writing), Lisandro, Olena - Увидимся завтра ☺ , Gosia (I will return your driving license theory book soon!!), Sara C.- for introducing me to Nationskören, Chen, Joanna, Viktoria , Hande, Erik T. (for the trips and good times we spent together),Ming, Christina S. (all the training and chat we had.. and also our encounter with “hehe” mannen), Sofia (For our teaching together!!!) Barbara (you are up next!), Elin I ., Miss bear-foot ☺, Tomas (for taking care of the beer corner now) the Drosophila/cancer gang; Linus, Chaz, Anna, Sa-Chen, Linn, Andreas, Sara R. and many more!!!! Big thanks! ☺ To my friends from the BacRNAs and Europathogenomics (NoE) as well. Special thanks to Marita , Ethel, Berit , Maria and Anitha for making my life easier in the department.. ☺ thank you for helping me with all the official stuffs …..Ethel- for our chats in your office. Thank you Media and Disken for making solutions and cleaning for us, without you guys, we could not survive in the department!!! Johnny for helping me with all the purchases. Mariella for cleaning and also making sure we are on our best behaviour in cleanliness.
I also would like to thank my Umeå gang that I usually hang out with- Mattias E. Mattias J., Sara, Catarina (I always forget whether is it C or K ☺), Johan S., Johan L.for always being there and all the crazy parties, movies and fika”s” we’ve had together… Nu ska jag byta till min "patetiska" Svenska, jag vill även tacka Eva och Micke för att jag fått bo hos er under sommar och vinter när jag hälsade på, tack för alla julklappar och julmiddagar vi har tillbringat tillsammans. Tack även till Kalle och Kickie för julklappar och julmiddagar i Hörnefors. Stort tack till Daniels farmor och mormor för att de tog hand om mig under alla dessa år och behandlade mig som om jag vore en i familjen. Tusen tack till Marie-Louise och Christer för alla middagar, julklappar, resan till sommarstugan i Sörmöjle och körens konsert i Hörnefors-kyrkan.
70
Finally but most important of all, I need thank my MOTHER, for bringing me up and encouraging me no matter what I have chosen to pursue for my future. My
gang of aunties and uncles (you know who you guys are☺), my cousins (Felicia-for the painting, Jason, Raymond, Steve, Fun etc), friends that I have made all around
the world. Without you guys, I am nothing. ALL THE BEST! THANK YOU!!!!!!!
71
References Agaisse, H., and Lereclus, D. (1996). STAB-SD: a Shine-Dalgarno sequence in the 5' untranslated region is a determinant of mRNA stability. Mol Microbiol 20, 633-643.
Aiba, H. (2007). Mechanism of RNA silencing by Hfq-binding small RNAs. Curr Opin Microbiol 10, 134-139.
Ali Azam, T., Iwata, A., Nishimura, A., Ueda, S., and Ishihama, A. (1999). Growth phase-dependent variation in protein composition of the Escherichia coli nucleoid. J Bacteriol 181, 6361-6370.
Alvarez-Dominguez, C., Vazquez-Boland, J.A., Carrasco-Marin, E., Lopez-Mato, P., and Leyva-Cobian, F. (1997). Host cell heparan sulfate proteoglycans mediate attachment and entry of Listeria monocytogenes, and the listerial surface protein ActA is involved in heparan sulfate receptor recognition. Infect Immun 65, 78-88.
Argaman, L., Hershberg, R., Vogel, J., Bejerano, G., Wagner, E.G., Margalit, H., and Altuvia, S.(2001). Novel small RNA-encoding genes in the intergenic regions of Escherichia coli. Curr Biol 11, 941-950.
Arluison, V., Hohng, S., Roy, R., Pellegrini, O., Regnier, P., and Ha, T. (2007). Spectroscopic observation of RNA chaperone activities of Hfq in post-transcriptional regulation by a small non-coding RNA. Nucleic Acids Res 35, 999-1006.
Auerbuch, V., Loureiro, J.J., Gertler, F.B., Theriot, J.A., and Portnoy, D.A. (2003). Ena/VASP proteins contribute to Listeria monocytogenes pathogenesis by controlling temporal and spatial persistence of bacterial actin-based motility. Mol Microbiol 49, 1361-1375.
Auger, S., Yuen, W.H., Danchin, A., and Martin-Verstraete, I. (2002). The metIC operon involved in methionine biosynthesis in Bacillus subtilis is controlled by transcription antitermination. Microbiology148, 507-518.
Azam, T.A., and Ishihama, A. (1999). Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity. J Biol Chem 274, 33105-33113.
Babitzke, P., and Romeo, T. (2007). CsrB sRNA family: sequestration of RNA-binding regulatory proteins. Curr Opin Microbiol 10, 156-163.
Barrick, J.E., and Breaker, R.R. (2007). The distributions, mechanisms, and structures of metabolite-binding riboswitches. Genome Biol 8, R239.
Beauregard, K.E., Lee, K.D., Collier, R.J., and Swanson, J.A. (1997). pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes. J Exp Med 186, 1159-1163.
Becroft, D.M., Farmer, K., Seddon, R.J., Sowden, R., Stewart, J.H., Vines, A., and Wattie, D.A.(1971). Epidemic listeriosis in the newborn. Br Med J 3, 747-751.
Begley, M., Gahan, C.G., and Hill, C. (2002). Bile stress response in Listeria monocytogenes LO28: adaptation, cross-protection, and identification of genetic loci involved in bile resistance. Appl Environ Microbiol 68, 6005-6012.
Begley, M., Hill, C., and Gahan, C.G. (2003). Identification and disruption of btlA, a locus involved in bile tolerance and general stress resistance in Listeria monocytogenes. FEMS Microbiol Lett 218, 31-38.
72
Begley, M., Sleator, R.D., Gahan, C.G., and Hill, C. (2005). Contribution of three bile-associated loci, bsh, pva, and btlB, to gastrointestinal persistence and bile tolerance of Listeria monocytogenes. Infect Immun 73, 894-904.
Benito, Y., Kolb, F.A., Romby, P., Lina, G., Etienne, J., and Vandenesch, F. (2000). Probing the structure of RNAIII, the Staphylococcus aureus agr regulatory RNA, and identification of the RNA domain involved in repression of protein A expression. RNA 6, 668-679.
Bierne, H., Garandeau, C., Pucciarelli, M.G., Sabet, C., Newton, S., Garcia-del Portillo, F., Cossart, P., and Charbit, A. (2004). Sortase B, a new class of sortase in Listeria monocytogenes. J Bacteriol 186, 1972-1982.
Bierne, H., Mazmanian, S.K., Trost, M., Pucciarelli, M.G., Liu, G., Dehoux, P., Jansch, L., Garcia-del Portillo, F., Schneewind, O., and Cossart, P. (2002). Inactivation of the srtA gene in Listeria monocytogenes inhibits anchoring of surface proteins and affects virulence. Mol Microbiol 43, 869-881.
Binnie, C., Lampe, M., and Losick, R. (1986). Gene encoding the sigma 37 species of RNA polymerase sigma factor from Bacillus subtilis. Proc Natl Acad Sci U S A 83, 5943-5947.
Birmingham, C.L., Canadien, V., Gouin, E., Troy, E.B., Yoshimori, T., Cossart, P., Higgins, D.E., and Brumell, J.H. (2007). Listeria monocytogenes evades killing by autophagy during colonization of host cells. Autophagy 3, 442-451.
Bocobza, S.E., and Aharoni, A. (2008). Switching the light on plant riboswitches. Trends Plant Sci 13, 526-533.
Bohn, C., Rigoulay, C., and Bouloc, P. (2007). No detectable effect of RNA-binding protein Hfq absence in Staphylococcus aureus. BMC Microbiol 7, 10.
Bohne, J., Sokolovic, Z., and Goebel, W. (1994). Transcriptional regulation of prfA and PrfA-regulated virulence genes in Listeria monocytogenes. Mol Microbiol 11, 1141-1150.
Borezee, E., Pellegrini, E., Beretti, J.L., and Berche, P. (2001). SvpA, a novel surface virulence-associated protein required for intracellular survival of Listeria monocytogenes. Microbiology 147, 2913-2923.
Boujemaa-Paterski, R., Gouin, E., Hansen, G., Samarin, S., Le Clainche, C., Didry, D., Dehoux, P., Cossart, P., Kocks, C., Carlier, M.F., et al. (2001). Listeria protein ActA mimics WASp family proteins: it activates filament barbed end branching by Arp2/3 complex. Biochemistry 40, 11390-11404.
Brantl, S. (2007). Regulatory mechanisms employed by cis-encoded antisense RNAs. Curr Opin Microbiol10, 102-109.
Braun, L., Ghebrehiwet, B., and Cossart, P. (2000). gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes. EMBO J 19, 1458-1466.
Braun, L., Ohayon, H., and Cossart, P. (1998). The InIB protein of Listeria monocytogenes is sufficient to promote entry into mammalian cells. Mol Microbiol 27, 1077-1087.
Brennan, R.G., and Link, T.M. (2007). Hfq structure, function and ligand binding. Curr Opin Microbiol10, 125-133.
Brock, J.E., Paz, R.L., Cottle, P., and Janssen, G.R. (2007). Naturally occurring adenines within mRNA coding sequences affect ribosome binding and expression in Escherichia coli. J Bacteriol 189, 501-510.
73
Brody, M.S., and Price, C.W. (1998). Bacillus licheniformis sigB operon encoding the general stress transcription factor sigma B. Gene 212, 111-118.
Cabanes, D., Dussurget, O., Dehoux, P., and Cossart, P. (2004). Auto, a surface associated autolysin ofListeria monocytogenes required for entry into eukaryotic cells and virulence. Mol Microbiol 51, 1601-1614.
Cabanes, D., Sousa, S., Cebria, A., Lecuit, M., Garcia-del Portillo, F., and Cossart, P. (2005). Gp96 is a receptor for a novel Listeria monocytogenes virulence factor, Vip, a surface protein. EMBO J 24, 2827-2838.
Cain, D.B., and McCann, V.L. (1986). An unusual case of cutaneous listeriosis. J Clin Microbiol 23, 976-977.
Callaghan, A.J., Marcaida, M.J., Stead, J.A., McDowall, K.J., Scott, W.G., and Luisi, B.F. (2005). Structure of Escherichia coli RNase E catalytic domain and implications for RNA turnover. Nature 437, 1187-1191.
Camilli, A., Goldfine, H., and Portnoy, D.A. (1991). Listeria monocytogenes mutants lacking phosphatidylinositol-specific phospholipase C are avirulent. J Exp Med 173, 751-754.
Camilli, A., Tilney, L.G., and Portnoy, D.A. (1993). Dual roles of plcA in Listeria monocytogenespathogenesis. Mol Microbiol 8, 143-157.
Carvajal, A., and Frederiksen, W. (1988). Fatal endocarditis due to Listeria monocytogenes. Rev Infect Dis 10, 616-623.
Chao, Y., and Vogel, J. (2010). The role of Hfq in bacterial pathogens. Curr Opin Microbiol 13, 24-33.
Cheng, L.W., and Portnoy, D.A. (2003). Drosophila S2 cells: an alternative infection model for Listeria monocytogenes. Cell Microbiol 5, 875-885.
Chico-Calero, I., Suarez, M., Gonzalez-Zorn, B., Scortti, M., Slaghuis, J., Goebel, W., and Vazquez-Boland, J.A. (2002). Hpt, a bacterial homolog of the microsomal glucose- 6-phosphate translocase, mediates rapid intracellular proliferation in Listeria. Proc Natl Acad Sci U S A 99, 431-436.
Christiansen, J.K., Larsen, M.H., Ingmer, H., Sogaard-Andersen, L., and Kallipolitis, B.H. (2004). The RNA-binding protein Hfq of Listeria monocytogenes: role in stress tolerance and virulence. J Bacteriol186, 3355-3362.
Christiansen, J.K., Nielsen, J.S., Ebersbach, T., Valentin-Hansen, P., Sogaard-Andersen, L., and Kallipolitis, B.H. (2006). Identification of small Hfq-binding RNAs in Listeria monocytogenes. RNA 12, 1383-1396.
Collins, J.A., Irnov, I., Baker, S., and Winkler, W.C. (2007). Mechanism of mRNA destabilization by the glmS ribozyme. Genes Dev 21, 3356-3368.
Condon, C. (2003). RNA processing and degradation in Bacillus subtilis. Microbiol Mol Biol Rev 67, 157-174, table of contents.
Cone, L.A., Leung, M.M., Byrd, R.G., Annunziata, G.M., Lam, R.Y., and Herman, B.K. (2003). Multiple cerebral abscesses because of Listeria monocytogenes: three case reports and a literature review of supratentorial listerial brain abscess(es). Surg Neurol 59, 320-328.
74
Conlan, J.W., and North, R.J. (1991). Neutrophil-mediated dissolution of infected host cells as a defense strategy against a facultative intracellular bacterium. J Exp Med 174, 741-744.
Corbino, K.A., Barrick, J.E., Lim, J., Welz, R., Tucker, B.J., Puskarz, I., Mandal, M., Rudnick, N.D., and Breaker, R.R. (2005). Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria. Genome Biol 6, R70.
Cossart, P., Vicente, M.F., Mengaud, J., Baquero, F., Perez-Diaz, J.C., and Berche, P. (1989). Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation. Infect Immun 57, 3629-3636.
Court, D. (1993). RNase III: A double strand processing enzyme. In Brawerman,G. and Belasco J. (eds) (New York, Academic Press).
Cousens, L.P., and Wing, E.J. (2000). Innate defenses in the liver during Listeria infection. Immunol Rev174, 150-159.
Dabiri, G.A., Sanger, J.M., Portnoy, D.A., and Southwick, F.S. (1990). Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly. Proc Natl Acad Sci U S A87, 6068-6072.
Domann, E., Leimeister-Wachter, M., Goebel, W., and Chakraborty, T. (1991). Molecular cloning, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene. Infect Immun 59, 65-72.
Domann, E., Zechel, S., Lingnau, A., Hain, T., Darji, A., Nichterlein, T., Wehland, J., and Chakraborty, T. (1997). Identification and characterization of a novel PrfA-regulated gene in Listeria monocytogenes whose product, IrpA, is highly homologous to internalin proteins, which contain leucine-rich repeats. Infect Immun 65, 101-109.
Dons, L., Weclewicz, K., Jin, Y., Bindseil, E., Olsen, J.E., and Kristensson, K. (1999). Rat dorsal root ganglia neurons as a model for Listeria monocytogenes infections in culture. Med Microbiol Immunol 188, 15-21.
Dramsi, S., Biswas, I., Maguin, E., Braun, L., Mastroeni, P., and Cossart, P. (1995). Entry of Listeria monocytogenes into hepatocytes requires expression of inIB, a surface protein of the internalin multigene family. Mol Microbiol 16, 251-261.
Dramsi, S., and Cossart, P. (2003). Listeriolysin O-mediated calcium influx potentiates entry of Listeria monocytogenes into the human Hep-2 epithelial cell line. Infect Immun 71, 3614-3618.
Dramsi, S., Dehoux, P., Lebrun, M., Goossens, P.L., and Cossart, P. (1997). Identification of four new members of the internalin multigene family of Listeria monocytogenes EGD. Infect Immun 65, 1615-1625.
Dramsi, S., Kocks, C., Forestier, C., and Cossart, P. (1993). Internalin-mediated invasion of epithelial cells by Listeria monocytogenes is regulated by the bacterial growth state, temperature and the pleiotropic activator prfA. Mol Microbiol 9, 931-941.
Drevets, D.A. (1999). Dissemination of Listeria monocytogenes by infected phagocytes. Infect Immun 67, 3512-3517.
Dreyfus, M. (1988). What constitutes the signal for the initiation of protein synthesis on Escherichia colimRNAs? J Mol Biol 204, 79-94.
75
DuBow, M.S., Ryan, T., Young, R.A., and Blumenthal, T. (1977). Host factor for coliphage Q beta RNA replication: presence in procaryotes and association with the 30S ribosomal subunit in Escherichia coli. Mol Gen Genet 153, 39-43.
Dussurget, O., Cabanes, D., Dehoux, P., Lecuit, M., Buchrieser, C., Glaser, P., and Cossart, P. (2002). Listeria monocytogenes bile salt hydrolase is a PrfA-regulated virulence factor involved in the intestinal and hepatic phases of listeriosis. Mol Microbiol 45, 1095-1106.
Dussurget, O., Pizarro-Cerda, J., and Cossart, P. (2004). Molecular determinants of Listeria monocytogenes virulence. Annu Rev Microbiol 58, 587-610.
Eiting, M., Hageluken, G., Schubert, W.D., and Heinz, D.W. (2005). The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA-binding affinity by stabilizing the HTH motif. Mol Microbiol 56, 433-446.
Engelbrecht, F., Chun, S.K., Ochs, C., Hess, J., Lottspeich, F., Goebel, W., and Sokolovic, Z. (1996). A new PrfA-regulated gene of Listeria monocytogenes encoding a small, secreted protein which belongs to the family of internalins. Mol Microbiol 21, 823-837.
Farber, J.M., and Peterkin, P.I. (1991). Listeria monocytogenes, a food-borne pathogen. Microbiol Rev55, 476-511.
Ferreira, A., Gray, M., Wiedmann, M., and Boor, K.J. (2004). Comparative genomic analysis of the sigB operon in Listeria monocytogenes and in other Gram-positive bacteria. Curr Microbiol 48, 39-46.
Fouet, A., Namy, O., and Lambert, G. (2000). Characterization of the operon encoding the alternative sigma(B) factor from Bacillus anthracis and its role in virulence. J Bacteriol 182, 5036-5045.
Fozo, E.M., Kawano, M., Fontaine, F., Kaya, Y., Mendieta, K.S., Jones, K.L., Ocampo, A., Rudd, K.E., and Storz, G. (2008). Repression of small toxic protein synthesis by the Sib and OhsC small RNAs. Mol Microbiol 70, 1076-1093.
Franklund, C.V., and Kadner, R.J. (1997). Multiple transcribed elements control expression of the Escherichia coli btuB gene. J Bacteriol 179, 4039-4042.
Franze de Fernandez, M.T., Eoyang, L., and August, J.T. (1968). Factor fraction required for the synthesis of bacteriophage Qbeta-RNA. Nature 219, 588-590.
Freitag, N.E., Port, G.C., and Miner, M.D. (2009). Listeria monocytogenes - from saprophyte to intracellular pathogen. Nat Rev Microbiol 7, 623-628.
Freitag, N.E., and Portnoy, D.A. (1994). Dual promoters of the Listeria monocytogenes prfAtranscriptional activator appear essential in vitro but are redundant in vivo. Mol Microbiol 12, 845-853.
Freitag, N.E., Rong, L., and Portnoy, D.A. (1993). Regulation of the prfA transcriptional activator of Listeria monocytogenes: multiple promoter elements contribute to intracellular growth and cell-to-cell spread. Infect Immun 61, 2537-2544.
Fuchs, R.T., Grundy, F.J., and Henkin, T.M. (2006). The S(MK) box is a new SAM-binding RNA for translational regulation of SAM synthetase. Nat Struct Mol Biol 13, 226-233.
Gaillard, J.L., Berche, P., Frehel, C., Gouin, E., and Cossart, P. (1991). Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci. Cell 65, 1127-1141.
76
Gaillard, J.L., Berche, P., Mounier, J., Richard, S., and Sansonetti, P. (1987). In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect Immun 55, 2822-2829.
Gaillard, J.L., Berche, P., and Sansonetti, P. (1986). Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes. Infect Immun 52, 50-55.
Garandeau, C., Reglier-Poupet, H., Dubail, I., Beretti, J.L., Berche, P., and Charbit, A. (2002). The sortase SrtA of Listeria monocytogenes is involved in processing of internalin and in virulence. Infect Immun 70, 1382-1390.
Gedde, M.M., Higgins, D.E., Tilney, L.G., and Portnoy, D.A. (2000). Role of listeriolysin O in cell-to-cell spread of Listeria monocytogenes. Infect Immun 68, 999-1003.
Geese, M., Loureiro, J.J., Bear, J.E., Wehland, J., Gertler, F.B., and Sechi, A.S. (2002). Contribution of Ena/VASP proteins to intracellular motility of listeria requires phosphorylation and proline-rich core but not F-actin binding or multimerization. Mol Biol Cell 13, 2383-2396.
Geissmann, T.A., and Touati, D. (2004). Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator. EMBO J 23, 396-405.
Geoffroy, C., Raveneau, J., Beretti, J.L., Lecroisey, A., Vazquez-Boland, J.A., Alouf, J.E., and Berche, P. (1991). Purification and characterization of an extracellular 29-kilodalton phospholipase C from Listeria monocytogenes. Infect Immun 59, 2382-2388.
Gerdes, K., and Wagner, E.G. (2007). RNA antitoxins. Curr Opin Microbiol 10, 117-124.
Gilot, P., Andre, P., and Content, J. (1999). Listeria monocytogenes possesses adhesins for fibronectin. Infect Immun 67, 6698-6701.
Giuliodori, A.M., Di Pietro, F., Marzi, S., Masquida, B., Wagner, R., Romby, P., Gualerzi, C.O., and Pon, C.L. (2010). The cspA mRNA is a thermosensor that modulates translation of the cold-shock protein CspA. Mol Cell 37, 21-33.
Glaser, P., Frangeul, L., Buchrieser, C., Rusniok, C., Amend, A., Baquero, F., Berche, P., Bloecker, H., Brandt, P., Chakraborty, T., et al. (2001). Comparative genomics of Listeria species. Science 294, 849-852.
Gold, L. (1988). Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem 57, 199-233.
Goldfine, H., Johnston, N.C., and Knob, C. (1993). Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes. J Bacteriol 175, 4298-4306.
Goldfine, H., and Knob, C. (1992). Purification and characterization of Listeria monocytogenes phosphatidylinositol-specific phospholipase C. Infect Immun 60, 4059-4067.
Gorke, B., and Vogel, J. (2008). Noncoding RNA control of the making and breaking of sugars. Genes Dev 22, 2914-2925.
Gottesman, S. (2004). The small RNA regulators of Escherichia coli: roles and mechanisms*. Annu Rev Microbiol 58, 303-328.
77
Gottesman, S. (2005). Micros for microbes: non-coding regulatory RNAs in bacteria. Trends Genet 21, 399-404.
Gouin, E., Welch, M.D., and Cossart, P. (2005). Actin-based motility of intracellular pathogens. Curr Opin Microbiol 8, 35-45.
Grundling, A., Gonzalez, M.D., and Higgins, D.E. (2003). Requirement of the Listeria monocytogenesbroad-range phospholipase PC-PLC during infection of human epithelial cells. J Bacteriol 185, 6295-6307.
Grundy, F.J., and Henkin, T.M. (1998). The S box regulon: a new global transcription termination control system for methionine and cysteine biosynthesis genes in gram-positive bacteria. Mol Microbiol 30, 737-749.
Gudapaty, S., Suzuki, K., Wang, X., Babitzke, P., and Romeo, T. (2001). Regulatory interactions of Csr components: the RNA binding protein CsrA activates csrB transcription in Escherichia coli. J Bacteriol 183, 6017-6027.
Hain, T., Steinweg, C., and Chakraborty, T. (2006). Comparative and functional genomics of Listeriaspp. J Biotechnol 126, 37-51.
Hammer, B.K., and Bassler, B.L. (2003). Quorum sensing controls biofilm formation in Vibrio cholerae. Mol Microbiol 50, 101-104.
Hankins, J.S., Denroche, H., and Mackie, G.A. (2010). Interactions of the RNA-binding protein, Hfq, with cspA mRNA encoding the major cold-shock protein. J Bacteriol.
Hantke, K. (2001). Iron and metal regulation in bacteria. Curr Opin Microbiol 4, 172-177.
Heidrich, N., Chinali, A., Gerth, U., and Brantl, S. (2006). The small untranslated RNA SR1 from the Bacillus subtilis genome is involved in the regulation of arginine catabolism. Mol Microbiol 62, 520-536.
Heymer, B., Wirsing von Konig, C.H., Finger, H., Hof, H., and Emmerling, P. (1988). Histomorphology of experimental listeriosis. Infection 16 Suppl 2, S106-111.
Hoe, N.P., Minion, F.C., and Goguen, J.D. (1992). Temperature sensing in Yersinia pestis: regulation of yopE transcription by lcrF. J Bacteriol 174, 4275-4286.
Hubbard, S.R., and Till, J.H. (2000). Protein tyrosine kinase structure and function. Annu Rev Biochem69, 373-398.
Huntzinger, E., Boisset, S., Saveanu, C., Benito, Y., Geissmann, T., Namane, A., Lina, G., Etienne, J., Ehresmann, B., Ehresmann, C., et al. (2005). Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. EMBO J 24, 824-835.
Ireton, K., Payrastre, B., and Cossart, P. (1999). The Listeria monocytogenes protein InlB is an agonist of mammalian phosphoinositide 3-kinase. J Biol Chem 274, 17025-17032.
Janzon, L., Lofdahl, S., and Arvidson, S. (1989). Identification and nucleotide sequence of the delta-lysin gene, hld, adjacent to the accessory gene regulator (agr) of Staphylococcus aureus. Mol Gen Genet 219, 480-485.
Johansson, J., Mandin, P., Renzoni, A., Chiaruttini, C., Springer, M., and Cossart, P. (2002). An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 110, 551-561.
78
Jonquieres, R., Bierne, H., Fiedler, F., Gounon, P., and Cossart, P. (1999). Interaction between the protein InlB of Listeria monocytogenes and lipoteichoic acid: a novel mechanism of protein association at the surface of gram-positive bacteria. Mol Microbiol 34, 902-914.
Jonquieres, R., Pizarro-Cerda, J., and Cossart, P. (2001). Synergy between the N- and C-terminal domains of InlB for efficient invasion of non-phagocytic cells by Listeria monocytogenes. Mol Microbiol42, 955-965.
Kajitani, M., and Ishihama, A. (1991). Identification and sequence determination of the host factor gene for bacteriophage Q beta. Nucleic Acids Res 19, 1063-1066.
Kajitani, M., Kato, A., Wada, A., Inokuchi, Y., and Ishihama, A. (1994). Regulation of the Escherichia coli hfq gene encoding the host factor for phage Q beta. J Bacteriol 176, 531-534.
Kathariou, S., Metz, P., Hof, H., and Goebel, W. (1987). Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes. J Bacteriol 169, 1291-1297.
Kawano, M., Aravind, L., and Storz, G. (2007). An antisense RNA controls synthesis of an SOS-induced toxin evolved from an antitoxin. Mol Microbiol 64, 738-754.
Kazmierczak, M.J., Mithoe, S.C., Boor, K.J., and Wiedmann, M. (2003). Listeria monocytogenes sigma B regulates stress response and virulence functions. J Bacteriol 185, 5722-5734.
Kim, H., Boor, K.J., and Marquis, H. (2004). Listeria monocytogenes sigmaB contributes to invasion of human intestinal epithelial cells. Infect Immun 72, 7374-7378.
Kirk, J. (1993). Diagnostic ultrastructure of Listeria monocytogenes in human central nervous tissue. Ultrastruct Pathol 17, 583-592.
Klein, D.J., Schmeing, T.M., Moore, P.B., and Steitz, T.A. (2001). The kink-turn: a new RNA secondary structure motif. EMBO J 20, 4214-4221.
Knobloch, J.K., Bartscht, K., Sabottke, A., Rohde, H., Feucht, H.H., and Mack, D. (2001). Biofilm formation by Staphylococcus epidermidis depends on functional RsbU, an activator of the sigB operon: differential activation mechanisms due to ethanol and salt stress. J Bacteriol 183, 2624-2633.
Kocks, C., Gouin, E., Tabouret, M., Berche, P., Ohayon, H., and Cossart, P. (1992). L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein. Cell 68, 521-531.
Kocks, C., Hellio, R., Gounon, P., Ohayon, H., and Cossart, P. (1993). Polarized distribution of Listeria monocytogenes surface protein ActA at the site of directional actin assembly. J Cell Sci 105 ( Pt 3), 699-710.
Köhler, S., Bubert, A., Vogel, M., and Goebel, W. (1991). Expression of the iap gene coding for protein p60 of Listeria monocytogenes is controlled on the posttranscriptional level. J Bacteriol 173, 4668-4674.
Köhler, S., Leimeister-Wachter, M., Chakraborty, T., Lottspeich, F., and Goebel, W. (1990). The gene coding for protein p60 of Listeria monocytogenes and its use as a specific probe for Listeria monocytogenes. Infect Immun 58, 1943-1950.
Konkel, M.E., and Tilly, K. (2000). Temperature-regulated expression of bacterial virulence genes. Microbes Infect 2, 157-166.
Kovacikova, G., and Skorupski, K. (2002). Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter. Mol Microbiol 46, 1135-1147.
79
Kreikemeyer, B., Boyle, M.D., Buttaro, B.A., Heinemann, M., and Podbielski, A. (2001). Group A streptococcal growth phase-associated virulence factor regulation by a novel operon (Fas) with homologies to two-component-type regulators requires a small RNA molecule. Mol Microbiol 39, 392-406.
Kudla, G., Murray, A.W., Tollervey, D., and Plotkin, J.B. (2009). Coding-sequence determinants of gene expression in Escherichia coli. Science 324, 255-258.
Kuhn, M., and Goebel, W. (1989). Identification of an extracellular protein of Listeria monocytogenespossibly involved in intracellular uptake by mammalian cells. Infect Immun 57, 55-61.
Kuhn, M., and Goebel, W. (2000). Internalization of Listeria monocytogenes by nonprofessional and professional phagocytes. Subcell Biochem 33, 411-436.
Lampidis, R., Gross, R., Sokolovic, Z., Goebel, W., and Kreft, J. (1994). The virulence regulator protein of Listeria ivanovii is highly homologous to PrfA from Listeria monocytogenes and both belong to the Crp-Fnr family of transcription regulators. Mol Microbiol 13, 141-151.
Lasa, I., Gouin, E., Goethals, M., Vancompernolle, K., David, V., Vandekerckhove, J., and Cossart, P.(1997). Identification of two regions in the N-terminal domain of ActA involved in the actin comet tail formation by Listeria monocytogenes. EMBO J 16, 1531-1540.
Laurent, V., Loisel, T.P., Harbeck, B., Wehman, A., Grobe, L., Jockusch, B.M., Wehland, J., Gertler, F.B., and Carlier, M.F. (1999). Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes. J Cell Biol 144, 1245-1258.
Lease, R.A., and Belfort, M. (2000). A trans-acting RNA as a control switch in Escherichia coli: DsrA modulates function by forming alternative structures. Proc Natl Acad Sci U S A 97, 9919-9924.
Lease, R.A., Cusick, M.E., and Belfort, M. (1998). Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci. Proc Natl Acad Sci U S A 95, 12456-12461.
Lease, R.A., Smith, D., McDonough, K., and Belfort, M. (2004). The small noncoding DsrA RNA is an acid resistance regulator in Escherichia coli. J Bacteriol 186, 6179-6185.
Lease, R.A., and Woodson, S.A. (2004). Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA. J Mol Biol 344, 1211-1223.
Lecuit, M., Dramsi, S., Gottardi, C., Fedor-Chaiken, M., Gumbiner, B., and Cossart, P. (1999). A single amino acid in E-cadherin responsible for host specificity towards the human pathogen Listeria monocytogenes. EMBO J 18, 3956-3963.
Lecuit, M., Nelson, D.M., Smith, S.D., Khun, H., Huerre, M., Vacher-Lavenu, M.C., Gordon, J.I., and Cossart, P. (2004). Targeting and crossing of the human maternofetal barrier by Listeria monocytogenes: role of internalin interaction with trophoblast E-cadherin. Proc Natl Acad Sci U S A 101, 6152-6157.
Lecuit, M., Ohayon, H., Braun, L., Mengaud, J., and Cossart, P. (1997). Internalin of Listeria monocytogenes with an intact leucine-rich repeat region is sufficient to promote internalization. Infect Immun 65, 5309-5319.
Lecuit, M., Vandormael-Pournin, S., Lefort, J., Huerre, M., Gounon, P., Dupuy, C., Babinet, C., and Cossart, P. (2001). A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier. Science 292, 1722-1725.
80
Leimeister-Wachter, M., Domann, E., and Chakraborty, T. (1991). Detection of a gene encoding a phosphatidylinositol-specific phospholipase C that is co-ordinately expressed with listeriolysin in Listeria monocytogenes. Mol Microbiol 5, 361-366.
Leimeister-Wachter, M., Haffner, C., Domann, E., Goebel, W., and Chakraborty, T. (1990). Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of Listeria monocytogenes. Proc Natl Acad Sci U S A 87, 8336-8340.
Lenz, D.H., Mok, K.C., Lilley, B.N., Kulkarni, R.V., W ingreen, N.S., and Bassler, B.L. (2004). The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae. Cell 118, 69-82.
Li, N., Xiang, G.S., Dokainish, H., Ireton, K., and Elferink, L.A. (2005). The Listeria protein internalin B mimics hepatocyte growth factor-induced receptor trafficking. Traffic 6, 459-473.
Lim, J., Winkler, W.C., Nakamura, S., Scott, V., and Breaker, R.R. (2006). Molecular-recognition characteristics of SAM-binding riboswitches. Angew Chem Int Ed Engl 45, 964-968.
Livny, J., Teonadi, H., Livny, M., and Waldor, M.K. (2008). High-throughput, kingdom-wide prediction and annotation of bacterial non-coding RNAs. PLoS One 3, e3197.
Loh, E., Dussurget, O., Gripenland, J., Vaitkevicius, K., Tiensuu, T., Mandin, P., Repoila, F., Buchrieser, C., Cossart, P., and Johansson, J. (2009). A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes. Cell 139, 770-779.
Loh, E., Gripenland, J., and Johansson, J. (2006). Control of Listeria monocytogenes virulence by 5'-untranslated RNA. Trends Microbiol 14, 294-298.
Mackaness, G.B. (1962). Cellular resistance to infection. J Exp Med 116, 381-406.
Majdalani, N., Cunning, C., Sledjeski, D., Elliott, T., and Gottesman, S. (1998). DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription. Proc Natl Acad Sci U S A 95, 12462-12467.
Mandal, M., and Breaker, R.R. (2004). Gene regulation by riboswitches. Nat Rev Mol Cell Biol 5, 451-463.
Mandin, P., Repoila, F., Vergassola, M., Geissmann, T., and Cossart, P. (2007). Identification of new noncoding RNAs in Listeria monocytogenes and prediction of mRNA targets. Nucleic Acids Res 35, 962-974.
Mansfield, B.E., Dionne, M.S., Schneider, D.S., and Freitag, N.E. (2003). Exploration of host-pathogen interactions using Listeria monocytogenes and Drosophila melanogaster. Cell Microbiol 5, 901-911.
Marco, A.J., Prats, N., Ramos, J.A., Briones, V., Blanco, M., Dominguez, L., and Domingo, M. (1992). A microbiological, histopathological and immunohistological study of the intragastric inoculation of Listeria monocytogenes in mice. J Comp Pathol 107, 1-9.
Marino, M., Braun, L., Cossart, P., and Ghosh, P. (1999). Structure of the lnlB leucine-rich repeats, a domain that triggers host cell invasion by the bacterial pathogen L. monocytogenes. Mol Cell 4, 1063-1072.
Marquis, H., Doshi, V., and Portnoy, D.A. (1995). The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells. Infect Immun 63, 4531-4534.
81
Martin-Farmer, J., and Janssen, G.R. (1999). A downstream CA repeat sequence increases translation from leadered and unleadered mRNA in Escherichia coli. Mol Microbiol 31, 1025-1038.
Masse, E., Escorcia, F.E., and Gottesman, S. (2003). Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli. Genes Dev 17, 2374-2383.
Masse, E., and Gottesman, S. (2002). A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci U S A 99, 4620-4625.
Masse, E., Salvail, H., Desnoyers, G., and Arguin, M. (2007). Small RNAs controlling iron metabolism. Curr Opin Microbiol 10, 140-145.
Masse, E., Vanderpool, C.K., and Gottesman, S. (2005). Effect of RyhB small RNA on global iron use in Escherichia coli. J Bacteriol 187, 6962-6971.
Mathy, N., Benard, L., Pellegrini, O., Daou, R., Wen, T., and Condon, C. (2007). 5'-to-3' exoribonuclease activity in bacteria: role of RNase J1 in rRNA maturation and 5' stability of mRNA. Cell129, 681-692.
McCarthy, J.E., and Brimacombe, R. (1994). Prokaryotic translation: the interactive pathway leading to initiation. Trends Genet 10, 402-407.
McDaniel, B.A., Grundy, F.J., Artsimovitch, I., and Henkin, T.M. (2003). Transcription termination control of the S box system: direct measurement of S-adenosylmethionine by the leader RNA. Proc Natl Acad Sci U S A 100, 3083-3088.
McDaniel, B.A., Grundy, F.J., and Henkin, T.M. (2005). A tertiary structural element in S box leader RNAs is required for S-adenosylmethionine-directed transcription termination. Mol Microbiol 57, 1008-1021.
McGann, P., Wiedmann, M., and Boor, K.J. (2007). The alternative sigma factor sigma B and the virulence gene regulator PrfA both regulate transcription of Listeria monocytogenes internalins. Appl Environ Microbiol 73, 2919-2930.
McLauchlin, J., Hall, S.M., Velani, S.K., and Gilbert, R.J. (1991). Human listeriosis and pate: a possible association. BMJ 303, 773-775.
Mengaud, J., Braun-Breton, C., and Cossart, P. (1991). Identification of phosphatidylinositol-specific phospholipase C activity in Listeria monocytogenes: a novel type of virulence factor? Mol Microbiol 5, 367-372.
Mengaud, J., Ohayon, H., Gounon, P., Mege, R.M., and Cossart, P. (1996). E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells. Cell 84, 923-932.
Mengaud, J., Vicente, M.F., and Cossart, P. (1989). Transcriptional mapping and nucleotide sequence of the Listeria monocytogenes hlyA region reveal structural features that may be involved in regulation. Infect Immun 57, 3695-3701.
Mey, A.R., Craig, S.A., and Payne, S.M. (2005). Characterization of Vibrio cholerae RyhB: the RyhB regulon and role of ryhB in biofilm formation. Infect Immun 73, 5706-5719.
Mikulecky, P.J., Kaw, M.K., Brescia, C.C., Takach, J.C., Sledjeski, D.D., and Feig, A.L. (2004). Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs. Nat Struct Mol Biol 11, 1206-1214.
82
Milohanic, E., Jonquieres, R., Cossart, P., Berche, P., and Gaillard, J.L. (2001). The autolysin Ami contributes to the adhesion of Listeria monocytogenes to eukaryotic cells via its cell wall anchor. Mol Microbiol 39, 1212-1224.
Moller, T., Franch, T., Hojrup, P., Keene, D.R., Bachinger, H.P., Brennan, R.G., and Valentin-Hansen, P. (2002). Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction. Mol Cell 9, 23-30.
Montange, R.K., and Batey, R.T. (2006). Structure of the S-adenosylmethionine riboswitch regulatory mRNA element. Nature 441, 1172-1175.
Montange, R.K., and Batey, R.T. (2008). Riboswitches: emerging themes in RNA structure and function. Annu Rev Biophys 37, 117-133.
Moors, M.A., Levitt, B., Youngman, P., and Portnoy, D.A. (1999). Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes. Infect Immun 67, 131-139.
Morfeldt, E., Taylor, D., von Gabain, A., and Arvidson, S. (1995). Activation of alpha-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII. EMBO J 14, 4569-4577.
Morita, M., Kanemori, M., Yanagi, H., and Yura, T. (1999a). Heat-induced synthesis of sigma32 in Escherichia coli: structural and functional dissection of rpoH mRNA secondary structure. J Bacteriol 181, 401-410.
Morita, M.T., Tanaka, Y., Kodama, T.S., Kyogoku, Y., Yanagi, H., and Yura, T. (1999b). Translational induction of heat shock transcription factor sigma32: evidence for a built-in RNA thermosensor. Genes Dev 13, 655-665.
Morita, T., Maki, K., and Aiba, H. (2005). RNase E-based ribonucleoprotein complexes: mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs. Genes Dev 19, 2176-2186.
Mounier, J., Ryter, A., Coquis-Rondon, M., and Sansonetti, P.J. (1990). Intracellular and cell-to-cell spread of Listeria monocytogenes involves interaction with F-actin in the enterocytelike cell line Caco-2. Infect Immun 58, 1048-1058.
Mylonakis, E., Paliou, M., Hohmann, E.L., Calderwood, S.B., and Wing, E.J. (2002). Listeriosis during pregnancy: a case series and review of 222 cases. Medicine (Baltimore) 81, 260-269.
Nadon, C.A., Bowen, B.M., Wiedmann, M., and Boor, K.J. (2002). Sigma B contributes to PrfA-mediated virulence in Listeria monocytogenes. Infect Immun 70, 3948-3952.
Nahvi, A., Sudarsan, N., Ebert, M.S., Zou, X., Brown, K.L., and Breaker, R.R. (2002). Genetic control by a metabolite binding mRNA. Chem Biol 9, 1043.
Narberhaus, F., Weiglhofer, W., Fischer, H.M., and Hennecke, H. (1996). The Bradyrhizobium japonicum rpoH1 gene encoding a sigma 32-like protein is part of a unique heat shock gene cluster together with groESL1 and three small heat shock genes. J Bacteriol 178, 5337-5346.
Nechooshtan, G., Elgrably-Weiss, M., Sheaffer, A., Westhof, E., and Altuvia, S. (2009). A pH-responsive riboregulator. Genes Dev 23, 2650-2662.
Newton, S.M., Klebba, P.E., Raynaud, C., Shao, Y., Jiang, X., Dubail, I., Archer, C., Frehel, C., and Charbit, A. (2005). The svpA-srtB locus of Listeria monocytogenes: fur-mediated iron regulation and effect on virulence. Mol Microbiol 55, 927-940.
83
Nielsen, J.S., Lei, L.K., Ebersbach, T., Olsen, A.S., Klitgaard, J.K., Valentin-Hansen, P., and Kallipolitis, B.H. (2010). Defining a role for Hfq in Gram-positive bacteria: evidence for Hfq-dependent antisense regulation in Listeria monocytogenes. Nucleic Acids Res 38, 907-919.
Nikulin, A., Stolboushkina, E., Perederina, A., Vassilieva, I., Blaesi, U., Moll, I., Kachalova, G., Yokoyama, S., Vassylyev, D., Garber, M., et al. (2005). Structure of Pseudomonas aeruginosa Hfq protein. Acta Crystallogr D Biol Crystallogr 61, 141-146.
North, C.S., Alpers, D.H., Helzer, J.E., Spitznagel, E.L., and Clouse, R.E. (1991). Do life events or depression exacerbate inflammatory bowel disease? A prospective study. Ann Intern Med 114, 381-386.
Novick, R.P., and Geisinger, E. (2008). Quorum sensing in staphylococci. Annu Rev Genet 42, 541-564.
Novick, R.P., Ross, H.F., Projan, S.J., Kornblum, J., Kreiswirth, B., and Moghazeh, S. (1993). Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J 12, 3967-3975.
Nudler, E., and Mironov, A.S. (2004). The riboswitch control of bacterial metabolism. Trends Biochem Sci 29, 11-17.
Oglesby, A.G., Murphy, E.R., Iyer, V.R., and Payne, S.M. (2005). Fur regulates acid resistance in Shigella flexneri via RyhB and ydeP. Mol Microbiol 58, 1354-1367.
Opdyke, J.A., Kang, J.G., and Storz, G. (2004). GadY, a small-RNA regulator of acid response genes inEscherichia coli. J Bacteriol 186, 6698-6705.
Personnic, N., Bruck, S., Nahori, M.A., Toledo-Arana, A., Nikitas, G., Lecuit, M., Dussurget, O., Cossart, P., and Bierne, H. (2010). The stress-induced virulence protein InlH controls interleukin-6 production during murine listeriosis. Infect Immun.
Pilgrim, S., Kolb-Maurer, A., Gentschev, I., Goebel, W., and Kuhn, M. (2003). Deletion of the gene encoding p60 in Listeria monocytogenes leads to abnormal cell division and loss of actin-based motility. Infect Immun 71, 3473-3484.
Pinner, R.W., Schuchat, A., Swaminathan, B., Hayes, P.S., Deaver, K.A., Weaver, R.E., Plikaytis, B.D., Reeves, M., Broome, C.V., and Wenger, J.D. (1992). Role of foods in sporadic listeriosis. II. Microbiologic and epidemiologic investigation. The Listeria Study Group. JAMA 267, 2046-2050.
Pizarro-Cerda, J., and Cossart, P. (2006). Subversion of cellular functions by Listeria monocytogenes. J Pathol 208, 215-223.
Poiata, E., Meyer, M.M., Ames, T.D., and Breaker, R.R. (2009). A variant riboswitch aptamer class for S-adenosylmethionine common in marine bacteria. RNA 15, 2046-2056.
Portnoy, D.A., Jacks, P.S., and Hinrichs, D.J. (1988). Role of hemolysin for the intracellular growth of Listeria monocytogenes. J Exp Med 167, 1459-1471.
Preis, H., Eckart, R.A., Gudipati, R.K., Heidrich, N., and Brantl, S. (2009). CodY activates transcription of a small RNA in Bacillus subtilis. J Bacteriol 191, 5446-5457.
Pron, B., Boumaila, C., Jaubert, F., Sarnacki, S., Monnet, J.P., Berche, P., and Gaillard, J.L. (1998). Comprehensive study of the intestinal stage of listeriosis in a rat ligated ileal loop system. Infect Immun 66, 747-755.
84
Pucciarelli, M.G., Calvo, E., Sabet, C., Bierne, H., Cossart, P., and Garcia-del Portillo, F. (2005). Identification of substrates of the Listeria monocytogenes sortases A and B by a non-gel proteomic analysis. Proteomics 5, 4808-4817.
Pung, O.J., Tucker, A.N., Vore, S.J., and Luster, M.I. (1985). Influence of estrogen on host resistance: increased susceptibility of mice to Listeria monocytogenes correlates with depressed production of interleukin 2. Infect Immun 50, 91-96.
Py, B.F., Lipinski, M.M., and Yuan, J. (2007). Autophagy limits Listeria monocytogenes intracellular growth in the early phase of primary infection. Autophagy 3, 117-125.
Racz, P., Tenner, K., and Mero, E. (1972). Experimental Listeria enteritis. I. An electron microscopic study of the epithelial phase in experimental listeria infection. Lab Invest 26, 694-700.
Raffelsbauer, D., Bubert, A., Engelbrecht, F., Scheinpflug, J., Simm, A., Hess, J., Kaufmann, S.H., and Goebel, W. (1998). The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice. Mol Gen Genet 260, 144-158.
Raveneau, J., Geoffroy, C., Beretti, J.L., Gaillard, J.L., Alouf, J.E., and Berche, P. (1992). Reduced virulence of a Listeria monocytogenes phospholipase-deficient mutant obtained by transposon insertion into the zinc metalloprotease gene. Infect Immun 60, 916-921.
Regulski, E.E., Moy, R.H., Weinberg, Z., Barrick, J.E., Yao, Z., Ruzzo, W.L., and Breaker, R.R.(2008). A widespread riboswitch candidate that controls bacterial genes involved in molybdenum cofactor and tungsten cofactor metabolism. Mol Microbiol 68, 918-932.
Renzoni, A., Cossart, P., and Dramsi, S. (1999). PrfA, the transcriptional activator of virulence genes, is upregulated during interaction of Listeria monocytogenes with mammalian cells and in eukaryotic cell extracts. Mol Microbiol 34, 552-561.
Renzoni, A., Klarsfeld, A., Dramsi, S., and Cossart, P. (1997). Evidence that PrfA, the pleiotropic activator of virulence genes in Listeria monocytogenes, can be present but inactive. Infect Immun 65, 1515-1518.
Repoila, F., and Gottesman, S. (2001). Signal transduction cascade for regulation of RpoS: temperature regulation of DsrA. J Bacteriol 183, 4012-4023.
Repoila, F., Majdalani, N., and Gottesman, S. (2003). Small non-coding RNAs, co-ordinators of adaptation processes in Escherichia coli: the RpoS paradigm. Mol Microbiol 48, 855-861.
Resch, A., Afonyushkin, T., Lombo, T.B., McDowall, K.J., Blasi, U., and Kaberdin, V.R. (2008). Translational activation by the noncoding RNA DsrA involves alternative RNase III processing in the rpoS 5'-leader. RNA 14, 454-459.
Ripio, M.T., Brehm, K., Lara, M., Suarez, M., and Vazquez-Boland, J.A. (1997). Glucose-1-phosphate utilization by Listeria monocytogenes is PrfA dependent and coordinately expressed with virulence factors. J Bacteriol 179, 7174-7180.
Ripio, M.T., Dominguez-Bernal, G., Suarez, M., Brehm, K., Berche, P., and Vazquez-Boland, J.A.(1996). Transcriptional activation of virulence genes in wild-type strains of Listeria monocytogenes in response to a change in the extracellular medium composition. Res Microbiol 147, 371-384.
Robbins, J.R., Barth, A.I., Marquis, H., de Hostos, E.L., Nelson, W.J., and Theriot, J.A. (1999). Listeria monocytogenes exploits normal host cell processes to spread from cell to cell. J Cell Biol 146, 1333-1350.
85
Rodionov, D.A., Dubchak, I., Arkin, A., Alm, E., and Gelfand, M.S. (2004). Reconstruction of regulatory and metabolic pathways in metal-reducing delta-proteobacteria. Genome Biol 5, R90.
Romeo, T. (1998). Global regulation by the small RNA-binding protein CsrA and the non-coding RNA molecule CsrB. Mol Microbiol 29, 1321-1330.
Ruhland, G.J., Hellwig, M., Wanner, G., and Fiedler, F. (1993). Cell-surface location of Listeria-specific protein p60--detection of Listeria cells by indirect immunofluorescence. J Gen Microbiol 139, 609-616.
Sauter, C., Basquin, J., and Suck, D. (2003). Sm-like proteins in Eubacteria: the crystal structure of the Hfq protein from Escherichia coli. Nucleic Acids Res 31, 4091-4098.
Schluter, D., Domann, E., Buck, C., Hain, T., Hof, H., Chakraborty, T., and Deckert-Schluter, M.(1998). Phosphatidylcholine-specific phospholipase C from Listeria monocytogenes is an important virulence factor in murine cerebral listeriosis. Infect Immun 66, 5930-5938.
Schumacher, M.A., Pearson, R.F., Moller, T., Valentin-Hansen, P., and Brennan, R.G. (2002). Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein. EMBO J 21, 3546-3556.
Schwab, U., Bowen, B., Nadon, C., Wiedmann, M., and Boor, K.J. (2005). The Listeria monocytogenes prfAP2 promoter is regulated by sigma B in a growth phase dependent manner. FEMS Microbiol Lett 245, 329-336.
Schwartz, B., Hexter, D., Broome, C.V., Hightower, A.W., Hirschhorn, R.B., Porter, J.D., Hayes, P.S., Bibb, W.F., Lorber, B., and Faris, D.G. (1989). Investigation of an outbreak of listeriosis: new hypotheses for the etiology of epidemic Listeria monocytogenes infections. J Infect Dis 159, 680-685.
Scortti, M., Monzo, H.J., Lacharme-Lora, L., Lewis, D.A., and Vazquez-Boland, J.A. (2007). The PrfA virulence regulon. Microbes Infect 9, 1196-1207.
Sheehan, B., Klarsfeld, A., Ebright, R., and Cossart, P. (1996). A single substitution in the putative helix-turn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence. Mol Microbiol 20, 785-797.
Shen, A., and Higgins, D.E. (2005). The 5' untranslated region-mediated enhancement of intracellular listeriolysin O production is required for Listeria monocytogenes pathogenicity. Mol Microbiol 57, 1460-1473.
Shen, Y., Naujokas, M., Park, M., and Ireton, K. (2000). InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase. Cell 103, 501-510.
Shetron-Rama, L.M., Marquis, H., Bouwer, H.G., and Freitag, N.E. (2002). Intracellular induction of Listeria monocytogenes actA expression. Infect Immun 70, 1087-1096.
Shimizu, T., Yaguchi, H., Ohtani, K., Banu, S., and Hayashi, H. (2002). Clostridial VirR/VirS regulon involves a regulatory RNA molecule for expression of toxins. Mol Microbiol 43, 257-265.
Silvaggi, J.M., Perkins, J.B., and Losick, R. (2006). Genes for small, noncoding RNAs under sporulation control in Bacillus subtilis. J Bacteriol 188, 532-541.
Skoble, J., Auerbuch, V., Goley, E.D., Welch, M.D., and Portnoy, D.A. (2001). Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility. J Cell Biol 155, 89-100.
86
Skoble, J., Portnoy, D.A., and Welch, M.D. (2000). Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility. J Cell Biol 150, 527-538.
Sleator, R.D., Wemekamp-Kamphuis, H.H., Gahan, C.G., Abee, T., and Hill, C. (2005). A PrfA-regulated bile exclusion system (BilE) is a novel virulence factor in Listeria monocytogenes. Mol Microbiol55, 1183-1195.
Sledjeski, D., and Gottesman, S. (1995). A small RNA acts as an antisilencer of the H-NS-silenced rcsAgene of Escherichia coli. Proc Natl Acad Sci U S A 92, 2003-2007.
Sledjeski, D.D., Gupta, A., and Gottesman, S. (1996). The small RNA, DsrA, is essential for the low temperature expression of RpoS during exponential growth in Escherichia coli. EMBO J 15, 3993-4000.
Smith, G.A., Marquis, H., Jones, S., Johnston, N.C., Portnoy, D.A., and Goldfine, H. (1995). The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread. Infect Immun 63, 4231-4237.
Smith, M.A., Takeuchi, K., Brackett, R.E., McClure, H.M., Raybourne, R.B., Williams, K.M., Babu, U.S., Ware, G.O., Broderson, J.R., and Doyle, M.P. (2003). Nonhuman primate model for Listeria monocytogenes-induced stillbirths. Infect Immun 71, 1574-1579.
Song, T., Mika, F., Lindmark, B., Liu, Z., Schild, S., Bishop, A., Zhu, J., Camilli, A., Johansson, J., Vogel, J., et al. (2008). A new Vibrio cholerae sRNA modulates colonization and affects release of outer membrane vesicles. Mol Microbiol 70, 100-111.
Soper, T.J., and Woodson, S.A. (2008). The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA. RNA 14, 1907-1917.
Soukup, G.A., and Breaker, R.R. (1999). Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A 96, 3584-3589.
Soukup, G.A., DeRose, E.C., Koizumi, M., and Breaker, R.R. (2001). Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes. RNA 7, 524-536.
Sprengart, M.L., Fatscher, H.P., and Fuchs, E. (1990). The initiation of translation in E. coli: apparent base pairing between the 16srRNA and downstream sequences of the mRNA. Nucleic Acids Res 18, 1719-1723.
Steiner, K., and Malke, H. (2002). Dual control of streptokinase and streptolysin S production by thecovRS and fasCAX two-component regulators in Streptococcus dysgalactiae subsp. equisimilis. Infect Immun 70, 3627-3636.
Steinweg, C., Kuenne, C.T., Billion, A., Mraheil, M.A., Domann, E., Ghai, R., Barbuddhe, S.B., Karst, U., Goesmann, A., Puhler, A., et al. (2010). Complete genome sequence of Listeria seeligeri, a nonpathogenic member of the genus Listeria. J Bacteriol 192, 1473-1474.
Stoll, R., Mertins, S., Joseph, B., Muller-Altrock, S., and Goebel, W. (2008). Modulation of PrfA activity in Listeria monocytogenes upon growth in different culture media. Microbiology 154, 3856-3876.
Stork, M., Di Lorenzo, M., Welch, T.J., and Crosa, J.H. (2007). Transcription termination within the iron transport-biosynthesis operon of Vibrio anguillarum requires an antisense RNA. J Bacteriol 189, 3479-3488.
87
Stritzker, J., Schoen, C., and Goebel, W. (2005). Enhanced synthesis of internalin A in aro mutants ofListeria monocytogenes indicates posttranscriptional control of the inlAB mRNA. J Bacteriol 187, 2836-2845.
Suarez, M., Gonzalez-Zorn, B., Vega, Y., Chico-Calero, I., and Vazquez-Boland, J.A. (2001). A role for ActA in epithelial cell invasion by Listeria monocytogenes. Cell Microbiol 3, 853-864.
Sue, D., Boor, K.J., and Wiedmann, M. (2003). Sigma(B)-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes. Microbiology 149, 3247-3256.
Sukhodolets, M.V., and Garges, S. (2003). Interaction of Escherichia coli RNA polymerase with the ribosomal protein S1 and the Sm-like ATPase Hfq. Biochemistry 42, 8022-8034.
Suzuki, K., Babitzke, P., Kushner, S.R., and Romeo, T. (2006). Identification of a novel regulatory protein (CsrD) that targets the global regulatory RNAs CsrB and CsrC for degradation by RNase E. Genes Dev 20, 2605-2617.
Tilney, L.G., and Portnoy, D.A. (1989). Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J Cell Biol 109, 1597-1608.
Toledo-Arana, A., Dussurget, O., Nikitas, G., Sesto, N., Guet-Revillet, H., Balestrino, D., Loh, E., Gripenland, J., Tiensuu, T., Vaitkevicius, K., et al. (2009). The Listeria transcriptional landscape from saprophytism to virulence. Nature 459, 950-956.
Tomsic, J., McDaniel, B.A., Grundy, F.J., and Henkin, T.M. (2008). Natural variability in S-adenosylmethionine (SAM)-dependent riboswitches: S-box elements in Bacillus subtilis exhibit differential sensitivity to SAM In vivo and in vitro. J Bacteriol 190, 823-833.
Tramonti, A., De Canio, M., and De Biase, D. (2008). GadX/GadW-dependent regulation of the Escherichia coli acid fitness island: transcriptional control at the gadY-gadW divergent promoters and identification of four novel 42 bp GadX/GadW-specific binding sites. Mol Microbiol 70, 965-982.
Trotochaud, A.E., and Wassarman, K.M. (2004). 6S RNA function enhances long-term cell survival. J Bacteriol 186, 4978-4985.
Trotochaud, A.E., and Wassarman, K.M. (2005). A highly conserved 6S RNA structure is required for regulation of transcription. Nat Struct Mol Biol 12, 313-319.
Tsan, M.F., and Gao, B. (2004). Heat shock protein and innate immunity. Cell Mol Immunol 1, 274-279.
Tsui, H.C., and Winkler, M.E. (1994). Transcriptional patterns of the mutL-miaA superoperon of Escherichia coli K-12 suggest a model for posttranscriptional regulation. Biochimie 76, 1168-1177.
Unoson, C., and Wagner, E.G. (2008). A small SOS-induced toxin is targeted against the inner membrane in Escherichia coli. Mol Microbiol 70, 258-270.
Valentin-Hansen, P., Eriksen, M., and Udesen, C. (2004). The bacterial Sm-like protein Hfq: a key player in RNA transactions. Mol Microbiol 51, 1525-1533.
Vazquez-Boland, J.A., Kocks, C., Dramsi, S., Ohayon, H., Geoffroy, C., Mengaud, J., and Cossart, P.(1992). Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread. Infect Immun 60, 219-230.
88
Vazquez-Boland, J.A., Kuhn, M., Berche, P., Chakraborty, T., Dominguez-Bernal, G., Goebel, W., Gonzalez-Zorn, B., Wehland, J., and Kreft, J. (2001). Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 14, 584-640.
Vecerek, B., Moll, I., Afonyushkin, T., Kaberdin, V., and Blasi, U. (2003). Interaction of the RNA chaperone Hfq with mRNAs: direct and indirect roles of Hfq in iron metabolism of Escherichia coli. Mol Microbiol 50, 897-909.
Vega, Y., Dickneite, C., Ripio, M.T., Bockmann, R., Gonzalez-Zorn, B., Novella, S., Dominguez-Bernal, G., Goebel, W., and Vazquez-Boland, J.A. (1998). Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA. J Bacteriol 180, 6655-6660.
Vega, Y., Rauch, M., Banfield, M.J., Ermolaeva, S., Scortti, M., Goebel, W., and Vazquez-Boland, J.A. (2004). New Listeria monocytogenes prfA* mutants, transcriptional properties of PrfA* proteins and structure-function of the virulence regulator PrfA. Mol Microbiol 52, 1553-1565.
Veiga, E., and Cossart, P. (2005). Listeria hijacks the clathrin-dependent endocytic machinery to invade mammalian cells. Nat Cell Biol 7, 894-900.
Velge, P., Herler, M., Johansson, J., Roche, S.M., Temoin, S., Fedorov, A.A., Gracieux, P., Almo, S.C., Goebel, W., and Cossart, P. (2007). A naturally occurring mutation K220T in the pleiotropic activator PrfA of Listeria monocytogenes results in a loss of virulence due to decreasing DNA-binding affinity. Microbiology 153, 995-1005.
Vitreschak, A.G., Rodionov, D.A., Mironov, A.A., and Gelfand, M.S. (2003). Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural element. RNA 9, 1084-1097.
Wagner, E.G., Altuvia, S., and Romby, P. (2002). Antisense RNAs in bacteria and their genetic elements. Adv Genet 46, 361-398.
Waldminghaus, T., Fippinger, A., Alfsmann, J., and Narberhaus, F. (2005). RNA thermometers are common in alpha- and gamma-proteobacteria. Biol Chem 386, 1279-1286.
Wassarman, K.M., Repoila, F., Rosenow, C., Storz, G., and Gottesman, S. (2001). Identification of novel small RNAs using comparative genomics and microarrays. Genes Dev 15, 1637-1651.
Wassarman, K.M., and Saecker, R.M. (2006). Synthesis-mediated release of a small RNA inhibitor of RNA polymerase. Science 314, 1601-1603.
Wassarman, K.M., and Storz, G. (2000). 6S RNA regulates E. coli RNA polymerase activity. Cell 101, 613-623.
Weinberg, E.D. (1984). Pregnancy-associated depression of cell-mediated immunity. Rev Infect Dis 6, 814-831.
Weinberg, Z., Barrick, J.E., Yao, Z., Roth, A., Kim, J.N., Gore, J., Wang, J.X., Lee, E.R., Block, K.F., Sudarsan, N., et al. (2007). Identification of 22 candidate structured RNAs in bacteria using the CMfinder comparative genomics pipeline. Nucleic Acids Res 35, 4809-4819.
Weinberg, Z., Regulski, E.E., Hammond, M.C., Barrick, J.E., Yao, Z., Ruzzo, W.L., and Breaker, R.R. (2008). The aptamer core of SAM-IV riboswitches mimics the ligand-binding site of SAM-I riboswitches. RNA 14, 822-828.
89
Wenger, J.D., Hightower, A.W., Facklam, R.R., Gaventa, S., and Broome, C.V. (1990). Bacterial meningitis in the United States, 1986: report of a multistate surveillance study. The Bacterial Meningitis Study Group. J Infect Dis 162, 1316-1323.
Whitford, P.C., Schug, A., Saunders, J., Hennelly, S.P., Onuchic, J.N., and Sanbonmatsu, K.Y. (2009). Nonlocal helix formation is key to understanding S-adenosylmethionine-1 riboswitch function. Biophys J96, L7-9.
Wilderman, P.J., Sowa, N.A., FitzGerald, D.J., FitzGerald, P.C., Gottesman, S., Ochsner, U.A., and Vasil, M.L. (2004). Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosainvolved in iron homeostasis. Proc Natl Acad Sci U S A 101, 9792-9797.
Winkler, W.C., Grundy, F.J., Murphy, B.A., and Henkin, T.M. (2001). The GA motif: an RNA element common to bacterial antitermination systems, rRNA, and eukaryotic RNAs. RNA 7, 1165-1172.
Winkler, W.C., Nahvi, A., Sudarsan, N., Barrick, J.E., and Breaker, R.R. (2003). An mRNA structure that controls gene expression by binding S-adenosylmethionine. Nat Struct Biol 10, 701-707.
Wong, K.K., Bouwer, H.G., and Freitag, N.E. (2004). Evidence implicating the 5' untranslated region ofListeria monocytogenes actA in the regulation of bacterial actin-based motility. Cell Microbiol 6, 155-166.
Wood, S., Maroushek, N., and Czuprynski, C.J. (1993). Multiplication of Listeria monocytogenes in a murine hepatocyte cell line. Infect Immun 61, 3068-3072.
Wu, S., de Lencastre, H., and Tomasz, A. (1996). Sigma-B, a putative operon encoding alternate sigma factor of Staphylococcus aureus RNA polymerase: molecular cloning and DNA sequencing. J Bacteriol178, 6036-6042.
Yanofsky, C. (1981). Attenuation in the control of expression of bacterial operons. Nature 289, 751-758.
Zhang, A., Wassarman, K.M., Ortega, J., Steven, A.C., and Storz, G. (2002). The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs. Mol Cell 9, 11-22.
Zhu, J., Miller, M.B., Vance, R.E., Dziejman, M., Bassler, B.L., and Mekalanos, J.J. (2002). Quorum-sensing regulators control virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci U S A 99, 3129-3134.
90
Appendix: Paper I-IV