literature review linked in
TRANSCRIPT
Resistance, persistence and the continued rise and threat of the superbugs; could bacterial
suicide modules have the answer?
Introduction
The pre-antibiotic era was a time when bacteria were able to thrive with doctors unable to offer much
in the way of treatment to patients suffering from bacterial infections. Even a small graze or cut could
result in an untreatable infection that could lead to sepsis and possibly death (Beatson and Walker,
2014). Today thankfully due to antibiotics most bacterial infections can be treated, however bacteria
have found a way to avoid the effects of antibiotics and now the rise of superbugs from antibiotic
resistance has become a real threat and a major public health problem. Resistance was known about
early on in antibiotic development, even before penicillin’s first clinical use in the early 1940s, an
enzyme that was able to destroy it had already been identified (Moellering, 2010). Alexander
Flemming famously mentioned during his Nobel Prize acceptance speech in 1945 that the careless
overuse or under-dosing of penicillin could result in bacteria becoming resistant to it (Cruickshank et
al., 2014). Now following decades of antibiotic overuse by doctors, veterinarians and the farming
community, resistance is now a very real and current issue. The possibility of entering a new post
antibiotic era similar to that of the pre-antibiotic era could be a possibility and many procedures such
as life-saving organ transplantation or cancer treatments that require immunosuppression would
become extremely difficult to carry out (Yap, 2013). No new classes of antibiotics have been
discovered since the 1970s and drug companies see antibiotics as a bad investment, so the search for
novel antimicrobials is now extremely important (Aminov, 2010).
Antibiotic use and resistance
Resistance is not a new phenomenon; it was present within natural ecosystems long before humans
began using antibiotic drug therapy. Actinomycetes bacteria found within soil produce many of the
naturally occurring antibiotics which are currently used, they also possess genes that confer resistance
to these antibiotics (Davies and Davies, 2010). Within an ecosystem bacteria may employ resistance
either as a form of self-protection if they are producers of antibiotics themselves or for protection
against other bacteria producing antibiotics as a means to co-exist (Martinez, 2012). Many resistance
genes conferring resistance to currently used antibiotics have been identified within environmental
biomes (D'Costa et al., 2006).
Beta-lactamase genes which confer resistance to beta-lactam antibiotics such as the penicillins are
ancient and have been isolated within environments unpolluted by human activity such as Alaskan
soils, demonstrating that resistance was already present within the environment long before humans
began using antibiotics (Allen et al., 2009). A study by Mindlin et al., (2008) tested resistance present
in bacteria isolated from 3 million year old Eastern Siberian permafrost sediments and found bacteria
resistant to kanamycin, streptomycin, gentamicin, chloramphenicol and tetracycline within their
samples, many of which were Gram positive species. A study by Bhullar et al., (2012) investigated the
antibiotic resistance occurring in bacteria which had been isolated for more than 4 million years within
the Lechuguilla Cave, New Mexico. 93 strains of the bacteria isolated from this cave were screened
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against 26 antibiotics which included naturally occurring, semisynthetic and completely synthetic
antibiotics. The results revealed that 70% of the Gram positive strains were resistant to an average of
3-4 antibiotic classes, with three strains of Streptomyces spp. showing resistance to 14 antibiotics.
Within the Gram negative samples 65% were resistant to 3-4 antibiotic classes that included the
antibiotics trimethoprim, sulfamethoxazole and fosfomycin. When looking at the actions of beta-
lactams against the samples, 22-62% of the strains were able to inactivate this class of antibiotic. Both
of these studies were able to reveal that resistance has deep evolutionary origins and that bacteria
living within secluded environments already possess a multitude of resistance genes.
A study by D’Costa (2006) looked to discover the levels of resistance present within 480 strains soil-
dwelling bacteria to 21 medically used antibiotics which included naturally occurring compounds and
both semisynthetic and completely synthetic derivatives. The results revealed that at least two of the
strains were resistance to 15 of the antibiotics with the rest being resistant to an average of 7 or 8
antibiotics. The antibiotic rifampicin was inactivated by 40% of the bacterial strains and the recently
approved daptomycin was inactivated by 80% of the strains. Both of these antibiotics play key roles in
the treatment of human pathogenic bacteria, with rifampicin used within mycobacterial infections and
daptomycin used against multidrug resistant strains of Gram positive bacteria. This study was able to
reveal the wealth of multidrug resistance present already within soil bacteria communities and could
offer insight into how clinical resistance may emerge within the future.
Antibiotics are not confined to fighting human pathogens, the application of commercially produced
antibiotics for this purpose account for less than half of their use (Davies and Davies, 2010). The use
within farming and the possible rise of resistance from this use has been studied extensively and has
caused a great deal of controversy and the implementation of both policy and regulation. There are
advantages from using antibiotics within animal husbandry and the results can include higher, more
efficient production levels, improved health of livestock, lower disease occurrence and low cost/high
quality and nutritious food production (Oliver et al., 2011). However with the increased levels of
resistance emerging, agricultural use has been seen as a possible route by which resistance can gain
access to the human population and it is now considered a severe public health problem (Landers et
al., 2012).
The realisation that antibiotics could be used as growth promoters within the farming industry was
discovered in the 1940s following research into what was then called the “animal protein factor” but is
now known as Vitamin B12 (Viola and DeVincent, 2006) (Stokstad and Jukes, 1950). A number of
further experiments showed great merit from mixing antibiotics into animal feed and following the
advent of confinement breeding and the increased risk of infection within herds, the antibiotic was
seen as both commercially advantageous and beneficial to animal health and welfare and has been
used intensely in farming since the 1950s (Gustafson and Bowen, 1997) (Chattopadhyay, 2014)
(Kemper, 2008). The practice of using antibiotics as growth promoters has now been banned within
the European Union (EU) since 2006, following the growing concern of resistance (Castanon, 2007).
The United States (US) still allows the use of antibiotics as growth promoters, however in 2012 the US
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Food and Drug Administration (FDA) released guidelines explaining how they would be making steps
to reduce the amount of antibiotics used for this purpose (FDA, 2012).
Trying to ascertain exact figures for antibiotic usage is challenging due to the limited documented
evidence available and the scarcity of pharmaceutical companies to release this information. However,
it is estimated that the release of millions of tonnes of antibiotics into the environment has occurred
following their use within agriculture, aquaculture and medicine over the last 50 years (Davies and
Davies, 2010). A report in 2001, estimated that out of the 24.6 million pounds of antibiotics consumed
within the US per year, a large proportion of this is used for growth promotion within farm animals
(Oliver et al., 2011). Spellberg et al. (2013) reported that around 13 million kg of antibiotics were used
in 2010 within farming, a large proportion of which were for growth promotion. The controversy over
both sides of the agricultural use argument appears to be a hot topic that remains unresolved. The ‘for’
argument believes it is uncertain how these antibiotics affect resistance at the “sub-therapeutic” doses
used and fear that banning the use of antibiotics as a prophylactic treatment, could actually increase
disease occurrences in humans from food-borne sources, along with causing an increase in animal
diseases (Chattopadhyay, 2014) (Casewell et al., 2003). The ‘against’ argument however argues that
even at very low concentrations of antibiotics, selection for resistance can still occur (Gullberg et al.,
2011). Furthermore a study by Couce and Blázquez (2009) revealed that the evolution of bacterial
resistance along with horizontal gene transfer (HGT) and recombinational events could be affected by
sub-inhibitory antibiotic use. There is also evidence to suggest that antibiotics can have an adverse
effect on the natural gut flora of the animal causing dysbiosis and an increase in pathogenic bacteria
such as Clostridium difficile, implying that antibiotic use could actually do more harm than good
(Chattopadhyay, 2014).
Attempts to use other antibiotics within farming that are not involved in human disease prevention
have also been shown to be problematic. Avoparcin an antibiotic not prescribed to humans but a close
relative of vancomycin was in use as a feed additive throughout the EU until the mid-1990s. EU
countries began to one by one ban the use of avoparcin, when it was found to be linked to increases in
the isolation of vancomycin-resistant enterococci (VRE) in both animals and humans (Marshall and
Levy, 2011). Studies in Italy, Hungary, Germany and Taiwan, all showed a decrease in VRE
prevalence among farm animals after the use of avoparcin had been discontinued (Pantosti et al.,
1999) (Kaszanyitzky et al., 2007) (Klare et al., 1999) (Lauderdale et al., 2007).
Another reason thought to be responsible for the emergence of antibiotic resistance is overprescribing
and over the counter purchasing of antibiotics worldwide. The combination of public demand and lack
of diagnosis have resulted in antibiotics being prescribed for illnesses such as upper respiratory tract
infections, acute bronchitis, lower respiratory tract infections, conjunctivitis and undiagnosed skin
complaints (Dallas et al., 2014) (Petersen et al., 2007) . Many of these infections are likely to not
require antibiotic treatment or are of viral origin. Medical practitioners however are placed in a difficult
position and empirical prescribing is carried out regardless of whether microbiological investigations
have been completed (Leibovici et al., 2012) (Vellinga, 2014). In Canada the results of a study
revealed that annually out of the 26 million antibiotics prescribed, up to half of them were deemed to
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be unnecessary (Heymann, 2006). The prescribing of an antibiotic not only affects the patient
receiving it, but also other people in the future who may require antibiotic treatment. Selective
pressures induced by antibiotic use have fuelled the rise of the resistant bacterium and in turn this has
induced tighter regulation on how antibiotics can and should be prescribed. The decision between not
prescribing and prophylactic treatment of a suspected bacterial infection is very much a medical and
ethical one, for both the current patient and the future population (Vellinga, 2014).
If large quantities of antibiotics, used within farming and human medicine enter the environment as
pollution they are at great risk of contaminating the environmental reservoir with large quantities of
resistance genes. Selection pressure due to high level antibiotic usage, results in increases in the
likelihood of resistant pathogenic bacteria appearing (Luis Martinez, 2009).
Becoming resistant
Bacteria gain their resistance to antibiotics predominantly by a process known as HGT, whereby
mobile genetic elements (MGE) are transferred between bacteria. Three main mechanisms exist for
HGT; natural transformation whereby competent bacteria take up DNA from the environment and
incorporate it into their own genome, transduction mediated by bacteriophage and conjugation which
requires the actions of a conjugative pilus to deliver MGE such as transposons or plasmids and even
entire chromosomes to a target cell (Norman et al., 2009) (Huddleston, 2014) (Guglielmini et al.,
2013).
Transduction mediated by bacteriophage has only recently begun to be understood. Transduction
occurs when a bacteriophage replicates and packages part of the host genome into its own genome. If
the cell is one that confers resistance to an antibiotic, upon infecting another cell the bacteriophage
can transfer the resistance. This process of genetic transfer is completely accidental and the genes
packaged into the bacteriophage can be either completely random (generalised transduction) or within
close proximity of the attachment site of the bacteriophage (specialised transduction). Transfer of
resistance genes mediated by transduction has only recently been considered to be of real
significance to the antibiotic resistance phenomenon. (Huddleston, 2014) (Muniesa et al., 2013b). An
experiment by Schmieger and Schicklmaier (1999) showed that transduction of resistance to
tetracycline, chloramphenicol and ampicillin was possible using bacteriophages within Salmonella
enterica serovar typhimurium DT104. A study by Fard et al. (2011) looked to demonstrate the ability of
bacteriophage to transfer resistance genes via transduction within the same and different
Enterococcus spp. Gentamicin was transduced between species of Enterococcus faecalis and also to
Enterococcus hirae ⁄ durans and Enterococcus casseliflavus and resistance to tetracycline was
transduced from Enterococcus gallinarum to E. faecalis. The results show interspecies transduction by
bacteriophage is a possibility. Both of these studies could offer better understanding about the
mechanisms by which resistance arises and how it can be mediated by bacteriophage. Transduction
does not require that the donor and recipient be present together and due to the nature of the capsid
of the bacteriophage protecting the transduced DNA and the long-lived nature of bacteriophages,
persistence within an environment can occur (Muniesa et al., 2013b). The event of bacteriophage-
4
mediated transduction of antibiotic resistance between different taxa is considered to be a rare event,
however the idea that bacteriophages could be a link between environmental reservoirs of resistance
and human or animal biomes is considered to be a possibility (Muniesa et al., 2013a).
Conjugation, whereby a plasmid is transferred via a conjugative pilus however is not a rare event and
occurs quite readily among bacterial populations (Huddleston, 2014). Plasmids often contain an array
of genes that contribute to the evolution of bacteria, aiding their survival and allowing them to exploit
particular niches or survive within hostile environments (Guglielmini et al., 2013) (Norman et al., 2009).
Plasmids are able to invade a variety of host bacteria; one example first identified within
Pseudomonas aeruginosa is the RP1 plasmid, which was shown to be able to invade almost any
Gram negative bacteria. Genes encoded by plasmids often confer resistance to heavy metals such as
silver, mercury or cadmium but they also encode antibiotic resistance genes allowing bacterial growth
even in the presence of antibiotics (Bennett, 2008).
Origins of resistance
Not all forms of resistance seen however in pathogenic bacteria can claim to have the same
biochemical function in nature. Genes acquired by processes such as HGT may have not necessarily
conferred resistance in the original host but may now serve that purpose in the pathogenic host. This
functional change due to HGT which does not alter the gene itself is known as exaptation (Martinez,
2012). Efflux pumps for example, are deployed by pathogenic bacteria to pump out antibiotics but they
are also used in physiological processes such as signalling or ridding the bacterial cell of toxic
substances such as bile, hormones and plant toxins (Piddock, 2006).
Within natural environments resistance genes are often chromosomally located but under strong
selective pressures, these genes can become integrated into plasmids which are then mobile and free
to enter other bacterial cells (Luis Martinez, 2009). The plasmid mediated resistance determinant QnrA
which confers resistance to quinolones, is widespread among pathogenic bacteria including
Enterobacteriaceae spp., Klebsiella pneumonia and Escherichia coli. The resistance determinant QnrA
is believed to have originated within Shewanella algae, a non-antibiotic producing marine and
freshwater bacterium. Within S. algae the gene encoding QnrA (qnrA) is chromosomally encoded but
continued quinolone pollution into water systems is believed to have encouraged integration of qnrA
into a plasmid (Poirel et al., 2005) (Luis Martinez, 2009). A study by Kim (2011) was able to show that
within its natural host QnrA has a very different physiological function. It was found that expression of
qnrA could be increased by induction of cold shock and the study concluded that the role of QnrA
within S. algae is likely to be in secondary structure stabilisation of both DNA and RNA and also
perhaps DNA binding proteins, which in turn allows for adaption to cold environmental conditions, a
function very different to that of conferring antibiotic resistance. Another example of resistance
determinants with differing functional origins are enzymes. Within their natural hosts they may be used
to modify bacterial peptidoglycan but upon transfer to pathogenic strains of bacteria, these enzymes
can be used to alter the structures of antibiotics (Martinez, 2012). The enzyme 2-N-acetyletransferase
within the bacteria Providencia struartti is able to alter both peptidoglycan and the antibiotic gentamicin
5
due to substrate similarity. Should a plasmid encoding this enzyme be acquired by another bacteria,
the enzyme’s role would now be simply in conferring gentamicin resistance as the enzyme is no longer
within its normal biochemical setting (Martinez, 2008) (Macinga and Rather, 1999).
Bacteria often already have the mechanisms to become resistant and their adept ability to share
genetic information can turn treatable pathogenic bacteria into multidrug resistant strains also
commonly called “superbugs”. Many superbugs are now treatable with only a few antibiotics and new
resistant strains continue to be isolated every year, some designated as pandrug-resistant are
resistant to every antibiotic currently available (Rossonlini et al., 2014) (Magiorakos et al., 2012).
Rise of the superbugs
Superbugs have become a global threat, causing huge costs and contributing to many deaths every
year worldwide. A report in 2009 by the European Centre for Disease Prevention and Control, placed
the death toll per year in Europe from multidrug resistance at 25,000 with costs of €1.5 billion (Uchil et
al., 2014). Gram positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and
VRE have for the most part been the most worrying culprits of antibiotic resistance. However, it is now
becoming clear that Gram negative bacteria such as those of the Enterobacteriaceae spp. could
possibly pose an even greater risk (Kumarasamy et al., 2010).
It was first reported in 1983 that beta-lactamase enzymes encoded on plasmids were able to
hydrolyse extended-spectrum cephalosporins now known as extended-spectrum beta-lactamases
(ESBLs). Carbapenams were the drug of choice against ESBLs and have since been used
extensively. Enterobacteriaceae spp. encoding resistance genes to carbapenems are now a major
public health problem and are grouped under the collective term carbapenem-resistant
Enterobacteriaceae (CRE). CRE confer resistance to carbapenems by using carbapenamase
enzymes to degrade the beta-lactam ring of beta-lactam antibiotics. The structures of the beta-lactam
classes of antibiotics along with the target for the beta-lactamase enzyme circled in red can be seen in
Figure 1.
Penicillin Cephalosporin Carbapenem Monobactam
Figure 1. Structures of the beta-lactam antibiotics. The red circle indicates the beta-lactam ring which is targeted and broken by beta-lactamase enzymes (Zervosen et al., 2012).
Beta-lactam antibiotics are often used against multidrug resistant strains of Gram negative bacteria,
sometimes as a last resort. A study by Conlan et al. (2014) investigated the diversity of CRE present
within a hospital environment and identified E. coli, Enterobacter cloacae, K. pneumoniae and
Klebsiella oxytoca as all possessing a variety of plasmids encoding the resistance gene. Several
bacteria encoding resistance genes to carbapenems are now categorised as CRE. K. pneumoniae
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carbapenemase (KPC) is a beta-lactamase class A penicillinase enzyme present within the bacterium
K. pneumoniae that confers resistance to not only carbapenems but also all other beta-lactam
antibiotics (Endimiani et al., 2008). The resistance gene known as blaKPC is located within a
transposon known as Tn4401, which can be taken up by a variety of plasmids and distributed among
many Gram negative bacteria (Arnold et al., 2011). Plasmids encoding blaKPC have been shown to
also encode genes such as qnrB or qnrA that confer resistance to quinolones and rmtB that confers
resistance to aminoglycosides. Both of these antibiotic classes are often used against beta-lactam
resistant infections (Endimiani et al., 2008) (Sheng et al., 2012). KPCs however are not only confined
to K. pneumoniae bacteria and have been found in other Enterobacteriaceae spp. such as Proteus,
Escherichia, Salmonella and Citrobacter. Resistance has also been found to be present within
Pseudomonas spp. and Acinetobacter baumannii which already harbour other resistance genes (Chen
et al., 2012). There remain only a few effective antibiotics such as tigecycline and colistin available to
treat KPC infections and it has now become a worldwide problem among nosocomial infections. More
worrying still infections that are resistant to tigecycline and colistin have also been reported. With
estimated mortality rates of between 22% and 59%, KPC is seen as formidable threat to public health
(Chen et al., 2012).
Another resistance enzyme causing multidrug resistance within Enterobacteriaceae spp. is the New
Delhi Metallo-beta-lactamase 1 (NDM-1), a class B metallo-beta-lactamase that possesses zinc at the
active site. The resistance gene responsible (blaNDM-1) is found on a plasmid and other resistance
genes have been shown to accompany it, such as those conferring resistance to chloramphenicol,
erythromycin, rifampicin and ciprofloxacillin. A further gene known as blaCMY-4 that encodes a broad
spectrum beta-lactamase, along with genes encoding an efflux pump have also been shown to be
present on the same mobile genetic element as blaNDM-1 (Charan et al., 2012). NDM-1 was first
identified in K. pneumoniae from a patient of Indian origin in Sweden in 2008, since then it has spread
to many countries including the US, Turkey, Israel, China, Australia, France, Japan and Britain.
Studies since its discovery have shown NDM-1 to be widespread among Enterobacteriaceae spp.
throughout India, Pakistan, Bangladesh and Britain. The high level use of non-prescription antibiotics
within India along with poor sanitation, overcrowding and widespread diarrheal disease make for a
good platform for this kind of resistance to develop (Moellering, 2010). Studies have shown the ability
of blaNDM-1 to spread is significant owing to the fact it can be found on plasmids of varying sizes, which
are extremely mobile and able to spread among bacterial populations. Another worrying finding is that
NDM-1 producing Enterobacteriaceae spp. resistant to colistin, one of the few antibiotics available to
treat these resistant infections have also been isolated (Bonomo, 2011) (Kumarasamy et al., 2010).
Bacteria classed as superbugs are a real threat to public health, their sheer numbers, adept ability at
sharing genetic information and the lack of new antibiotics to treat them, make finding an alternative to
traditional antimicrobial therapy an extremely urgent requirement (Kumarasamy et al., 2010).
Targeting novel physiological systems of bacteria could provide a new form of therapy, one such area
is the toxin-antitoxin (TA) system, which can act as a suicide module, inducing apoptosis of the
bacterial cell.
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Toxin-antitoxin systems – bacterial suicide modules.
The first TA system discovered within E. coli was the ccd (control of cell death) system encoded on the
F plasmid. The ccd TA system consists of the antitoxin CcdA and the toxin CcdB, under normal
cellular conditions, the toxin forms a complex together with the antitoxin stabilising it. It was found that
the antitoxin alone was unstable and could be readily degraded by Lon proteases, which freed the
toxin allowing it to interact with intracellular targets (Ogura and Hiraga, 1983) (Hu et al., 2010). TA
systems came to be known as plasmid addiction modules following the discovery that they are
involved in post-segregational killing of plasmid free cells. TA systems that were discovered to be
chromosomally encoded however were found to have other functions other than simply just plasmid
maintenance and TA systems targeting other cellular functions such as translation, cell division and
DNA replication were also soon discovered (Schuster and Bertram, 2013). TA systems have been
found to be present within many different species of bacteria and can be classified into groups
denoted as type I – V (Ghafourian et al., 2014).
Type I TA systems consist of an antisense RNA antitoxin which is able to bind the toxin mRNA,
resulting in regulation of antitoxin translation. A good example of this system is Hok/Sok which is
encoded on the plasmid R1, hok encodes the toxin and sok encodes the antitoxin. This type of TA
system is involved in post-segregational killing of plasmid free cells (Franch et al., 1997) (Schuster and
Bertram, 2013) (Ghafourian et al., 2014).
Type II TA systems consist of a long-lived sequence-specific endoribonuclease and a labile antitoxin
protein. Both the toxin and antitoxin form a complex together which renders the toxin inactive due to
the antitoxin obscuring essential sites required for the toxin’s activity. Degradation of the antitoxin by
proteases allows the toxin to interact with intracellular targets, which will eventually lead to cell death.
The RelBE TA system which is chromosomally encoded is a good example a Type II TA system, the
antitoxin RelB binds to the relBE promoter and acts as an autoregulator of relBE transcription, the
toxin RelE acts as a corepressor (Tashiro et al., 2012).
Type III TA systems consist of an endonuclease toxin which is inhibited by an RNA antitoxin. The
toxin gene is transcriptionally regulated by a short palindromic repeat, acting as a terminator. The
ToxIN TA system which can be both chromosomally and plasmid encoded is an example of a type III
TA system and consists of the toxin ToxN which is inhibited by the RNA antitoxin ToxI by formation of
a protein/RNA complex. The ToxIN system was first identified within the Gram negative
phytopathogen Pectobacterium atrosepticum encoded on the plasmid pECA1039 (Blower et al., 2012)
(Schuster and Bertram, 2013) (Ghafourian et al., 2014). ToxIN has been shown to be activated during
bacteriophage infection and is involved in bacteriophage defence via “altruistic suicide” whereby
individual bacterial cells infected with bacteriophages are killed to prevent the spread of the
bacteriophage infection (Fineran et al., 2009).
The type IV TA system is unlike other TA systems as the toxin and antitoxin do not form a complex.
The CbtA/CbeA is an example of a type IV TA system. The toxin CbtA targets two cytoskeleton
proteins MreB and FtsZ by inhibiting polymerisation and cellular growth. The antitoxin CbeA works as
8
an antagonist to CbtA by stabilising the toxin’s target and shielding it. CbeA has also been shown to
neutralise other inhibitors of MreB and FtsZ such as MinC, A22 and SulA (Masuda et al., 2012a)
(Masuda et al., 2012b) (Schuster and Bertram, 2013).
The type V TA system consists of a sequence specific endoribonuclease antitoxin and a protein toxin,
which are thought to form a TA pair. GhoST is an example of a type V TA system where the activity of
the toxin produces ghost cells through cell lysis. GhoS the antitoxin post-transcriptionally regulates the
toxin gene ghoT by cleaving mRNA, preventing production of the membrane lytic protein toxin GhoT.
GhoST has also been shown to increase persister cells. The GhoST type V TA system shows some
similarity to the Type II TA system however GhoS is not labile under stress and does not act as a
transcriptional regulator (Wang et al., 2012) (Schuster and Bertram, 2013) (Unterholzner et al., 2013).
By far the most abundant TA system found in nature is the type II system, one such system belonging
to this group, which has been studied extensively is the MazEF TA system. The genes encoding the
MazEF TA system (mazEF) are present on the chromosome of many species of bacteria, though
many of the studies conducted have been within E. coli (Engelberg-Kulka et al., 2006). The mazEF
genes lay adjacent to each other and are negatively autoregulated via the actions of both the MazE
and MazF proteins. The mazEF genes are upstream of the relA gene that encodes the RelA protein,
which under stressful conditions such as nutrient depletion releases the amino acid signal molecule
ppGpp (3’ 5’ guanosine bispyrophosphate), leading to mazEF expression inhibition. Other inhibitors of
translation and transcription leading to programmed cell death (also termed bacterial suicide)
mediated by the MazF toxin, include the use of antibiotics such as chloramphenicol, spectinomycin
and rifampicin, the prophage P1 Doc protein, thymine starvation leading to DNA damage, oxidative
stress and UV irradiation (Hazan et al., 2004) (Hayes, 2003) (Van Melderen, 2010). A diagram
detailing the pathways of MazEF can be seen in Figure 2.
Figure 2. Pathways of MazEF: 1. The mazE and mazF genes lay adjacent to each other and upstream of the relA gene. mazEF are negatively regulated by the MazEF proteins at the P2 promoter. 2. Continued expression of MazE is required to form the stable complex with MazF. 3. Stress such as nutrient depletion or antibiotic use can lead to MazEF inhibition. 4. Under stressful conditions MazE is degraded by ClpAP proteases. 5. Leaving free uncomplexed MazF toxin within the cell. 6. MazF targets ACA sequences of mRNA. 7. mRNA is degraded. 8. Translation is terminated leading to growth arrest and eventual cell death.
9
Harnessing the ability to control the toxic component of the TA system by artificially activating it could
provide a new novel form of antimicrobial therapy. Homologs of the proteins found in the TA system
are not present in humans, so they serve as good targets for antimicrobial drugs. One idea for an
antimicrobial target that could be used against TA systems such as RelBE and MazEF, is to disrupt
the formation of the TA complex, resulting in the toxin being unable to complex with the antitoxin
leading to cell death, this is known as direct activation. Another approach known as indirect activation
would be to introduce a molecule known as a sequence specific DNA binder that binds to the
promoter, modulating TA expression and inhibiting transcription of the antitoxin. The inducible
activation of Lon and Clp proteases which degrade the antitoxin and in turn release the toxin has also
been suggested (Williams and Hergenrother, 2012). A diagram detailing the modes of actions of these
proposed antimicrobial targets can be seen in Figure 3.
Figure 3. Direct and indirect activation of the TA complex. (Williams and Hergenrother, 2012).
There has been much interest in finding ways to target the TA system, however studies are still in the
early stages and many challenges are faced before exploitation of TA systems as a form of therapy
can be achieved. As MazEF and RelBE or homologs of them have been found to be present in many
pathogenic bacteria such as S. aureus (including MRSA), Mycobacterium tuberculosis and P.
aeruginosa these TA systems look promising as possible targets for novel antimicrobial therapy
development (Williams and Hergenrother, 2012) (Fu et al., 2007) (Park et al., 2013).
Conclusion
Antibiotics used within clinical therapy have been around less than a century and in that time
resistance and the rise of superbugs has become a major issue, resulting in many deaths every year.
It is evident though that antibiotic resistance is ancient and a problem that will continue even if new
antibiotics are developed. Bacteria are adept at finding ways to render the antibiotics produced to kill
them useless and with human and veterinary medicine heavily reliant on antibiotics, they will continue
to be used in vast numbers and resistance will continue to arise unless an alternative is found. Without
antibiotics however, bacterial infections previously curable could be treated with palliative care at best.
Now more than ever the requirement to find a new approach and to develop novel therapies is
essential to ensure movement into a pre-antibiotic era is prevented.
10
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