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The Role of Bps Polysaccharide in Bordetella Resistance to Host Innate
Defenses
BY
CHERATON FABRICE LOVE
A Dissertation Submitted to the Graduate Faculty of
WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES
in Partial Fulfillment of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
Microbiology and Immunology
August 2010
Winston-Salem, North Carolina
Approved By:
Rajendar Deora, Ph.D., Advisor __________________________
Examining Committee:
Linda McPhail, Ph.D., Chairman __________________________
Martha Alexander-Miller, Ph.D. __________________________
Griffith Parks, Ph.D. __________________________
Nancy Kock, D.V.M, Ph.D. __________________________
Table of Contents List of Figures....................................................................................................................iv List of Tables.....................................................................................................................vi List of Abbreviations.......................................................................................................vii Abstract..............................................................................................................................x Chapter One: Introduction...............................................................................................1 The Bordetella genus……………………………………………………………………...1 Bordetella pertussis course of illness…………………………………………………......2 The BvgAS signal transduction system……………………………………………….......6 BvgAS regulation of Bordetella phenotypes......................................................................9 BvgAS regulation of Bordetella virulence factors……………………………………….10
Adhesins………………………………………………………………………….10
Toxins……………………………………………………………………………12
Biofilms………………………………………………………………………………….15
Bordetella biofilms………………………………………………………………………16
Bacterial Exopolysaccharides……………………………………………………………19
Biofilm relevance to human infection……………………………………………………21
Innate immune responses to B. bronchiseptica…………………………………………..22
Antimicrobial peptides…………………………………………………………...22
Neutrophils and macrophages…………………………………………………....24
Cytokines………………………………………………………………………...24
Animal models…………………………………………………………………………...25
ii
Limitations of studies reported in the literature……..…..……………………………….26
Chapter Two: Materials and Methods...……………………………………………...28
Chapter Three: The Role of Bps in Resistance to Host Innate Defenses….………..35
The Bps polysaccharide protects B. bronchiseptica from killing by polymyxin B...........35
The Bps polysaccharide protects biofilms formed by the Δbps strain from polymyxin B
killing in trans....................................................................................................................41
Overproduction of Bps prevents binding of polymyxin B to B. bronchiseptica
biofilms..............................................................................................................................44
The Bps polysaccharide directly binds polymyxin B........................................................45
The bps locus promotes colonization of B. bronchiseptica in the mouse respiratory tract
during the persistent stage of infection..............................................................................51
The Bps polysaccharide enhances colonization of B. bronchiseptica in the mouse
respiratory tract at 3 days post-inoculation........................................................................51
Neutrophils are an important host factor in the clearance of the Δbps strain in vivo........54
The Bps polysaccharide does not play a role in cellular infiltration following a
B. bronchiseptica infection...............................................................................................64
The Δbps mutant strain does not have increased uptake by neutrophils...........................67
The Δbps strain induces greater cytokine production in mouse lungs at 2 and 3 days post-
inoculation..........................................................................................................................67
Chapter Four: Discussion……………………………………………………………...75
References……………………………………………………………………………..…86
CurriculumVitae………………………………………………………………………..104
iii
List of Figures
Chapter One
Figure 1: Bacterial load of B. pertussis during classical pertussis illness...........................3
Figure 2: The BvgAS signal transduction system of Bordetella species............................7
Figure 3: Biofilm formation is a multi-step process..........................................................17
Chapter Three
Figure 1: The Bps polysaccharide protects planktonic B. bronchiseptica cells from killing by polymyxin B.......................................................................................36
Figure 2: The Bps polysaccharide protects B. bronchiseptica biofilms from killing by
polymyxin B.......................................................................................................39 Figure 3: The Bps polysaccharide increases resistance of biofilms formed by the Δbps
mutant strain to polymyxin B killing in trans.....................................................42 Figure 4: Overproduction of Bps prevents binding of polymyxin B to B. bronchiseptica
biofilms...............................................................................................................46 Figure 5: The Bps polysaccharide directly binds polymyxin B.........................................49 Figure 6: The bps locus promotes persistent colonization of B. bronchiseptica in the
mouse respiratory tract.......................................................................................52 Figure 7: Kinetics of colonization of the wild-type B. bronchiseptica and isogenic mutant,
Δbps, in the murine respiratory tract..................................................................55 Figure 8: Colonization of the wild-type and Δbps strains in neutropenic and wild-type
C57BL/6 mice at 2 days post-inoculation..........................................................62 Figure 9: Colonization of the wild-type and Δbps strains in neutropenic and wild-type
C57BL/6 mice at 3 days post-inoculation..........................................................65 Figure 10: Cellular infiltration in bronchoalveolar lavage (BAL) fluid of mice infected
with the wild-type B. bronchiseptica or the Δbps strain................................68 Figure 11: Phagocytosis of the wild-type and Δbps strains by neutrophils.......................70
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Figure 12: Cytokine detection in lungs of mice infected with the wild-type and Δbps strains...............................................................................................................73
Chapter Four Figure 1: Model of potential roles of Bps polysaccharide in evasion of host defenses....84
v
List of Tables
Chapter Three Table 1: Histopathology scores of lungs of mice infected with the wild-type or Δbps strain.................................................................................................................................57 Table 2: Presence of neutrophils in lungs of mice infected with the wild-type or Δbps strain.................................................................................................................................60
vi
List of Abbreviations
ADP..................................................................................................Adenosine diphosphate ATP..................................................................................................Adenosine triphosphate AMPs.................................................................................................Antimicrobial peptides BAL.................................................................................................Bronchoalveolar lavage BipA.......................................................................Bordetella intermediate phase protein A BMDCs.....................................................................................Bone marrow dendritic cells BG.................................................................................................................Bordet-Gengou Bps...............................................................................................Bordetella polysaccharide Bpshyp...................................................................................................Bps hyper-expressing Δbpsvec.................................................................................................................Δbps vector Bvg................................................................................................Bordetella virulence gene cAMP.................................................................................................................Cyclic AMP CFU.......................................................................................................Colony forming unit CHO..................................................................................................Chinese hamster ovary CPS.................................................................................................Capsular polysaccharide CR3..................................................................................................Complement receptor 3 CSLM...........................................................................Confocal scanning laser microscopy dPNAG...................................................................deacetylated Poly N-acetylglucosamine DNA...................................................................................................Deoxyribonucleic acid DNT......................................................................................................Dermonecrotic toxin DTaP.........................................................................Diphtheria-Tetanus-acellular Pertussis
vii
ELISA........................................................................Enzyme-linked immunosorbent assay EPS..........................................................................................................Exopolysaccharide FHA............................................................................................Filamentous hemagglutinin Fim...........................................................................................................................Fimbriae H & E................................................................................................Hematoxylin and eosin HBD.....................................................................................................Human beta defensin HNP-1.......................................................................................Human neutrophil peptide-1 HPD.................................................................................Histidine phosphotransfer domain HTE.............................................................................................Hamster tracheal epithelial IFNγ...........................................................................................................Interferon gamma Ig.................................................................................................................Immunoglobulin IL..........................................................................................................................Interleukin IS..............................................................................................................Insertion sequence LPS.........................................................................................................Lipopolysaccharide LRI/ IAP.......................................Leukocyte response integrin/ integrin-associated protein MgSO4.....................................................................................................Magnesium sulfate MIC................................................................................Minimum inhibitory concentration NAD...............................................................................Nicotinamide adenine dinucleotide OD.................................................................................................................Optical density PAMPs....................................................................Pathogen-associated molecular patterns PBS...............................................................................................Phosphate buffered saline PIA.............................................................................Polysaccharide intercellular adhesion PNAG...........................................................................................Poly N-acetylglucosamine
viii
PRN..........................................................................................................................Pertactin PT...................................................................................................................Pertussis toxin RGD..........................................................................................Arginine-Glycine-Aspartate SEM.......................................................................................Scanning electron microscopy SM....................................................................................................................Streptomycin TAP.......................................................................................Tracheal antimicrobial peptide TCT..........................................................................................................Tracheal cytotoxin TNFα.........................................................................................Tumor necrosis factor alpha TTSS.........................................................................................Type three secretion system WFUHS.................................................................Wake Forest University Health Sciences WT.........................................................................................................................Wild-type
ix
Abstract
Bacteria face many challenges during infection of the host. Initiation of infection
requires attachment to host surfaces. Bordetella species possess several virulence factors
that mediate binding to the host such as filamentous hemagglutinin and pertactin. A
crucial component of successful colonization is evasion of host defenses. Biofilms have
been proposed to promote bacterial persistence by suppressing the effectiveness of host
responses. A hallmark of mature biofilms is the production of a matrix composed of
extracellular polymers including proteins, DNA and polysaccharides. We have shown
that the bps locus is required for biofilm formation and production of the Bordetella
polysaccharide (Bps), a major constituent of Bordetella biofilms.
Bacterial polysaccharides have been attributed to promote bacterial resistance
to host responses. We chose to explore the role of Bps in resistance to antimicrobial
peptides, a key host factor involved in the first line of defense against microbial infection.
We used polymyxin B, an amphipathic molecule with considerable bactericidal activity,
as a model of antimicrobial peptides. Planktonic cells as well as biofilms formed by the
isogenic Δbps mutant were extremely susceptible to killing by polymyxin B. Addition of
purified Bps to biofilms formed by the Δbps strain in trans significantly increased
survival to polymyxin B treatment. Our findings indicate that Bps mediates resistance to
polymyxin B by binding and sequestering the molecule in the biofilm matrix.
We hypothesized that Bps may be required for long term survival of B.
bronchiseptica in the mouse respiratory tract. At 38 days post-inoculation, the wild-type
strain was recovered in higher numbers than the Δbps strain, suggesting that Bps
contributes to persistence at a late stage of Bordetella infection. Based on these results,
x
we hypothesized that the bps locus would contribute to the establishment of Bordetella
within the host. We observed that the wild-type strain colonized the murine respiratory
tract more efficiently than the Δbps strain at early stages of Bordetella infection.
Histopathological analysis of mouse lungs led us to investigate the role of neutrophils in
the enhanced clearance of the ∆bps strain. We observed that, in neutropenic mice, the
∆bps mutant strain colonization was restored to similar CFUs as the wild-type strain at 3
days post-inoculation. Bronchoalveolar lavage fluid of mice infected with the wild-type
or the ∆bps strain contained similar absolute numbers of neutrophils. In vitro neutrophil
uptake assays revealed that the ∆bps strain was not more susceptible to phagocytosis.
Thus we conclude that neutrophils play a role in the enhanced clearance of the ∆bps
strain at 3 days post-inoculation by a mechanism independent of rapid influx or
phagocytosis. We have demonstrated that Bps facilitates B. bronchiseptica colonization
in the mouse respiratory tract by successful evasion of host defenses. The Bordetella
polysaccharide is produced by the three classical Bordetella species, B. pertussis, B.
bronchiseptica and B. parapertussis. Our studies highlight the need for therapeutics that
can eliminate bacterial polysaccharide production in vivo to promote an effective host
response and subsequent clearance of the pathogen.
xi
Chapter One: Introduction
The Bordetella genus
Bordetellae are small, aerobic, Gram-negative bacteria that preferentially bind to
the ciliated epithelium of the respiratory tract of mammals. The Bordetella genus
contains nine species: B. pertussis, B bronchiseptica, B. parapertussishuman(hu), B.
parapertussisovine(ov), B. holmesii, B. hinzii, B. trematum, B. petrii and B. avium (125).
The three classical Bordetella species, B. pertussis, B. bronchiseptica and B.
parapertussis, remain thoroughly studied in the field for their role in pathogenesis.
Bordetella pertussis is a strict human pathogen and the etiological agent of whooping
cough or pertussis (24, 125). Although whooping cough is not life-threatening to
adolescents and adults, it has been proposed that these age groups serve as reservoirs of
transmission to infants and young children in whom the disease is quite serious and often
lethal (37). B. bronchiseptica infects a broad range of animals including cats, dogs and
laboratory animals such as mice and rats. Infection by B. bronchiseptica has been shown
to cause bronchopneumonia in laboratory animals and asymptomatic infection in most
animals (62). Although not often attributed with causing disease in humans, B.
bronchiseptica has been shown to infect immunocompromised individuals (47). B.
parapertussis exists as two genetically distinct strains. B. parapertussisov infection of
sheep leads to a chronic disease in the respiratory tract. B. parapertussishu is a human
adapted strain that causes a milder form of pertussis (9, 15, 47).
The three classical species of Bordetella have been classified as a subspecies
based on their significant DNA sequence similarity. B. bronchiseptica is proposed to be
the evolutionary progenitor of the human adapted strains B. pertussis and B.
1
parapertussishu (159). Interestingly, the genomes of B. pertussis and B. parapertussishu
contain approximately one megabase less DNA than B. bronchiseptica. In addition to
gene deletion, the human adapted strains contain several pseudogenes that have been
inactivated by insertion of insertion sequence (IS) elements, and frameshift mutations
(39). Though B. pertussis and B. parapertussishu have evolved from B. bronchiseptica
through genomic decay, they do not lack any genes that have been associated with
pathogenesis.
Bordetella pertussis course of illness
Classic illness caused by infection with B. pertussis lasts up to 12 weeks or longer
and consists of the following stages: catarrhal, paroxysmal and convalescent (Fig 1).
After an incubation period of approximately 7 to 10 days, the infected individual enters
the catarrhal stage with symptoms such as a mild cough and sometimes a slightly
elevated fever. Unfortunately, the symptoms associated with the catarrhal stage resemble
a common cold, making diagnosis of pertussis difficult. Bacterial numbers reach the peak
during the first stage of pertussis illness. The paroxysmal stage begins around the second
week of the illness and lasts up to 8 weeks. A hallmark of the paroxysmal stage is
repeated coughing fits with up to 10 forceful coughs during a single expiration followed
by an inhalation with the characteristic “whoop” sound. Vomiting and weight loss are
common symptoms in infected individuals. Unlike in the catarrhal stage, bacterial
numbers are relatively low during this second stage of pertussis illness. The coughing fits
2
Fig. 1: Bacterial load of B. pertussis during classical pertussis illness. Following an
incubation period of 7 to 10 days, pertussis illness begins with the catarrhal phase. During
this stage, the infected individual has cold-like symptoms and has a high chance of
transmitting B. pertussis to other individuals due to a large bacterial burden. At the
second week of illness, the paroxysmal stage is characterized by the presence of the
“whooping cough” and a decreased bacterial load. This phase lasts from 2 to 8 weeks. In
the last stage of pertussis illness, the convalescent stage, the infected individual has less
frequent and severe coughing fits. This stage lasts 1 to 2 weeks or sometimes longer.
3
Stage of pertussis illness
Catarrhal Paroxysmal Convalescent
Time
1 to 2 weeks 2 to 8 weeks 1 to 2 weeks
Bac
teria
l num
bers
Stage of pertussis illness
Catarrhal Paroxysmal Convalescent
Time
1 to 2 weeks 2 to 8 weeks 1 to 2 weeks
Bac
teria
l num
bers
4
decrease in frequency and severity in the convalescent stage which usually lasts about 1
to 2 weeks but can be longer (125).
It is estimated that there are approximately 48.5 million pertussis cases a year
worldwide with as many as 295,000 deaths (38). The spread of pertussis is limited in
areas of the world with extensive vaccine coverage. The first whole-cell pertussis vaccine
became available in the United States in the 1940s. Due to numerous reports of children
experiencing neurological distress after receiving the whole-cell vaccine, new acellular
vaccines were distributed in the 1990s that consisted of two or more B. pertussis
components (5, 23). The current acellular pertussis vaccine available in the U.S. is
combined with the diphtheria and tetanus toxoids and known as diphtheria-tetanus-
acellular pertussis vaccine (DTaP). B. pertussis virulence factors included in DTaP are
filamentous hemagglutinin (FHA), pertussis toxin (PT), pertactin (PRN) and two distinct
forms of fimbraie (Fim), Fim2 and Fim3. The recommended regimen of DTaP is four
doses at 2, 4, 6 and 15-18 months of age and a booster before the child enters school (20,
21).
As a result of vaccination, incidence of reported pertussis cases shifted from
unvaccinated children between 1 and 9 to vaccinated adolescents and adults (7, 60).
Some reasons for this shift in carriage of B. pertussis include incomplete immunity in
children who have not received all doses of the vaccine as well as the loss of protection in
vaccinated individuals over time due to waning immunity (194). Adolescents and adults
act as carriers of pertussis and serve as reservoirs, transmitting B. pertussis to
unvaccinated infants or children who have not received all doses of the vaccine (8, 41). A
5
booster vaccine is now available for adults to help prevent spread of pertussis to
susceptible infants and children.
The BvgAS signal transduction system.
Bacteria often use two-component regulatory systems to sense their environment
and subsequently change gene expression. A majority of Bordetella virulence factors
have been shown to be regulated by the BvgAS two-component system. BvgAS is unique
due its His-Asp-His-Asp phosphorelay system (Fig. 2) (183-185). BvgAS consists of the
sensor kinase BvgS and the response regulator BvgA. BvgA contains a receiver domain
and helix-turn-helix domains. BvgS includes a periplasmic domain connected by a linker
region to transmitter, receiver and histidine phosphotransfer domains (HPD). The
environmental stimuli of BvgAS remain unknown although nicotinic acid, sulfate anion
and temperature conditions are used to control activation of BvgAS in laboratory settings
(91, 136)
The periplasmic domain of BvgS recognizes an environmental signal which leads
to autophosphorylation of a histidine on the transmitter domain. This histidine transfers
the phosphoryl group to an aspartate on the BvgS receiver domain. To promote BvgAS-
regulated gene expression, the aspartate then donates its phosphate group to a histidine
located on the histidine phosphotransfer domain which, in turn, phosphorylates an
aspartic acid residue on the receiver domain of BvgA. Once phosphorylated, BvgA
activates transcription by binding to promoter regions of Bordetella genes.
Phosphorylated BvgA also activates the repressor protein BvgR, which represses
transcription of certain Bordetella genes (133-135).
6
Fig. 2: The BvgAS signal transduction system of Bordetella species. Expression of the
majority of Bordetella virulence factors is regulated by the BvgAS two component
system. BvgS is the transmembrane sensor kinase that exists as dimers of periplasmic
domains, transmitters, receivers and histidine phosphotransferase domains. Detection of
an environmental signal leads to phosphorylation of the transmitter domain. Several
phosphotransfers occur, leading ultimately to the phosphorylation of BvgA, the response
regulator. Phosphorylated BvgA can activate or repress Bordetella genes. When the
BvgAS system is active, Bordetella is in the Bvg+ phase in which several well
characterized virulence factors are expressed such as FHA, pertactin, adenylate cyclase or
pertussis toxin. When the BvgAS system is inactive, Bvg- phase factors such as flagella
and urease are expressed. A third phase, Bvgi, exists in which Bordetella expresses many
unknown factors not observed in the Bvg+ or the Bvg- phases. One factor expressed by
Bordetella during the Bvgi phase is Bordetella intermediate phase protein A (BipA).
MgSO4, nicotinic acid and varying temperatures are modulators used in the laboratory to
regulate the three Bordetella phases controlled by the BvgAS system.
7
Outer membrane
Inner membrane
BvgS
P P
P P
P PBvgA
Sulfate, Nicotinic acid, Temperature<30oC
Bvg+ Bvg-
Bvgi BipA
MotilityUrease
FHA
Pertactin
CyaA
Outer membrane
Inner membrane
BvgS
Sulfate, Nicotinic acid, Temperature<30oC
PP PP
PP PP
PP PPBvgA
Bvg+ Bvg-
Bvgi BipA
MotilityUrease
FHA
Pertactin
CyaA
8
BvgAS regulation of Bordetella phenotypes
The BvgAS system controls the three phenotypic stages of Bordetella species (31, 114).
BvgAS is active at 37oC in the absence of magnesium sulfate (MgSO4) or nicotinic acid.
During this phenotypic phase, known as the Bvg+ phase, BvgA activates transcription of
various Bordetella toxins and adhesins such as adenylate cyclase, pertussis toxin,
filamentous hemagglutinin and fimbriae. If the temperature is at or below 25oC or at 37oC
in the presence of millimolar concentrations of MgSO4 or nicotinic acid, BvgAS becomes
inactive and switches to the Bvg- phase in which transcription of Bordetella toxins and
adhesins is repressed. In the Bvg- phase, genes responsible for flagellin are transcribed
for B. bronchiseptica and BvgAS repressed genes of B. pertussis are activated (125).
The BvgAS system does not simply turn on or off in response to environmental
stimuli. A Bvg-intermediate (Bvgi) phase exists in which Bordetella are grown in
concentrations of MgSO4 or nicotinic acid that are less than needed to induce Bvg-
conditions (43, 175). In the Bvgi phase, several adhesins are produced including several
unknown, surface-associated factors. BipA, Bordetella intermediate phase protein A, was
identified as a protein that is maximally expressed during the Bvgi phase. The Bordetella
phenotypic phases are thought to exist for different conditions. The Bvg+ phase is
necessary and required for Bordetella survival during respiratory tract colonization. The
Bvg- phase may allow survival of Bordetella during nutrient starved conditions (32). The
role of the Bvgi phase is less known but it is thought that this phase may be involved in
transmission (58).
9
BvgAS regulation of Bordetella virulence factors
BvgAS controls the expression of the majority of the genes involved in Bordetella
virulence including adhesins that promote attachment to the host and toxins that modulate
host responses to allow efficient colonization.
Adhesins
Filamentous hemagglutinin (FHA): FHA is expressed in all three Bordetella species.
This highly immunogenic protein has been found to be surface associated as well as
secreted. FHA is considered a dominant Bordetella attachment factor in animal models
and has been shown to contain several binding domains (43, 162). The Arginine-Glycine-
Aspartate (RGD) domain of FHA allows adherence to monocytes and macrophages as
well as other leukocytes by the leukocyte response integrin/ integrin-associated protein
(LRI/ IAP) complex and complement receptor 3 (CR3) (161). The carbohydrate
recognition domain (CRD) mediates attachment of FHA to ciliated respiratory epithelial
cells and macrophages in vitro (158). FHA-mediated hemagglutination and adherence to
nonciliated epithelial cell lines is achieved by the lectin-like activity of FHA that binds to
heparin and other sulfated carbohydrates (132).
Studies suggest that FHA may act as an immunomodulatory molecule for
Bordetella. B. pertussis required FHA to inhibit T cell proliferation to exogenous antigen
(11). Anti-FHA antibodies inhibit phagocytosis of B. pertussis by neutrophils (138). In
addition to its role in immunomodulation, FHA has been found to be crucial for
Bordetella colonization in vivo. FHA of B. bronchiseptica is required for efficient
colonization of the trachea in mice (34). FHA is commonly included as a component in
10
acellular pertussis vaccines including the diphtheria-tetanus-acellular pertussis (DTaP)
vaccine (21, 23). Individuals that receive a vaccine containing FHA mount substantial
antibody responses to the protein (49). Acellular vaccines that contain FHA as well as
pertussis toxin (PT) are more effective than vaccines that contain PT alone (177). Thus,
FHA is an essential factor for B. bronchiseptica colonization and immunomodulation of
the host response.
Fimbriae (Fim): All three classical Bordetella species, B. pertussis, B. parapertussis and
B. bronchiseptica, express fimbriae- filamentous, polymeric, cell surface-associated
protein structures. Fimbriae have been shown to mediate Bordetella colonization in vivo.
B. pertussis fimbriae mutants were defective in their ability to multiply in the nose and
trachea of mice (59, 141). In B. bronchiseptica, fimbriae were shown to contribute to the
establishment of tracheal colonization in rats and mice (127). Fim acts as an
immunomodulatory molecule that serves as a T-independent antigen for early IgM
production and induction of a Th2 response in Bordetella (126). The acellular vaccine that
contains the two fimbriae serotypes, Fim2 and Fim3, in addition to pertussis toxin, FHA
and pertactin has higher efficacy than a vaccine that excludes fimbriae (71).
Pertactin (PRN): Pertactin, expressed by all three classical Bordetella species, is a
member of the autotransporter family. PRN is proposed to mediate attachment because it
contains both an RGD motif and proline-leucine motifs that are commonly involved in
promoting protein-protein interactions in eukaryotic cell binding (52). Purified PRN
allows attachment of chinese hamster ovary (CHO) cells to tissue culture wells.
11
Expression of PRN in E. coli and Salmonella increased adherence to various mammalian
cell lines (56). Vaccine trials have shown that pertactin antibodies are important for
protection from B. pertussis (42) Anti-pertactin antibodies are required for effective
phagocytosis of B. pertussis by host cells (83). Additionally, diphtheria toxin-acellular
pertussis (DTaP) vaccines that include pertactin have higher efficacy in prevention of B.
pertussis infection (72).
Toxins
Adenylate cyclase (CyaA): CyaA is maximally expressed in the Bvg+ phase. All
Bordetella species capable of infecting mammals secrete CyaA which functions as a
calmodulin-sensitive adenylate cyclase. CD11b serves as the host receptor for secreted
CyaA. When CyaA enters eukaryotic cells, it is activated by calmodulin and catalyzes the
production of supraphysiological amounts of cyclic AMP (cAMP) from ATP to
intoxicate the cell and prevent proper function (16, 25). Secretion of CyaA is required to
cause intoxication of eukaryotic cells (65) CyaA also acts as a hemolysin because of its
ability to lyse sheep red blood cells (85).
CyaA has been demonstrated to have several immunomodulatory functions such
as inhibition of chemotaxis and superoxide production by neutrophils in vitro (155). B.
pertussis CyaA inhibits phagocytosis of Bordetella by human neutrophils (190). CyaA is
responsible for inhibition of CD40 expression and IL-12 production by bone marrow
dendritic cells (BMDCs) of C57BL/6 mice that were infected by B. bronchiseptica (170,
171). In vivo, mutants that lack CyaA are defective in causing lethal infection in infant
mice or growth in the lungs of older mice (63, 67). Due to its ability to alter host
12
responses and promote bacterial colonization, CyaA is not present in current DTaP
vaccines.
Dermonecrotic Toxin (DNT): B. pertussis, B. parapertussis and B. bronchiseptica all
express a nearly identical DNT. DNT is a heat-labile toxin that, when injected
intradermally, induces localized necrotic lesions in mice. DNT has been found to be
lethal for mice at low doses intravenously (90, 154). DNT is an A-B toxin with an
unknown receptor although it has been demonstrated to be internalized by dynamin-
dependent endocytosis (123). In vitro, B. bronchiseptica DNT induces morphological
changes and stimulates DNA replication and impairs differentiation of MC 3T3
osteoblasts (87, 88). B. bronchiseptica strains with reduced DNT production cause
decreased turbinate atrophy in infected mice (163). B. pertussis transposon mutants that
do not produce DNT are less virulent in mice (191).
Tracheal cytotoxin (TCT): TCT is a disaccharide-tetrapeptide monomer of
peptidoglycan that Bordetella releases into the environment instead of incorporating into
the cell wall (153, 164). Since TCT is constantly expressed, it is a BvgAS independent
virulence factor. TCT has been shown to cause loss of ciliated cells, cell blebbing and
mitochondrial damage in biopsy samples of human epithelial cells (192). TCT alone can
reproduce the ciliated-cell cytopathogical effects in tracheal tissue explants specific to a
B. pertussis infection (61). Destruction of ciliated cells is proposed to be caused by TCT-
dependent increase in nitric oxide. TCT triggers the production of IL-1α which has been
13
demonstrated to increase nitric oxide production in hamster tracheal epithelial (HTE)
cells (81, 82).
Pertussis Toxin (PT): B. pertussis is the only member of the Bordetella species to
synthesize and secrete PT, an ADP-ribosylating toxin. Although B. parapertussis and B.
bronchiseptica contain the loci that codes for PT expression (ptx), the genes have
mutations in the promoter region, rendering the loci transcriptionally silent in these two
species (2, 66, 121). Pertussis toxin is an A-B toxin with six polypeptides named S1
through S5 with S1 acting as the A subunit and S2 through S5 acting as the pentameric B
subunit (116, 143). The A subunit sits on top of the ring-like B structure that binds
eukaryotic cell membranes to increase the efficiency with which S1 can enter the cell
(168, 181). When the B oligomer reaches the host cell cytosol, it intercalates in the
cytoplasmic membrane and binds ATP to release the enzymatic S1 subunit that becomes
active upon reduction of its disulfide bond (102). The S1 subunit transfers an ADP-ribose
from NAD to the alpha subunit of guanine nucleotide-binding proteins as a method of
inactivation of these proteins (10, 102). Observed effects of PT include inhibition of
chemotaxis, oxidative responses and lysosomal enzyme release in neutrophils and
macrophages (12, 14, 107, 130, 131). Although it has been shown to be involved in
immunosuppression, PT is attributed as being the major virulence factor of B. pertussis
and proposed as being responsible for symptoms observed in pertussis-infected children
such as leukocytosis and lymphocytosis (80, 193). PT is included in all available acellular
pertussis vaccines.
14
Type III secretion system (TTSS). Type III secretion systems are used by gram-
negative bacteria to inject effector proteins into the cytoplasm of eukaryotic cells (106).
These effector proteins often disturb signaling and other functions that may promote
bacterial clearance (109). The three members of the Bordetella subspecies contain the bsc
locus which contains genes required for the production of the TTSS. However, only B.
bronchiseptica and B. parapertussisov display phenotypes associated with TTSS in vitro
such as inhibition of translocation of NFκB to the nucleus and rapid cell death of
macrophages (195). In vivo, the TTSS of B. bronchiseptica promotes persistent tracheal
colonization in rats and mice (195, 196).
Biofilms
Traditionally, bacteria are grown and studied as planktonic cultures in laboratory
settings. However, it is becoming increasingly evident that bacteria exist predominately
in complex communities called biofilms instead of individual cells (29). Biofilms can be
defined as communities of bacteria attached to a surface and encased in a self-produced
extracellular polymeric matrix. The biofilm matrix can be composed of various materials
including proteins, polysaccharides and nucleic acids (13).
The use of confocal scanning laser microscopy (CSLM) has shown that biofilm
formation is a multi-step process that is illustrated in Fig. 3 (86, 151). Biofilm formation
begins when individual bacteria reversibly attach to a surface. In this first stage of biofilm
formation, the bacteria contain only minute amounts of exopolysaccharide on their
surface and can detach to move to another location (147). The production of extracellular
polymers, such as polysaccharides, promotes the transition of the bacterium from a
15
reversible, transient attachment to a permanent, irreversible association. Alginate, an
exopolysaccharide produced by Pseudomonas aeruginosa, is synthesized within fifteen
minutes of attachment. Production of alginate leads to early biofilm formation of P.
aeruginosa in which monolayers of bacteria stack vertically to form microcolonies (40).
An important characteristic of a mature biofilm is the formation of water channels that
allow entry of nutrients and oxygen and diffusion of waste products. Another hallmark of
a mature biofilm is the production of a matrix, composed of various polymers such as
nucleic acids, polysaccharides and proteins, that encases the bacterial cells (26, 176). The
last stage of biofilm formation is detachment in which individual cells or large clumps of
cells are released from the biofilm. The exact mechanism of detachment is not completely
understood. However, some studies reveal that dispersed bacteria are phenotypically
similar to planktonic cells. These detached cells may be released due to physical
deterioration of the biofilm, to find new nutrients or to populate another area to promote
nascent biofilm formation (110, 146, 146, 166).
Bordetella biofilms
We and others have demonstrated that the BvgAS system controls biofilm
formation in Bordetella (91, 136). Wild-type B. bronchiseptica as well as phase-locked
Bvg+ and Bvgi strains were capable of forming biofilms in microtiter wells while the Bvg-
strain was defective in biofilm formation. Chemical modulators that inhibit BvgAS
activity, such as nicotinic acid and sulfate anion, have also been shown to drastically
reduce Bordetella biofilm formation. Scanning electron microscopy (SEM) revealed that
both the wild-type B. bronchiseptica and Bvg- strains could adhere to glass coverslips but
16
Fig 3: Biofilm formation is a multi-step process. Biofilm formation includes five
distinct stages: 1) Reversible attachment of bacteria to a surface; 2) Increased production
of extracellular polymers, such as polysaccharides, leading to irreversible attachment;
3) Early biofilm development that contains microcolonies and water channels; 4) Mature
biofilm development in which the biofilm is encased in a matrix composed of various
molecules such as proteins, nucleic acids or polysaccharides; 5) Dispersal of the bacteria
within the biofilm.
17
18
1 2 3 4 5
only the wild-type strain developed into a biofilm, indicating that the BvgAS system
regulates biofilm formation after initial attachment. We have observed that B.
parapertussis and B. pertussis are also capable of forming biofilms in vitro and the
BvgAS system regulates biofilms in these Bordetella species (136).
Bordetellae are capable of forming biofilms on biotic surfaces as well. B.
bronchiseptica forms robust biofilms on the nasal septa of C57BL/6 mice at 15 and 38
days post-infection (172). We believe that Bordetella biofilm formation in the nose
allows efficient immune evasion and long-term colonization and promotes transmission
to other individuals.
Bacterial Exopolysaccharides (EPS)
One defining characteristic of mature biofilms is the production of an
extracellular polymeric matrix (13, 136). Pseudomonas aeruginosa has been studied
extensively as a model of biofilm formation due to its frequent colonization of
individuals with cystic fibrosis (13, 64, 142, 160). P. aeruginosa biofilms consist of
numerous polysaccharides including alginate, Pel and Psl (95, 148, 165). Alginate has
been found to contribute to resistance of P. aeruginosa to many host defenses such as
phagocytosis by macrophages and production of reactive oxygen species by phagocytes
in vitro (118, 157).
Two Staphylococcal species, S. epidermidis and S. aureus, produce a linear
polymer of β-1,6- linked N-acetylglucosamine known as polysaccharide intercellular
adhesion (PIA) or poly N-acetylglucosamine (PNAG), respectively. Although PIA/
PNAG were discovered at different times, it was later elucidated that these two
19
exopolysaccharides are identical structurally and are both encoded for by the icaADBC
locus. PIA/ PNAG are required for Staphylococcal biofilm formation and structural
integrity (36, 117, 118, 129, 157, 187). PIA/ PNAG have also been demonstrated to play
a role in resistance of Staphylococcus to innate responses. PIA of S. epidermidis is
required for resistance to the antimicrobial peptides LL-37 and HBD-3 and phagocytosis
by neutrophils (187). S. epidermidis biofilms that contained PNAG were shown to be
more resistant to opsonophagocytosis than planktonic cells (22, 165).
The PGA polysaccharide of E. coli, encoded by the pgaABCD locus, is
structurally similar to PIA/ PNAG and cross-reacts with anti-PIA/ PNAG antibodies. The
pgaABCD locus is required for optimal biofilm formation in E. coli (188). Homologues
of the icaADBC and pgaABCD loci have been identified in several other bacterial
genuses such as Actinobacillus actinomycetemcomitans, Yersinia pestis and Bordetella
species (108, 165). The three classical species of Bordetella all produce a β-1,6- linked
N-acetylglucosamine polysaccharide known as Bordetella polysaccharide (Bps) that is
cross-reactive to PNAG antiserum (108, 152). Bps is produced by the proteins encoded
for by the bpsABCD locus. Our laboratory has shown that Bps is required for in vitro
biofilm development following the initial attachment of Bordetella cells to a surface (38).
Additionally, we have demonstrated that Bps is a crucial component of Bordetella
biofilms in vivo. B. bronchiseptica was found to form extensive biofilms in the mouse
nose while the Δbps mutant strain existed as only minute clusters (108, 172) . Thus, Bps
plays a crucial role in Bordetella biofilm formation both in vitro and in vivo.
20
Biofilm relevance to human infection
Adherence to a surface is a defining characteristic of biofilms. The use of
microscopy has revealed biofilm formation on medical devices as well as tissues from the
site of chronic infections (27, 30). The formation of biofilms within the human body is a
cause of major concern. Biofilms have been shown to be extremely resistant to antibiotic
treatment compared to planktonic cells (29, 136, 174). One mechanism of resistance is
ineffective penetration of antimicrobials into biofilms. The biofilm matrix has been
shown to retard the diffusion of molecules such as antibiotics, antibodies and reactive
oxygen species (46, 89, 178). Surprisingly, in some instances, when antimicrobial factors
do gain entry into biofilms, they are rendered inactive and unable to kill. Jesiatis et al.
observed that, although neutrophils were able to penetrate a S. aureus biofilm, they were
ineffective at phagocytosis of the bacteria (97).
Bacteria in biofilms have been shown to exhibit a reduced metabolic activity. The
cells in the center of a biofilm may have limited nutrient availability, thus putting them in
a starved state. These bacterial cells that are not actively growing will have decreased
sensitivity to many antimicrobial agents (1, 54, 55). Biofilms formed in the human body
often lead to chronic infection due to the ineffective clearance by antimicrobials.
Antibiotic treatment often continues indefinitely until, in some cases, the biofilm is
surgically removed (27, 30).
21
Innate immune responses to B. bronchiseptica
Antimicrobial peptides (AMPs)
Antimicrobial peptides are a major component of the host innate response to
respiratory pathogens. AMPs can be divided into two families- defensins and
cathelicidins- based on structure. Defensins contain a β-sheet and three disulfide bridges.
These peptides can be further classified as α-defensins or β-defensins based on the
location of the disulfide bridges. Human neutrophil peptide-1 (HNP-1) is an α-defensin
produced in azurophilic granules of neutrophils. Human β-defensins (hBDs) are produced
by keratinocytes in the skin as well as by epithelial cells. Cathelicidins are synthesized as
precursor molecules and become active upon cleavage of the cathelin domain on the
amino terminus. Examples of cathelicidins are LL-37 in humans and cathelin-related
antimicrobial peptide (CRAMP) in mice. These peptides are produced by epithelial cells,
neutrophils and skin cells (150, 156).
Most AMPs are cationic in order to bind to the anionic bacterial surface.
Hydrophobic regions located on AMPs are also involved in the binding of these peptides
to bacterial cell wall components (3, 122). The exact mechanistic action of AMPs
remains unclear. AMPs are thought to form pores in the bacterial membrane that leads to
subsequent cell lysis. Three models of pore formation by AMPs are the barrel stave pore
model, the thoroidal model and the carpet model. According to the barrel stave model,
AMPs form multimers after binding the bacterial membrane. The AMP multimers cross
the cell membrane with hydrophobic portions facing the lipid bilayer and hydrophilic
portions facing the inside of the pore. These aggregated peptides form barrel-like
channels that appear similar to staves. The thoroidal model resembles the barrel-stave
22
model except that AMP multimers cause the lipid bilayer to form a monolayer in which
the outer and inner lipid bilayers of the bacterial membrane become connected. Lastly, in
the carpet model, AMPs completely cover the bacterial membrane like a carpet. The
result of bound AMPs is disruption of the charge of the membrane bilayer leading to pore
formation at the “carpet” site. Once AMPs are bound to the bacterial membrane, the
pores lead to leakage of the contents of the cell and death (75, 150). In addition to
directly killing bacteria, AMPs can serve as chemotactic factors to induce infiltration of
host cells such as neutrophils and macrophages to the site of infection (3).
Polymyxin B has been used extensively as a model of AMPs due to its
bactericidal activity and amphipathic structure consisting of hydrophobic fatty acids, a
tripeptide side chain and polycationic ring (3, 197). Studies using polymyxin B have
provided insight into the genes that are required by Salmonella enterica to allow
resistance to AMP killing (68, 69). Mathur et al.used polmyxin B as a model AMP to
demonstrate that Vibrio cholerae uses a porin to mediate resistance to AMP killing (122).
Interestingly, polymyxin B is produced by the bacterium Bacillus polymyxa which
emphasizes the important role of AMPs in defense in both eukaryotes as well as
prokaryotes (137).
B. bronchiseptica has evolved mechanisms to overcome the bactericidal
properties of AMPs (51). The type III system of B. bronchiseptica was shown to inhibit
NFκB activity and downregulate tracheal antimicrobial peptide (TAP) mRNA expression
in bovine tracheal epithelial cells (111). LPS protects B. bronchiseptica from killing by
protamine and the β-defensin HNP1. Studies indicate that B. bronchiseptica, which
23
naturally colonizes rabbits, is extremely resistant to AMPs produced in rabbit alveolar
macrophages and rabbit peritoneal granulocytes (112, 169). Thus, B. bronchiseptica
utilizes many virulence factors in order to resist killing mechanisms by antimicrobial
peptides.
Neutrophils and macrophages
Phagocytic cells are important in recognition and removal of pathogens from the host.
Inoculation of BALB/c mice with B. bronchiseptica results in the large infiltration of
neutrophils to the lungs. Neutrophils have been shown to be a crucial host component
involved in clearance of B. bronchiseptica. Neutropenic mice succumb to B.
bronchiseptica one to four days post-infection (76). Upon infection with B.
bronchiseptica, macrophages are also recruited to the lungs of mice in fewer numbers. In
vitro cytotoxicity assays revealed that B. bronchiseptica is extremely cytotoxic to J774
cells, a macrophage-like cell line (76).
Cytokines
Cytokines initiate host responses after invasion by pathogens. Microarray analysis
of bone marrow macrophages derived from C57BL/6 mice that were infected with B.
bronchiseptica revealed upregulation of TNFα mRNA. Further in vivo studies showed
that TNFα is required for survival of C57BL/6 mice infected with B. bronchiseptica.
TNFα-/- mice succumbed to a B. bronchiseptica infection as early as three days post-
infection. Neutralization of TNFα with an anti-TNFα antibody within two hours post-
infection resulted in mice developing lethal bordetellosis. These mice were sacrificed to
24
prevent suffering due to their severe reaction to B. bronchiseptica infection in the absence
of TNFα (120).
The type III secretion system of B. bronchiseptica causes decreased production of
IFNγ and increased production of IL-10. The use of knockout mice revealed that B.
bronchiseptica has reduced colonization in mice that do not produce IL-10, suggesting a
role for this cytokine in promoting optimal Bordetella persistence in vivo (170).
Animal models
Laboratory animals such as rabbits, rats and mice serve as excellent natural
models of infection for B. bronchiseptica (33, 35, 76, 78, 173, 179, 180). A B.
bronchiseptica strain, RB50, was isolated from the nasal cavity of a naturally infected
New Zealand white rabbit. RB50 has been used extensively in the laboratory to provide
insight into the pathogenesis of B. bronchiseptica in an animal host. The 50% infective
dose of intranasal inoculation of rabbits, rats and mice is less than 200, 25 and 5 colony-
forming units (CFU), respectively (124). The use of knockout mice has facilitated the
understanding of host factors that are crucial in clearance of B. bronchiseptica (76, 78,
103).
B. pertussis and B. parapertussishu have been studied with the use of mouse
models (18, 19, 186, 189). However, unlike B. bronchiseptica, these Bordetella species
are restricted to humans and mice may not serve as appropriate models of infection to
understand pathogenesis. Indeed, often large infectious doses of B. pertussis and B.
parapertussis are required to colonize mice, suggesting that the sensitivity of these
animals is not reflective of infection in humans (124). Nonetheless, intranasal inoculation
25
of mice with B. pertussis and B. parapertussis has provided copious amounts of
information concerning virulence factors required for colonization during host-pathogen
interactions. Overall, the use of mice has provided valuable insights into the colonization
of the respiratory tract by all three classical Bordetella species.
Limitations of studies reported in the literature. Although AMP killing has been used
extensively to study resistance by several bacterial species, few studies have been
performed using Bordetella. Elahi et al. demonstrated that B. bronchiseptica is resistant
to porcine and human beta-defensins while B. pertussis was significantly more
susceptible to killing by equivalent concentrations of these molecules. The authors
speculate that, as natural flora found in the respiratory tract of pigs, B. bronchiseptica
may have evolved to become resistant to killing by porcine AMPs. Additionally, the
authors predicted that B. pertussis was killed by AMPs due to direct bactericidal activity
as well as immunomodulatory effects such as recruitment of leukocytes (51). Banemann
et al. suggest that B. bronchiseptica may be more resistant to AMP killing due to its LPS
structure, specifically the presence of the O antigen acts as a protective shield to prevent
AMP killing (4). We hypothesized that Bps may also serve as a barrier to prevent AMP
killing of B. bronchiseptica. Since there were no studies reported that examined the role
of Bps, or any other Bordetella polysaccharide, in Bordetella resistance to AMPs, we
chose to perform AMP killing assays with the wild-type B. bronchiseptica strain as well
as the Δbps isogenic mutant.
We and others have demonstrated that Bps is required for Bordetella biofilm
formation in vitro and in vivo (92, 92, 94, 152, 172, 172). Biofilms are often thought to
26
promote bacterial persistence in vivo. However, we predicted that Bps may contribute to
the establishment of B. bronchiseptica in the murine respiratory tract as well. Therefore,
we performed in vivo studies using the wild-type B. bronchiseptica and the Δbps strains
to understand the contribution of Bps to Bordetella colonization during early time points
after infection. We also used these strains at early times post infection to determine the
host components that are required to clear B. bronchiseptica.
27
Chapter Two: Materials and Methods
Bacterial strains, media and growth conditions. The wild-type B. bronchiseptica
strain, Δbps isogenic mutant, Bps hyper-expressing (Bpshyp) and Δbps vector (Δbpsvec)
were maintained on Bordet-Gengou (BG) agar (Difco) supplemented with 7.5%
defibrinated sheep’s blood and 50 µg/mL of streptomycin (SM). For animal inoculations
and in vitro experiments, the B. bronchiseptica strains were grown in Stainer-Scholte
broth overnight at 37oC followed by subculture at OD600. The Bpshyp and Δbps vec strains
were grown in media containing chloramphenicol to maintain plasmids (151).
Polymyxin B killing assay. Overnight cultures were subcultured to an OD600 of 0.05 in
fresh medium and 1.5 mL was aliquoted in 12 well plates (Corning). The plates were
incubated at 37o C to allow biofilm formation. After 24 hours, the wells were washed
with water. Fresh media containing increasing concentrations of polymyxin B was added
and the plates were incubated at 37o C for 2 hours. The wells were washed with water
followed by addition of 1.5 ml of sterile PBS. Biofilms were harvested by vigorous
pippeting and serial dilutions were made to enumerate colony forming units (CFUs).
Percent survival was determined by dividing the number of CFUs recovered after
polymyxin B treatment by the number of CFUs recovered from the non-treated controls.
Exogenous addition of the Bps polysaccharide to Δbps cultures and biofilms. Log-
phase Δbps planktonic cells or 24 hour pre-formed biofilms were incubated with the 50
µg/ mL of the Bps or mock preparations for one hour. The bacterial cells were washed
28
gently with PBS and incubated with 1% formalin (EMD) for fifteen minutes. After
incubation with 1% formalin, the cells were washed with PBS and resuspended in
Stainer-Scholte media containing varying concentrations of polymyxin B. Percent
survival was determined by dividing the number of CFUs recovered after polymyxin B
treatment by the number of CFUs recovered from the non-treated controls.
Purification of the Bps polysaccharide. The Bpshyp and the Δbps vec strains were spun
down and pellets were resuspended in 0.5M EDTA (Fisher) and boiled for five minutes.
The cells were then treated with 1 mg/mL Pronase at 60oC overnight. The samples were
treated with phenol-chloroform followed by ethanol precipitation. After the pellets were
dried, they were stored in sterile water and treated with .1 mg/mL DNase and RNase
overnight, followed by another phenol-chloroform extraction and ethanol precipitation.
The samples were stored at -20oC or 4oC (151).
Polymyxin B binding assay. 1:2, 1:5 and 1:10 dilutions of 10 mg/ mL of the Bps or the
mock preparations were aliquoted in 10 µL and spotted on a nitrocellulose membrane and
allowed to dry overnight. The blot was blocked in 5% milk for thirty minutes then probed
with 25 µg/mL of polymyxin B (Sigma) for one hour. Anti-polymyxin B IgM primary
antibody (Abcam) was added at a dilution of 1:1000 for one hour followed by three
washes with TBST. The polyclonal IgG+IgM horseradish peroxidase-conjugated
secondary antibody (Abcam) was added at a dilution of 1:5000 for one hour and the blot
was washed three times with TBST and detected by the Amersham Enhanced
Chemiluminescence Western blot system. To detect other Bordetella polysaccharides
29
present in the preparations, the Bps or mock preparations were run on a 12% SDS-PAGE
gel followed by transfer to a nitrocellulose membrane. The membrane was treated using
the protocol described above and detected with the Amersham ECL Western blot system.
To perform a competition Western blot, the anti-polymyxin B primary antibody was
incubated with 25 µg/mL of polymyxin B for one hour prior to starting the blot. As a
negative control, the Western blot was performed as above without the addition of
polymyxin B.
Detection of poly-β-1,6-N-acetyl glucosamine. 1:2, 1:5 and 1:10 dilutions of 10 mg/ mL
of the Bps or the mock preparations were aliquoted in 10 µL and spotted on a
nitrocellulose membrane and allowed to dry overnight. The blot was blocked with 5%
milk for thirty minutes and incubated with an antibody raised against S. aureus dPNAG
at a dilution of 1:5000 in 5% milk. After washing three times with TBST, the secondary
horseradish peroxidase-conjugated IgG antibody was used at a dilution of 1:20,000. The
blot was washed three times with TBST and detected with the Amersham Enhanced
Chemiluminescence Western blot system (151).
Titration of polymyxin B in Bordetella biofilms. 24 hour biofilms formed by the wild-
type and Bpshyp strains were treated with 50 µg/ mL of polymyxin B for 2 hours. The
supernatant from these biofilms was transferred to 24 hour pre-formed biofilms formed
by the Δbps and Δbpsvec strains (respectively) for 2 hours. The biofilms were washed
with water and harvested to determine percent survival of the Δbps and Δbpsvec biofilms
30
determined by dividing the number of CFUs recovered after supernatant treatment by the
number of CFUs recovered from the non-treated controls.
Animal experiments. Six to 8-week old female C57/BL6 mice (Jackson Laboratories)
were lightly sedated with isoflurane and intranasally inoculated with 50 µL of 5 X 105
CFU of the wild-type B. bronchiseptica or the Δbps mutant strain. At designated times
post-inoculation, mice were euthanized and the nasal septum, trachea and lungs were
excised, homogenized in PBS and plated on BG-blood agar plates containing
streptomycin (50 µg/mL) (172). Colonies were enumerated after 2 days of growth at
37oC. All animal experiments were carried out in accordance with institutional guidelines
and approved by the animal care and use committee of WFUHS.
Histopathological examination of the mouse lung. Three of five lobes of the mouse
lung were immediately fixed in 10% formalin following sacrifice for 24 hours. The lungs
were then trimmed, embedded in paraffin, cut at 4 to 6 µm and stained with hematoxylin
and eosin (H & E). Examination of pathology of the lungs was examined using a light
microscope. The lung sections were qualitatively assessed blindly by a board certified
veterinary pathologist, Dr. Nancy Kock, for overall cellularity, vascular changes, edema,
hemorrhage, presence of alveolar and interstitial neutrophils, intrabronchial neutrophils,
perivascular and peribronchiolar lymphocytes, alveolar macrophages and condition of
alveolar walls.
31
Depletion of neutrophils. Neutrophils were depleted by intraperitoneal injection of 1
mg/mL of the anti-Gr1 antibody 24 hours before bacterial inoculation. Mice were
inoculated with 5 X 105 CFUs of the wild-type or the Δbps strains and sacrificed at 2 or 3
days p.i. to homogenize the nasal septum, trachea and lungs for enumeration of CFUs
(179). Confirmation of neutropenia was determined by IDEXX laboratories by
differential cell counts of blood samples retrieved from cardiac puncture.
Quantification of mouse lung leukocyte infiltration. Following B. bronchiseptica
inoculation and sacrifice (as described above), mice were dissected to expose the trachea
and chest cavity. Tubing was placed in a small incision in the trachea and held with
surgical sutures. The lungs were perfused with 1 mL of sterile PBS two times. The total
number of cells present in the bronchoalveolar lavage (BAL) was quantified by trypan
blue exclusion using a hemacytometer. Cell suspensions were pelleted using Cytospin
centrifugation and stained with Hema 3 Stain. The leukocyte population was determined
by counting four fields of 100 cells with a light microscope (77) .
Neutrophil phagocytosis assay. The wild-type and Δbps mutant strain were grown to
logarithmic phase and opsonized with heat-inactivated mouse serum for 30 minutes at
37oC prior to incubation with human PMNs. The neutrophils and bacteria were incubated
at an MOI of 1 for twenty minutes or one hour in the wells of a 24 well plate (Corning).
After incubation, the supernatant was collected and the adherent neutrophils were washed
two times with sterile PBS. The neutrophils were lysed by vigourous pipetting in water to
harvest the bacterial cells (140). Percent uptake was determined by dividing the number
32
of bacteria recovered after incubation with neutrophils over the non-treated bacteria that
were recovered at the same time point.
Isolation of human neutrophils. Draw 30 mL of human blood into a heparinized syringe
and overlay over 6mL of Isolymph. Mix blood and Isolymph gently and let settle for
approximately 30 minutes. Transfer the white cell enriched supernatant to a clean 50 mL
tube and centrifuge at 1000 rpm for ten minutes at room temperature. Discard supernatant
and resuspend cell pellet in 1X PBS that is 1/10 the volume of blood drawn. Overlay
resuspended pellet on 3 mL Isolymph and centrifuge at 1350 rpm for 40 minutes at room
temperature. Aspirate the supernatant down to the cell pellet. Perform hypotonic lysis of
cell pellet by adding 5 mL of 0.2% NaCl for 25 seconds on ice then add 5 mL of 1.6%
NaCl and mix. Centrifuge at 1000 rpm for 10 minutes at 4oC. Repeat hypotonic lysis
until all residual red blood cells are removed. Resuspend cells in 3 mL of cold 1X HBSS
and count using hemocytometer. Adjust cell concentration according to experiment.
(Obtained neutrophil isolation assay from McPhail laboratory).
Detection of cytokines in mouse lung homogenates. Lung homogenates of mice
infected with the wild-type and the Δbps mutant strains were stored in -80oC until use for
cytokine assays. To detect cytokines, lung homogenates were thawed and used in ELISA
assays to detect TNFα and IL-10 (BD Biosciences). 96 well plates were coated with anti-
TNFα or anti-IL-10 antibodies at 4oC overnight. The wells were washed three times with
phosphate buffered saline containing 0.05% Tween 20 (PBST) and blocked with 200 µL
of 5% milk for one hour at 37 oC. 100 µL of 1:10 dilution of lung homogenates was
33
added to the wells and incubated at 37oC for two hours. Wells were washed three times
with PBST and incubated with anti-mouse antibodies conjugated to horseradish
peroxidase. Plates were washed five times with PBST and 3,3’, 5,5’-Tetramethyl
benzidine was added. Absorbance at OD450 was determined using a plate reader.
34
Chapter Three: The Role of Bps in Resistance to Host Innate Defenses
The Bps polysaccharide protects B. bronchiseptica from killing by polymyxin B.
Exopolysaccharides have been shown to provide resistance to killing by multiple
components of the innate response including antimicrobial peptides (AMPs). PIA
protects S. epidermidis from killing by the antimicrobial peptides human-β-defensin-3
(HBD-3), LL-37 and dermcidin (187). Alginate of P. aeruginosa binds and inhibits
bactericidal activity of the antimicrobial peptides LL-37 and SMAP-29 (84). We sought
to determine if Bps can protect Bordetella bronchiseptica from killing by polymyxin B,
an amphipathic peptide antibiotic that is often used as a model compound representative
of the bactericidal actions of AMPs (17, 182). We first tested the susceptibility of
planktonic cultures of the wild-type and the Δbps strains to polymyxin B. At 25 µg/ mL
of polymyxin B, there was no statistical difference observed in the survival of the wild-
type and the Δbps strain. However, the Δbps strain was recovered in extremely lower
numbers following incubation with 50 and 75 µg/mL of polymyxin B. In contrast, the
wild-type strain had approximately 30% and 10% survival at 50 and 75 µg/mL
polymyxin B, respectively (Fig. 1).
It is becoming increasingly evident that bacteria do not exist as individual, free-
swimming cells that are often studied in the laboratory but rather as biofilms (28, 29, 44).
Biofilms can be defined as a complex community of cells that are attached to a surface
and encased in a matrix that is composed of self-produced polymeric substances (13,
145). We and others have reported the prevalence of B. bronchiseptica in the biofilm
form in which the Bps polysaccharide is a major constituent of the Bordetella biofilm
35
Fig. 1: The Bps polysaccharide protects planktonic B. bronchiseptica cells from
killing by polymyxin B. Planktonic cells of the wild-type and the Δbps strains were
grown to mid-logarithmic phase and incubated with 25, 50 and 75 µg/ mL of polymyxin
B for 2 hours at 37oC. Results are expressed as percent survival as determined by
dividing the number of CFUs recovered after polymyxin B treatment by the number of
CFUs observed in the mock-treated controls. Bars represent the average of three
independent experiments performed in triplicate with error bars representative of ±
standard deviation.
36
0
10
20
30
40
50
60
70
25 50 75
Polymyxin B concentration (µg/mL)
% s
urvi
val
WTΔbps
0
10
20
30
40
50
60
70
25 50 75
Polymyxin B concentration (µg/mL)
% s
urvi
val
WTΔbps
37
matrix (13, 93, 151). Our preliminary results suggest that Bps is expressed at higher
levels in the biofilm state than during planktonic conditions (172). Therefore, we
hypothesized that biofilms formed by the wild-type strain would be resistant to killing by
polymyxin B due to higher levels of Bps. The wild-type B. bronchiseptica and the Δbps
strain were grown as biofilms for 24 hours. The pre-formed biofilms were then incubated
with various concentrations of polymyxin B for two hours at 37oC. As shown in Fig. 2A,
the biofilm formed by the wild-type strain had 100% survival when incubated with 25
µg/mL polymyxin B. At 50 and 75 µg/mL polymyxin B, survival of the biofilm formed
by the wild-type strain remained high averaging at approximately 80-90%. In contrast,
the Δbps mutant strain was susceptible to killing at 25 µg/mL polymyxin B with a
survival of approximately 60%. At 50 and 75 µg/mL polymyxin B, biofilms formed by
the Δbps had approximately 20% survival.
These results suggest that Bps protects B. bronchiseptica from killing by
polymyxin B. To confirm this, we performed polymyxin B killing assays using biofilms
formed by a Bps-hyperexpressing strain (Bpshyp) and a Δbps vector strain (Δbpsvec). The
Bps-hyperexpressing strain contains the entire bps locus complemented on a plasmid in
the Δbps background. The Δbps vector strain is a control that contains only the vector
plasmid in the Δbps strain. When incubated with 25, 50 and 75 µg/mL polymyxin B, the
biofilms formed by the Bpshyp strain had 95% survival (Fig. 2B). Biofilms formed by the
Δbpsvec strain were killed in a dose-dependent manner with 80%, 60% and 50% survival
at 25, 50 and 75 µg/mL polymyxin B, respectively. We concluded that the presence of
the Bps polysaccharide protects B. bronchiseptica biofilms from killing by polymyxin B.
38
Fig. 2: The Bps polysaccharide protects B. bronchiseptica biofilms from killing by
polymyxin B. The wild-type or the Δbps strains (A) or the Bpshyp or the Δbpsvec control
strains (B) were grown as biofilms for 24 hours in 12 well plates then incubated with 25,
50 and 75 µg/ mL polymyxin B for 2 hours at 37oC. Results are expressed as percent
survival as determined by dividing the number of CFUs recovered after polymyxin B
treatment by the number of CFUs observed in the mock-treated controls. Bars represent
the average of three independent experiments performed in triplicate with error bars
representative of ± standard deviation. Unpaired two-tailed Student t test was used to
determine statistical significance. Asterisk indicates the P value of <0.01.
39
020406080
100120
25 50 75
Polymyxin B concentration (µg/mL)
% s
urvi
val
WTΔbps
* *
0
20
40
60
80
100
120
25 50 75Polymyxin B concentration (µg/mL)
% s
urvi
val
Bpshyp
* *
A
B
Δbpsvec
020406080
100120
25 50 75
Polymyxin B concentration (µg/mL)
% s
urvi
val
WTΔbps
* *
0
20
40
60
80
100
120
25 50 75Polymyxin B concentration (µg/mL)
% s
urvi
val
Bpshyp
* *
A
B
Δbpsvec
40
The Bps polysaccharide protects biofilms formed by the Δbps strain from polymyxin
B killing in trans. We next wanted to determine if the addition of exogenous Bps could
increase resistance of the Δbps mutant strain to killing by polymyxin B. This will allow
us to test the hypothesis that purified Bps is able to rescue the Δbps strain from killing by
polymyxin B. To examine this, we purified Bps from the Bpshyp strain by EDTA
extraction to remove the polysaccharide from the bacterial surface followed by pronase,
DNase and RNase treatment to remove all molecules -proteins, DNA and RNA- from the
preparation except polysaccharide. We purified mock samples from the Δbpsvec strain
using the same protocol described above.
Polymyxin B has been shown to bind LPS. It is important to note that the Bps and
mock preparations that were externally added to the Δbps strain contained equivalent
amounts of LPS. We used the mock preparation as a control in this experiment to rule out
any effects that LPS may have on our results. We incubated planktonic Δbps cells with
the Bps or mock preparation for one hour. After the hour incubation, the cells were gently
washed and fixed with 1% formalin to stabilize the interaction between the bacterial cells
and the externally added Bps or mock preparation. We did not observe any effects of 1%
formalin on viability of Δbps cells (data not shown). After Δbps cells were incubated
with 1% formalin, the Δbps cells were incubated with polymyxin B for two hours.
Addition of the Bps preparation to Δbps planktonic cells did not significantly increase
resistance to killing by polymyxin B compared to Δbps planktonic cells treated with the
mock preparation (Fig 3A).
Studies indicate that biofilms are more resistant to killing by antimicrobial agents
than planktonic cells (45, 136, 174). Biofilms have been shown to have different gene
41
Fig. 3: The Bps polysaccharide increases resistance of biofilms formed by the Δbps
mutant strain to polymyxin B killing in trans. The Bps or mock preparations were
incubated with Δbps planktonic cells (A) or the Δbps strain grown as a biofilm for 24
hours (B) for one hour. The Δbps cells were washed in PBS and incubated with 1%
formalin for 15 minutes followed by treatment with polymyxin B for two hours. Results
are expressed as fold survival (A) or percent survival (B) as determined by dividing the
number of CFUs recovered after polymyxin B treatment by the number of CFUs
observed in the mock-treated controls. Bars represent the average of three independent
experiments performed in triplicate with error bars representative of ± standard deviation.
Unpaired two-tailed Student t test was used to determine statistical significance. Asterisk
indicates the P value of <0.01.
42
0
10
20
30
40
50
60
Mock Bps
% s
urvi
val
A
B
0
0.5
1
1.5
2
2.5
Mock BpsSurv
ival
(fold
ove
r vec
tor)
**
75 µg/mL Pol B
Pol B0
10
20
30
40
50
60
Mock Bps
% s
urvi
val
A
B
0
0.5
1
1.5
2
2.5
Mock BpsSurv
ival
(fold
ove
r vec
tor)
**
75 µg/mL Pol B
Pol B
43
expression profiles compared to their planktonic counterparts (176). It is possible that this
distinct gene expression allows better attachment of the purified Bps to the Δbps strain
grown as a biofilm. We sought to determine if Bps could protect 24 hour pre-formed
biofilms formed by the Δbps strain from the bactericidal effects of polymyxin B. The
biofilms formed by the Δbps strain incubated with polymyxin B alone or polymyxin B
and the mock preparation had approximately 10% survival. In contrast, biofilms formed
by the Δbps strain that were incubated with polymyxin B and the Bps preparation had a
significantly increased survival of approximately 50% (Fig. 3B). These results indicate
that the addition of exogenous Bps can protect the biofilms formed by the Δbps strain
from killing by polymyxin B.
Overproduction of Bps prevents binding of polymyxin B to B. bronchiseptica
biofilms. We wanted to determine the mechanism of Bps-mediated resistance of B.
bronchiseptica to polymyxin B killing. Bps may provide resistance by acting as a
physical barrier that shields Bordetella and prevents binding by polymyxin B. We
hypothesized that Bps provides resistance to killing by polymyxin B by sequestering it in
the biofilm matrix. To test this, we incubated polymyxin B with media alone or with a 24
hour biofilm formed by the wild-type strain for 2 hours at 37oC. We then transferred the
supernatant from the polymyxin B alone control or the biofilm formed by the wild-type
strain to a biofilm formed by the Δbps strain for an additional 2 hour incubation. We
predicted that if we first incubated polymyxin B with the biofilm formed by the wild-type
strain, the polymyxin B would be bound by the Bps, resulting in an increase in survival of
the biofilm formed by the Δbps strain. Surprisingly, there was no difference observed in
44
survival when the biofilm formed by the Δbps strain was treated with supernatant from
the polymyxin B alone control or the biofilm formed by the wild-type strain (Fig. 4A).
We hypothesized that the amount of Bps produced in the matrix of biofilms
formed by the wild-type strain was not sufficient to sequester polymyxin B. We predicted
that the Bpshyp strain that overproduces Bps would adequately sequester polymyxin B in
the matrix, thus leading to increased survival of the biofilm formed by the Δbpsvec strain.
As shown in Fig. 4B, when polymyxin B was incubated with the biofilm formed by the
Bpshyp strain, the biofilm formed by the Δbpsvec strain was protected from killing with
survival of approximately 100%. To ensure that Bps was responsible for the increased
survival of the Δbpsvec strain, we incubated polymyxin B with the biofilm formed by the
Δbpsvec strain for two hours followed by transferring the supernatant to another biofilm
formed by the Δbpsvec strain for an additional two hours. Percent survival did not increase
when polymyxin B was first incubated with a biofilm formed by the strain that does not
produce Bps. We conclude that Bps binds polymyxin B in the biofilm matrix to prevent it
from reaching the bacterial surface.
The Bps polysaccharide directly binds polymyxin B. We predicted that polymyxin B
would bind to purified Bps. Bps and PIA/PNAG of Staphylococcal species produce
antigenically similar polysaccharides consisting of poly-β-1,6-N-acetyl glucosamine. The
homology of Bps to PIA/PNAG has allowed us to use an anti-dPNAG antibody to detect
the Bps polysaccharide in Western blots. To ensure that our Bps preparation contains
poly-β-1,6-N-acetylglucosamine and that our mock preparation lacks this polysaccharide,
we probed the Bps and mock preparations with the anti-dPNAG antibody. Results of the
45
Fig. 4: Overproduction of Bps prevents binding of polymyxin B to B. bronchiseptica
biofilms. The wild-type or BPShyp strains were grown as biofilms in 12 well plates for 24
hours then incubated with 50 µg/ mL of polymyxin B for two hours at 37oC. The
supernatant from biofilms formed by the wild-type or BPShyp strains was transferred to
the 24 hour pre-formed biofilms formed by the Δbps mutant strain or Δbpsvec strain
(respectively) for two hours at 37oC. Results are expressed as percent survival of the
Δbps strain (A) or the Δbpsvec strain (B) as determined by the number of CFUs recovered
after supernatant treatment by the number of CFUs observed in the mock-treated
controls. Bars represent the average of three independent experiments performed in
triplicate with error bars representative of ± standard deviation. Unpaired two-tailed
Student t test was used to determine statistical significance. The asterisk indicates the P
value of <0.01
46
01020304050607080
% s
urvi
val o
f the
Δbp
sst
rain
Polymyxin B alone
0
20
40
60
80
100
120
140 *
Polymyxin B alone
Supernatant from Δbpsvec
Supernatant from Bpshyp
A
Supernatant fromwild-type
% s
urvi
val o
f the
Δbp
svec
stra
in
01020304050607080
% s
urvi
val o
f the
Δbp
sst
rain
Polymyxin B alone
0
20
40
60
80
100
120
140 *
Polymyxin B alone
Supernatant from Δbpsvec
Supernatant from Bpshyp
A
Supernatant fromwild-type
% s
urvi
val o
f the
Δbp
svec
stra
in
47
immunoblot assay revealed that the anti-dPNAG antibody recognizes the Bps preparation
but not the mock preparation (Fig. 5A).
To determine if polymyxin B binds purified Bps, we spotted the Bps and mock
preparations on nitrocellulose and incubated the blot with polymyxin B followed by a
mouse primary antibody raised against polymyxin B and a secondary antibody
conjugated to horseradish peroxidase. We observed that polymyxin B bound the Bps
preparation but not the mock, suggesting that Bps binds polymyxin B (Fig. 5B). A caveat
of these dot blots is that we cannot directly demonstrate that Bps is the only
polysaccharide that is binding to polymyxin B. Therefore, we separated the Bps and
mock preparations on an SDS-PAGE gel and probed with either the anti-dPNAG
antibody or polymyxin B. Our results indicated that polymyxin B recognized the same
band that was detected by the anti-dPNAG antibody, revealing its specificity for Bps and
not other Bordetella polysaccharides (Fig. 5C). Based on these findings, we concluded
that B. bronchiseptica Bps, but not other polysaccharides, binds polymyxin B.
To confirm the specificity of the anti-polymyxin B antibody, we performed a
competition immunoblot in which the antibody was incubated with polymyxin B prior to
its use as the primary antibody in the Western blot. No cross-reactivity was observed,
indicating that the anti-polymyxin B antibody is specific for polymyxin B. To ensure that
polymyxin B does bind the Bps preparation, we performed the Western blot without
addition of polymyxin B. We did not obtain any cross-reactivity when polymyxin B was
not added, indicating that the Bps preparation is only bound by polymyxin B (data not
shown).
48
Fig. 5: The Bps polysaccharide directly binds polymyxin B. 1:2, 1:5 and 1:10 dilutions
of 10 mg/ mL of the Bps or the mock preparations were aliquoted in 10 µL and spotted
on nitrocellulose and probed with a 1:5000 dilution of anti-dPNAG to detect the presence
of poly-β-1,6-N-acetylglucosamine (A). Equivalent dilutions of the Bps or mock
preparations were spotted on nitrocellulose and incubated with 25 µg/mL polymyxin B
followed by a dilution of 1:1000 of the primary anti-polymyxin B primary and 1:5000
dilution of the secondary antibody conjugated to horseradish peroxidase and detected
with enhanced chemiluminescence. (B). To detect if other Bordetella polysaccharides
present in the preparations are bound by polymyxin B, the Bps or mock preparations
were run on a 12% SDS-PAGE gel followed by transfer to a nitrocellulose membrane.
The blot was incubated with polymyxin B as described above (C). Blots shown are
representative images of at least three independent experiments.
49
B
Bps
Mock
Polymyxin B αPNAG
BpsMock Bps
Mock
A C
Bps
Mock
B
Bps
Mock
Polymyxin B αPNAG
BpsMock Bps
Mock
A C
Bps
Mock
50
The bps locus promotes colonization of B. bronchiseptica in the mouse respiratory
tract during the persistent stage of infection. Biofilms have been hypothesized to
promote bacterial persistence in vivo (29, 73, 115). Since Bps is necessary for Bordetella
biofilm formation, we predicted that it would be required for long-term survival of B.
bronchiseptica in the murine respiratory tract. To investigate this, we inoculated mice
intranasally with approximately 5 x 105 CFUs of either the wild-type or the Δbps mutant
strain in 50 µL to sufficiently seed the entire respiratory tract. We chose to sacrifice the
mice at 15 and 38 days post-inoculation as time points indicative of persistent
colonization. At 15 days post-inoculation, the wild-type and Δbps mutant strain colonized
the trachea, nasal septum and lungs of C57BL/6 mice similarly (Fig. 6). However, by 38
days post-inoculation, the wild-type strain was recovered in significantly higher numbers
from both the nasal septum and lungs of mice compared to the Δbps mutant strain. Both
the wild-type and Δbps strains were cleared from the trachea at this late time point. We
concluded that, at a late time in B. bronchiseptica infection, the presence of the Bps
polysaccharide promotes persistent colonization in the mouse respiratory tract.
The Bps polysaccharide enhances colonization of B. bronchiseptica in the mouse
respiratory tract at 3 days post-inoculation. We have shown that Bps promotes
persistent colonization in the murine respiratory tract at 38 days post-inoculation (Fig. 6).
Biofilm matrix polysaccharides have been shown to allow bacterial evasion of the host
immune response. Purified PNAG inhibits opsonization of S. aureus (22). Alginate
protects P. aeruginosa from phagocytosis and hydrogen peroxide production by rat
51
Fig. 6: The bps locus promotes persistent colonization of B. bronchiseptica in the
mouse respiratory tract. Groups of five C57BL/6 mice were inoculated with
approximately 5 x 105 CFUs of the wild-type or the Δbps mutant strain. At 15 and 38
days post-inoculation, the mice were sacrificed. The trachea, nasal septum and lungs
were removed and homogenized and the resident bacteria were plated to measure
colonization. Horizontal bars represent the numerical mean for each group. The dashed
line represents the lower limit of detection. Unpaired two-tailed Student t test was used to
determine statistical significance. One asterisk indicates the P value of <0.05. Two
asterisks indicate the P value of <0.005.
52
53
alveolar macrophages (105). We hypothesized that Bps may allow B. bronchiseptica to
subvert host defenses. We performed a time course to observe colonization of the wild-
type and Δbps strains from as early as three hours up to 14 days post-inoculation. Both
the wild-type and the Δbps mutant strain were recovered in similar numbers in all tissues
at 3, 6 and 24 hours post-inoculation, indicating that Bps is not required for initial
colonization (Fig. 7). The Δbps strain was recovered in lower numbers in the trachea,
nasal septum and lungs at day 3 post-inoculation compared to the wild-type strain.
Colonization of wild-type and the Δbps strains were similar at 9 and 14 days post-
inoculation. We concluded that Bps is not required for initial establishment of B.
bronchiseptica in the murine respiratory tract. However, our data suggest that Bps may
contribute to optimal colonization of B. bronchiseptica at three days post-inoculation.
Neutrophils are an important host factor in the clearance of the Δbps strain in vivo.
B. bronchiseptica infection of mice results in host inflammation and pathology (77). We
sought to determine host cells, such as macrophages and neutrophils, that may be
involved in the response to the wild-type and the Δbps strains. At 6 and 24 hours, 3 and
14 days post-inoculation, the lungs were excised, fixed and stained with hematoxylin and
eosin (H&E). The lungs received qualitative scores based on the pathology parameters
listed in Table 1. Mice infected with the Δbps strain received a higher pathology score at
6 hours although this score was not significantly higher than mouse lungs infected with
the wild-type strain. However, at this time point, mice infected with the Δbps strain were
reported as having exacerbated bronchopneumonia compared to the wild-type strain.
Mice infected with the wild-type and Δbps strains received similar pathology scores at
54
Fig. 7: Kinetics of colonization of the wild-type B. bronchiseptica and isogenic
mutant, Δbps, in murine respiratory tract. Groups of five C57BL/6 mice were
inoculated with approximately 5 x 105 CFUs of the wild-type strain (diamonds) or the
Δbps mutant strain (squares). At the indicated time point, the mice were sacrificed. The
trachea, nasal septum and lungs were removed and homogenized and resident bacteria
were plated to measure colonization. Each point represents the average of five mice per
group and error bars indicate ± standard deviation. The dashed line represents the lower
limit of detection. Unpaired two-tailed Student t test was used to determine statistical
significance. The asterisk indicates the P value of <0.05.
55
Trachea
0123456
WT
Δbps
Nasal Septum
0123456
Lungs
012345678
0.125 0.25 1 3 9 14
Time (days)
*
*
*
Log 1
0C
FULo
g 10
CFU
Log 1
0C
FU
Trachea
0123456
Trachea
0123456
WT
Δbps
Nasal Septum
0123456
Nasal Septum
0123456
Lungs
012345678
0.125 0.25 1 3 9 14
Time (days)
Lungs
012345678
0.125 0.25 1 3 9 14
Time (days)
*
*
*
Log 1
0C
FULo
g 10
CFU
Log 1
0C
FU
56
Table 1: Histopathology scores of the lungs of mice infected with the wild-type or
Δbps strain. Four mice were inoculated with 5 x 105 CFU of the wild-type or Δbps strain
and sacrificed at 6 and 24 hours and 14 days post-inoculation. The right lung was
harvested and processed for H & E staining. The sections were examined blindly by Dr.
Nancy Kock. Results expressed for each pathology parameter are an average score of
four mice with + standard deviations. The total average score for each group represents
the summation of all nine pathology parameters with + standard deviations. No statistical
significance was observed between the wild-type and the Δbps strain at any time point
following use of the unpaired two-tailed student t test.
57
Pathology parameters WT, 6h Δbps, 6h WT, 24h Δbps, 24h WT, 14d Δbps, 14doverall cellularity 2 ± 0 2.5 ± 0.58 1.25 ± 0.5 2.5 ± 0.58 2.75 ± 0.5 2 ± 0.81 vascular changes(degeneration) 0 ± 0 0.75 ± 0.96 0 ± 0 0 ± 0 0 ± 0 0 ± 0 edema 1.25 ± 0.96 1.5 ± 0.58 1.5 ± 0.58 1.25 ± 0.96 0 ± 0 0 ± 0 hemorrhage 1 ± 0 2 ± 0.8 1.25 ± 0.5 1 ± 0 0 ± 0 0 ± 0 (alveolar/interstitial neutrophils) 1.25 ± 0 2 ± 0.8 1.25 ± 0.5 2.25 ± 0.96 0.5± 0.58 0 ± 0 (intrabronchial neutrophils) 1.75 ± 0.5 1.75 ± 0.96 1 ± 0.5 1.5 ± 1 0.25 ± 0.5 0.25 ± 0.5 (perivascular/peribronchiolar lymphocytes) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 2.25 ± 0.96 2.25 ± 1.3 (alveolar macrophages) 0.25 ± 0.5 0.75 ± 0.5 0.25 ± 0.5 0 ± 0 0 ± 0 0 ± 0 alveolar walls (edema/cellularity) 1.75 ± 0.5 2.75 ± 0.5 1.5 ± 0.58 2.5 ± 0.58 2.5 ± 0.58 1.5 ± 0.58 total average score 9.25 ± 0.78 14 ± 0.90 7.95 ± 0.62 11 ± 1 8.25 ± 1.2 6 ± 0.96
58
later time points. The histopathology indicated that neutrophils were involved in response
to both the wild-type and Δbps strains (Tables 1 and 2). Neutrophils have been shown to
be a crucial host factor required to control Bordetella infections B (77). Therefore, we
sought to determine the role neutrophils play in the clearance of the Δbps strain by
depleting these cells from the blood of C57BL/6 mice. We rendered mice neutropenic by
intraperitoneal injection of the anti-Gr1 antibody which has been shown to deplete Gr-1
positive cells, including neutrophils, for one to two weeks in mice (104). To confirm
neutropenia in the mice, blood collected from cardiac puncture was analyzed by IDEXX
laboratories for differential leukocyte blood count. It is important to note that the
treatment was effective in depletion of neutrophils but the presence of other host cells,
such as macrophages and lymphocytes, was not altered (data not shown).
We inoculated mice with the wild-type and Δbps mutant strain and sacrificed at 2
and 3 days post-inoculation due to the consistent colonization defect we observed by the
Δbps mutant strain in wild-type C57BL/6 mice. At 2 days post-inoculation, there was
only a slight defect in colonization of the Δbps mutant compared to the wild-type strain in
the nasal septum of wild-type C57BL/6 mice (Fig. 8). Both the wild-type and the Δbps
mutant strain were recovered in similar numbers in the trachea and the lungs of wild-type
C57BL/6 mice. When C57BL/6 mice were rendered neutropenic, colonization of the
wild-type and the Δbps mutant strain was not significantly different than from
colonization observed by these strains in wild-type C57BL/6 mice. These results suggest
that neutrophils do not play a significant role in clearance of B. bronchiseptica from the
mouse respiratory tract at 2 days post-inoculation.
59
Table 2: Presence of neutrophils in the lungs of mice infected with the wild-type or
Δbps strain at 3 days post-inoculation. Five mice were inoculated with 5 x 105 CFUs
of the wild-type or Δbps strain and sacrificed at 3 days post-inoculation. The right lung
was harvested and processed for H & E staining. The sections were reported and
examined blindly by Dr. Nancy Kock.
60
Wild-type
(3 days p.i.)
Δbps
3 days p.i.)
Mild neutrophilic
pneumonia
2/5 mice
(40%)
3/5 mice
(60%)
Mild to moderate
neutrophilic
pneumonia
2/5 mice
(40%)
2/5 mice
(40%)
Moderate to
severe
neutrophilic
pneumonia
1/5 mice
(20%)
0/5 mice
(0%)
61
Fig. 8: Colonization of the wild-type and Δbps strains in neutropenic and wild-type
C57BL/6 mice at 2 days post-inoculation.
Groups of five C57BL/6 mice were rendered neutropenic by intraperitoneal injection of
the anti-Gr1 antibody. One day later the mice were infected with the wild-type (solid red
diamonds) or the Δbps strain (open red diamonds). At two days p.i., the trachea, nasal
septum and lungs were harvested and homogenized. Resident bacteria were plated to
measure colonization. Colonization of neutropenic mice is compared to colonization of
groups of five wild-type C57BL/6 mice infected with wild-type B. bronchiseptica (solid
black diamonds) or the Δbps strain (open black diamonds). Horizontal bars represent the
numerical mean for each group. The dashed line represents the lower limit of detection.
Unpaired two-tailed Student t test was used to determine statistical significance. The
asterisk indicates a P value of < 0.05.
62
Trachea
0
1
2
3
4
5
6
Log 1
0C
FU
Nasal Septum
01234567
Log 1
0C
FU
Lungs
0123456789
Log 1
0C
FU
Neutropenic mice WT C57BL/6
WT Δbps WT Δbps
*
Trachea
0
1
2
3
4
5
6
Log 1
0C
FU
Nasal Septum
01234567
Log 1
0C
FU
Lungs
0123456789
Log 1
0C
FU
Neutropenic mice WT C57BL/6
WT Δbps WT Δbps
*
63
Consistently at 3 days post-inoculation, the Δbps mutant strain had significantly
lower colonization in the trachea, nasal septum and lungs compared to the wild-type B.
bronchiseptica strain in C57BL/6 mice (Fig. 9). In neutropenic mice, colonization of the
Δbps mutant strain increased approximately two logs in the trachea compared to
colonization in wild-type C57BL/6 mice. In the nasal septum, both the wild-type and the
Δbps mutant strain increased slightly in colonization in neutropenic mice compared to
colonization in wild-type C57BL/6 mice. Surprisingly, the Δbps mutant strain colonized
approximately five logs higher in the lungs of neutropenic mice compared to colonization
of the Δbps strain in lungs of wild-type C57BL/6 mice. The wild-type strain colonized
similarly in neutropenic or wild-type C57BL/6 mice in the trachea and lungs at this time
point. From these results, we conclude that neutrophils play a crucial role in clearance of
the Δbps mutant strain from the murine respiratory tract at 3 days post-inoculation.
The Bps polysaccharide does not play a role in cellular infiltration following a B.
bronchiseptica infection. Our neutropenic mice data suggest a role for neutrophils in the
clearance of the Δbps strain. We predicted that there will be a more rapid recruitment of
neutrophils to the lungs of mice infected with the Δbps strain. We also hypothesized that
there will be a greater number of neutrophils in the lungs of mice infected with the Δbps
strain. After bacterial inoculation with the wild-type and Δbps strains, we sacrificed mice
at 6, 24 hours and 2, 3 days post-inoculation to perform bronchoalveolar lavage (BAL) in
order to determine cellular infiltration to the lungs. At almost all time points tested, there
was no difference observed in the absolute numbers of neutrophils and macrophages
found in the lungs of mice inoculated with the wild-type or Δbps strain
64
Fig. 9: Colonization of the wild-type and Δbps strains in neutropenic and wild-type
C57BL/6 mice at 3 days post-inoculation. Mice were rendered neutropenic with
intraperitoneal injection of the anti-Gr1 antibody. One day later the mice were infected
with the wild-type (solid red diamonds) or the Δbps strain (open red diamonds). At three
days post-inoculation., the trachea, nasal septum and lungs were harvested and
homogenized. Resident bacteria were plated to measure colonization. Colonization of
neutropenic mice is compared to colonization of groups of five wild-type C57BL/6 mice
infected with wild-type B. bronchiseptica (solid black diamonds) or the Δbps strain (open
black diamonds). Horizontal bars represent the numerical mean for each group. The
dashed line represents the lower limit of detection. Unpaired two-tailed Student t test was
used to determine statistical significance. One asterisk indicates a P value of < 0.01. Two
asterisks indicates a P value of <0.05.
65
Trachea
0123
456
Log 1
0C
FU* *
Nasal Septum
0
1
2
3
4
5
6
Log 1
0C
FU
* ****
Lungs
0123456789
Log 1
0C
FU
* **
Neutropenic mice WT C57BL/6
WT Δbps WT Δbps
Trachea
0123
456
Log 1
0C
FU* *
Nasal Septum
0
1
2
3
4
5
6
Log 1
0C
FU
* ****
Lungs
0123456789
Log 1
0C
FU
* **
Neutropenic mice WT C57BL/6
WT Δbps WT Δbps
66
(Fig. 10). At 24 hours post-inoculation, there were slightly more neutrophils recruited to
the lungs of mice infected with the wild-type strain. It is important to note that there may
be more neutrophils found in the parenchymal lung tissue of mice than in the vessels.
However, there were no significant differences observed in the pathology scores of mice
infected with the wild-type or the Δbps strain of the interstitial or intrabronchial
neutrophils which reflect infiltration in the lung tissue and vessels, respectively. We
conclude that the enhanced clearance of the Δbps strain at 3 days post-inoculation is not
due to a rapid, large influx of neutrophils into the mouse lung.
The Δbps mutant strain does not have increased uptake by neutrophils. We have
observed that the Δbps strain is recovered in significantly higher numbers in the lungs of
neutropenic mice than wild-type C57BL/6, suggesting a role for neutrophils in clearance
of the mutant strain. Biofilm exopolysaccharides have been shown to mediate resistance
to phagocytosis by neutrophils. We hypothesized that, due to the absence of the Bps
polysaccharide, the Δbps mutant strain may be taken up more by neutrophils. We tested
the ability of both the wild-type strain and the Δbps strain to be taken up by neutrophils
after twenty minutes and one hour. At both time points, there was no significant
difference in the percent uptake of the wild-type or Δbps strain after incubation with
neutrophils (Fig. 11). We concluded that the Δbps strain is not more susceptible to
phagocytosis by neutrophils.
The Δbps strain induces greater cytokine production in mouse lungs at 2 and 3 days
post-inoculation. Cytokines play a significant role in the innate response to bacterial
67
Fig. 10: Cellular infiltration in bronchoalveolar lavage (BAL) fluid of mice infected
with the wild-type B. bronchiseptica or the Δbps strain. Groups of five C57BL/6 mice
were inoculated with 5 x 105 CFUs of the wild-type (black bars) or the Δbps strain (white
bars). Mice were sacrificed at the indicated time point and BAL was harvested by
perfusion of the lungs with sterile PBS. Results are expressed as the absolute numbers of
neutrophils (A) or macrophages (B) determined from differential cell counts of the BAL.
Bars represent the average of five mice and error bars indicate the ± standard deviation.
Unpaired two-tailed Student t test was used to determine statistical significance. The
asterisk indicates the P value of <0.01.
68
WT
Δbps
0
2000000
4000000
6000000
8000000
10000000
Abs
olut
e N
umbe
rs*
A
Time (days)
0
500000
10000001500000
2000000
2500000
3000000
Abs
olut
e N
umbe
rs
0.25 1 2 3
B
WT
Δbps
0
2000000
4000000
6000000
8000000
10000000
Abs
olut
e N
umbe
rs*
A
0
2000000
4000000
6000000
8000000
10000000
Abs
olut
e N
umbe
rs*
A
Time (days)
0
500000
10000001500000
2000000
2500000
3000000
Abs
olut
e N
umbe
rs
0.25 1 2 3
B
Time (days)
0
500000
10000001500000
2000000
2500000
3000000
Abs
olut
e N
umbe
rs
0.25 1 2 3Time (days)
0
500000
10000001500000
2000000
2500000
3000000
Abs
olut
e N
umbe
rs
0.25 1 2 3
B
69
Fig. 11: Phagocytosis of the wild-type and Δbps strains by neutrophils. The wild-type
and Δbps strains were opsonized with heat-inactivated Bordetella serum for thirty
minutes at 37oC prior to a one hour incubation with neutrophils. The neutrophils were
lysed in water by vigorous pipetting. Results are expressed as percent uptake determined
by the number of CFUs recovered after incubation with neutrophils divided by the
number of CFUs recovered from mock-treated controls. Bars represent the average of
three independent experiments performed in triplicate. Error bars indicate ± standard
deviation.
70
012345678
20 60
% u
ptak
eWT
Δbps
Time (min.)
012345678
20 60
% u
ptak
eWT
Δbps
012345678
20 60
% u
ptak
eWT
Δbps
Time (min.)
71
infection. We performed ELISAs with lung homogenates from mice infected with either
the wild-type or the Δbps strain to observe the production of cytokines in response to
infection. TNFα is required for mice to clear a B. bronchiseptica infection (119).
Therefore, we tested the production of TNFα in lung homogenates at 6 and 24 hours as
well as 2 and 3 days. TNFα production was substantial in mice infected with either strain
at 6 and 24 hours (Fig. 12A). By 2 and 3 days post-inoculation, the lungs of mice infected
with the Δbps strain had significantly more TNFα present than the wild-type strain.
IL-10 has been shown to be involved in the dampening of the immune response.
We hypothesized that the wild-type strain would induce more IL-10 production in the
lungs of mice to prevent clearance. We observed no difference in IL-10 production in
lungs of mice infected with the wild-type or Δbps strain at 6 hours post-inoculation (Fig.
12B). However, mice infected with the wild-type strain did have significantly higher IL-
10 production at 24 hours post-inoculation. By day 2 and 3 post-inoculation, infection
with the Δbps mutant strain produced more IL-10 in the mouse lungs. We conclude that
TNFα and IL-10 are produced in the mouse lung due to infection by B. bronchiseptica
and the Δbps strain induces higher production of these cytokines at days 2 and 3 post-
inoculation.
72
Fig. 12: Cytokine detection in lungs of mice infected with the wild-type and Δbps
strains. Mice were infected with the wild-type and the Δbps strains and sacrificed at the
indicated time points to harvest and homogenize lungs to measure cytokine production.
ELISA kits were used to detect the presence of TNFα (A) or IL-10 (B) in lung
homogenates. Bars represent the average of five mice and error bars indicate the ±
standard deviation. Unpaired two-tailed Student t test was used to determine statistical
significance. One asterisk indicates the P value of 0.01.
73
02000400060008000
100001200014000
0.25 1 2 3
TNFα
(pg/
mL)
WT
Δbps
* *
01000200030004000500060007000
0.25 1 2 3
Time (days)
IL-1
0 (p
g/m
L) * * *
A
B
02000400060008000
100001200014000
0.25 1 2 3
TNFα
(pg/
mL)
WT
Δbps
* *
01000200030004000500060007000
0.25 1 2 3
Time (days)
IL-1
0 (p
g/m
L) * * *
A
B
74
Chapter Four: Discussion
The mammalian host innate response comprises the first line of defense against
invading pathogens. Physical barriers such as the mucociliary escalator strive to prevent
binding of microorganisms to the host epithelial surface. However, when physical
barriers fail to restrict binding of pathogens, the host has a vast arsenal of compounds and
cells to attack microorganisms such as antimicrobial peptides and phagocytic cells such
as neutrophils and macrophages. If these mechanisms are unsuccessful in elimination of a
pathogen, adaptive immunity becomes crucial in clearance of the microbe from the host.
The respiratory tract serves as a prevalent site of infection by various bacteria. B.
bronchiseptica is a respiratory pathogen that often causes asymptomatic infection in
mammals including horses, pigs, companion animals such as dogs and cats as well as
laboratory animals such as rabbits, mice and rats. A major concern of B. bronchiseptica
infection is potential transmission to other animals or zoonotic transfer to
immunocompromised individuals. B. bronchiseptica has been reported to cause illness in
individuals who are infected with the human immunodeficiency virus (HIV) (48).
Investigating host-pathogen interactions provides insight into virulence factors necessary
for efficient colonization by B. bronchiseptica as well as host components required for
clearance of this unique pathogen. We have explored the role of the Bordetella
polysaccharide (Bps) in resistance to antimicrobial peptides (AMPs) in vitro and host
innate components, specifically neutrophils, in vivo.
We chose to examine the interaction of B. bronchiseptica with polymyxin B as a
model of antimicrobial peptides. AMPs, which are abundant in the lung epithelium and
neutrophils, are one of the first host components to respond to a B. bronchiseptica
75
infection. Most AMPs are both cationic and hydrophobic to allow effective binding to
bacterial surfaces. Although the structure of AMPs is quite diverse, the mechanism of
action is universal. AMPs aggregate to form a pore in the bacterial membrane, leading to
leakage of cellular contents and death (156).
It has been demonstrated that AMPs bind the anionic lipid A portion of Gram-
negative bacteria (53). Bacteria have evolved several mechanisms in order to resist
killing by AMPs. Proteus mirabilis incorporates aminoarabinose into its lipid A to reduce
the negative charge of the outer membrane and prevent AMP binding (128). Salmonella
enterica includes additional fatty acids into the lipid A structure to make the outer
membrane more stable and less prone to permeabilization by AMPs (70). Bacteria have
been shown to use efflux pumps and proteases as additional measures to oppose the
powerful killing capacity of AMPs (149, 156).
Although some bacteria have evolved multiple methods of resistance, most are
still susceptible to AMP killing. Because of this, AMPs can serve as a template for new
antimicrobial agents. Biochemists have synthesized AMPs in the laboratory in order to
study the role of structure in AMP bactericidal activity (6). Cationic peptides are also
being produced for use in clinical applications. Nisin, a cationic peptide made by AMBI,
has successfully passed Phase I clinical trials and is being considered for treating stomach
ulcers caused by Helicobacter pylori. Intrabiotics has synthesized the peptide IB-367 to
begin Phase I clinical trial with the hopes of using this peptide against infections caused
by P. aeruginosa in cystic fibrosis patients in the future (74).
Due to its amphipathic nature and demonstrated bacteriocidal activity, polymyxin
B has been used extensively to study the role of AMPs in bacterial clearance. Use of
76
polymyxins has demonstrated that several bacteria such as P. aeruginosa, E. coli,
Helicobacter pylori and Yersinia pestis modify lipid A in order to evade AMP killing
(197). In vivo studies performed by Miyajima et al. observed that mice inoculated with
multi-drug resistant (MDR) P. aeruginosa had significantly reduced bacteremia at 6
hours post-inoculation after treatment with polymyxin B (137). Due to the vast use of
polymyxin B in studies involving AMPs, we chose to use this molecule as a model of
AMP interaction with B. bronchiseptica.
Bps allows resistance of B. bronchiseptica to AMPs. The use of a polysaccharide to
sequester AMPs and inhibit killing is a novel mechanism of bacterial resistance. Campos
et al. observed that a capsular polysaccharide (CPS) mutant of Klebsiella pneumoniae
was susceptible to killing by a variety of AMPs including human neutrophil defensin-1
(HNP-1), human β-defensin-1 (hBD-1) and polymyxin B. Moreover, this study
demonstrated that serotypes that produced higher amounts of CPS on their surface had
increased resistance to polymyxin B (17). Encapsulated Neisseria meningitidis had
significantly more survival than an isogenic capsule-deficient mutant following LL-37
treatment (99).
We have shown that Bps binds polymyxin B in order to prevent killing of B.
bronchiseptica biofilms. An obvious consequence of Bps-mediated B. bronchiseptica
resistance to polymyxin B is the reduced effectiveness of AMP killing. It is important to
reiterate that all three classical species, B. bronchiseptica, B. parapertussis and B.
pertussis, produce Bps. Therefore, it is plausible that our findings, indicating that B.
77
bronchiseptica Bps promotes resistance to polymyxin B, will be observed in B.
parapertussis and B. pertussis as well.
Dispersal of the biofilm matrix may prove useful as a method to circumvent
bacterial resistance to AMPs. Treatment of alginate with alginate lyase, an enzyme that
cleaves the glycosidic linkages of this polysaccharide, increases diffusion of antibiotics
into alginate. Alginate lyase has been proposed as a potential therapeutic agent capable of
removing alginate from lungs of cystic fibrosis patients (79). Dispersin B (DspB) is an
enzyme produced by Actinobacillus actinomycetemcomitans that specifically cleaves the
glycosidic linkage of poly-β-1,6-N-acetyl glucosamine (100, 101, 151). We have
previously shown that DspB treatment of Bordetella biofilms leads to dissolution of the
extracellular polymeric substances in the biofilm (151). Treatment of Bordetella biofilms
with DspB may serve as a method to increase susceptibility to AMPs in vivo.
Potential roles of Bps in evasion of host immunity to promote efficient colonization
of B. bronchiseptica in the mouse respiratory tract.
We have shown that, at 38 days post-inoculation, Bps promotes persistence of B.
bronchiseptica in the nasal septum and lungs of C57BL/6 mice. Additionally, Bps was
found to be a major component of B. bronchiseptica biofilms formed in the respiratory
tract, suggesting that Bps plays a significant role in biofilm formation in vivo (108, 172).
We hypothesized that Bps may contribute to the establishment of Bordetella colonization
in vivo by acting as an attachment factor or promoting biofilm formation. Inoculation of
C57BL/6 mice revealed no significant differences in colonization of the wild-type or the
Δbps strain except a consistent defect observed by the Δbps strain in the nasal septum,
78
trachea and lungs at 3 days post-inoculation. Histopathological analysis of mouse lungs
led us to investigate the role of neutrophils in the enhanced clearance of the Δbps strain at
3 days post-inoculation. The utilization of neutropenic mice revealed that, at 3 days post-
inoculation, the Δbps strain was recovered in significantly higher numbers in the nasal
septum, trachea and lungs compared to colonization of the Δbps strain in wild-type
C57BL/6 mice. These results suggest that neutrophils contribute to the enhanced
clearance of the Δbps strain at 3 days post-inoculation.
It is important to note that both the wild-type and the Δbps strain were recovered
at high numbers in the mouse respiratory tract at 3 and 6 hours post-inoculation.
Therefore, we believe that Bps does not play a role in the initial establishment of B.
bronchiseptica infection in vivo. One possible explanation of the striking defect in
colonization observed by the Δbps strain at 3 days post-inoculation could be its inability
to form biofilms. We have demonstrated that Bps is required for B. bronchiseptica to
form biofilms both in vitro and in vivo (108, 151, 172). We hypothesize that the wild-type
strain may form biofilms as early as 3 days post-inoculation to avoid clearance by the
host response. Jesaitis et al. observed that neutrophils that attempted to phagocytose
biofilms formed by Pseudomonas aeruginosa had impaired killing activity including
reduced degranulation and hydrogen peroxide production (98). Furthermore, biofilms
have been attributed to causing several chronic human infections including periodontitis,
otitis media, and cystic fibrosis pneumonia, indicating their role in evasion of host
immune responses (29, 151). The presence of Bps in a maturing biofilm could further
increase resistance of B. bronchiseptica to host immunity. S. epidermidis biofilms that
contain PNAG had significantly reduced killing by opsonophagocytosis (22).
79
In stark contrast to the wild-type, the Δbps strain is incapable of biofilm formation
or production of a protective matrix. Thus, the Δbps strain may be more susceptible to
phagocytosis by neutrophils. Our in vitro neutrophil uptake assay does not demonstrate
increased susceptibility of the Δbps strain to phagocytosis by neutrophils. However, this
assay does not necessarily reflect true conditions in vivo, such as neutrophil activation
signals produced by cytokines. TNFα has been demonstrated to increase neutrophil
antimicrobial activity (57, 139). Although inoculation of mice with either the wild-type or
the Δbps strain resulted in considerable TNFα production, we observed significantly
higher amounts of TNFα produced in the lungs of mice inoculated with the Δbps strain at
2 and 3 days post-inoculation. We believe that the increased production of TNFα allows
the enhanced clearance of the Δbps mutant. Additionally, it is possible that the TNFα-
mediated activation of neutrophils is time-dependent. This hypothesis could explain our
interesting finding that the Δbps mutant has enhanced clearance at 3 days post-
inoculation when high amounts of TNFα have accumulated in the mouse lung. Our
mouse data at 2 days post-inoculation, in which colonization of the Δbps strain is similar
in mice in the presence or absence of neutrophils, could also be due to the fact that a high
amount of TNFα is required for a long duration of time for effective clearance of B.
bronchiseptica by neutrophils. Additionally, in vitro studies suggest that neutrophils are
defective in phagocytosis of bacteria that exist as biofilms. Leid et al. demonstrated that
neutrophils can penetrate biofilms formed by Staphylococcus aureus but do not
internalize bacterial cells (113). In the future, we would like to investigate neutrophil
phagocytosis of biofilms formed by the wild-type and the Δbps strain.
80
We believe that neutrophils are a crucial host component involved in the
clearance of the Δbps strain. One important point to note is that we used human
neutrophils in our in vitro phagocytosis assay. We believe that human cells are
appropriate for use with B. bronchiseptica since this pathogen can infect humans as well
as other mammals. However, human neutrophils produce beta-defensins while mouse
neutrophils do not (50). It may be necessary to use purified beta-defensins with both the
wild-type and the Δbps strain to understand the contribution of this molecule in killing B.
bronchiseptica. In the future, we would like to further investigate the role of neutrophils
in killing the Δbps strain. A limitation from our studies is that we conclude that Bps does
not play a role in migration of neutrophils to the lungs. This conclusion was based on
performing differential cell counts of BAL from mice infected with the wild-type or the
Δbps strain in which we saw no differences in absolute numbers of neutrophils in the
lavage fluid. Our assay observes neutrophils that have traveled in the bloodstream but
resident neutrophils in the lungs or other tissues are certainly important as well. One
important assay we would like to perform in the future is to separate neutrophils that
travel to the lungs from the bloodstream from neutrophils that have migrated to the lungs
from other host tissues.
In our in vitro assays, we refer to structures formed by the Δbps strain as
“biofilms” because similar numbers are recovered from 12 well plates for both the wild-
type and the Δbps strain after 24 hours of growth. Therefore, we are confident that our
results reflect the finding that Bps mediates resistance to AMPs. However, it may be a
misnomer to define the structures formed by the Δbps strain as “biofilms” since all of our
published data indicates that the Δbps strain does not form biofilms. Despite the
81
nomenclature, our in vitro studies have demonstrated that the Δbps strain is more
susceptible than the wild-type strain to killing by polymyxin B and possibly others
AMPs. One mechanism of killing by neutrophils that we have not explored is AMP
production. AMPs have been shown to be upregulated after bacterial infection (144, 167).
The production of Bps in the biofilm matrix may mask virulence factors on the surface of
the wild-type strain which results in reduced recognition and upregulation of AMPs by
neutrophils as well as other host cells such as epithelial cells. However, since the
virulence factors on the surface of the Δbps strain are not obstructed by the presence of
Bps, we hypothesize that neutrophils recognize PAMPs and thus serve as a predominant
source of AMPs in the mouse lung following infection by the Δbps strain. One future
experiment we propose to explore this hypothesis is infection of mouse neutrophils with
the wild-type and the Δbps strain followed by use of an immunofluorescent antibody to
detect the presence of CRAMP- the homolog of LL-37 produced in mice (96). We predict
that more AMPs would be produced in the neutrophils infected with the Δbps strain.
Isolation of AMPs from neutrophils could reveal if the Δbps strain has increased
susceptibility to AMP killing. In addition to direct bactericidal activity, AMPs also act as
immunomodulatory molecules that increase cytokine production and chemotaxis of
neutrophils and lymphocytes (3). We believe that AMPs produced in the lungs of a
mouse infected with the Δbps strain may not only kill the Δbps mutant but contribute to
the increased production of cytokines observed at 3 days post-inoculation.
It is important to note that Bps is not the only virulence factor of Bordetella that
will allow effective subversion of the host immune response. The B. bronchiseptica type
III secretion system induces increased production of IL-10 to promote persistence in vivo.
82
We have observed IL-10 production in the lungs of mice inoculated with the wild-type
and the Δbps strains, which both have an intact type III secretion system. We hypothesize
that the type III secretion system mey be involved in production of IL-10 to promote
persistent colonization of the wild-type and allow increased colonization of the Δbps
mutant after 3 days post-inoculation. Alternatively, the host may produce IL-10 to
alleviate the elevated inflammation due to Bordetella infection. This project has provided
insight into the role of Bps in allowing Bordetella to evade host immune responses. The
study of host-pathogen interactions will further our understanding of the countermeasures
used by bacteria to resist host defenses so that we can create new combative agents to
eliminate bacterial colonization and persistence.
83
Fig. 1: Model of potential roles of Bps polysaccharide in evasion of host defenses.
We believe that the presence of Bps promotes biofilm formation by B. bronchiseptica at
early time points post-inoculation. Bps production in the biofilm matrix leads to efficient
colonization of the wild-type strain in the mouse respiratory tract by several possible
mechanisms: 1) Bps sequesters AMPs in the biofilm matrix to prevent killing; 2) Bps
inhibits phagocytosis of the wild-type strain by neutrophils; 3) Bps masks virulence
factors on the surface of the wild-type strain, leading to reduced recognition by host cells
and decreased AMP production.
84
wild-type strain
B. bronchiseptica biofilm formation
Bps
Effective phagocytosis by neutrophils
AMP production
AMP killing
wild-type strain
AMP killing
B. bronchiseptica biofilm formation
Bps
B. bronchiseptica biofilm formation
Bps
Effective phagocytosis by neutrophils
AMP production
85
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C U R R I C U L U M V I T A E
C H E R A T O N L O V E
ADDRESS 1216 Ebert St., Winston-Salem, NC 27103
Cell: (704) 280-9610
Work: (336) 716-1211
e-mail: [email protected]
EDUCATION 2005- 2010 Wake Forest University Winston-Salem, NC Ph.D., Microbiology and Immunology Advisor: Dr. Rajendar Deora
2001-2005 Winston-Salem State University Winston-Salem, NC B.S., Molecular Biology
RESEARCH SKILLS Immunology: ELISAs, proteome profiling for cytokine production.
Bacteriology: Biofilm assays, killing assays using host immune components and therapeutic agents, cloning, western blots.
Animal Models: Tissue harvesting, bronchoalveolar lavage (BAL), nasal washes, peritoneal exudate washes, intranasal/ intraperitoneal inoculations.
Tissue Culture: Maintenance of cell lines, opsonophagocytosis assays.
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PUBLICATIONS Cheraton F. Love, Rajendar Deora. The Role of Bordetella Bps Polysaccharide in Resito Antimicrobial Peptides. Manuscript in preparation. Neelima Sukumar, Gina Sloan, Matt Conover, Cheraton F. Love, Seema Mattoo, NancyKock, and Rajendar Deora. Cross-species protection mediated by a Bordetella bronchiseptica slacking antigenic homologs present in acellular pertussis vaccines. 2010. Infection and Immunity, 78: 2008-2016. Chelsie E. Armbruster, Wenzhou Hong , Bing Pang, Kristin E. Dew, Richard A. Juneau,Matthew S. Byrd, Cheraton F. Love, Nancy D. Kock, and W. Edward Sword. LuxS promotes biofilm maturation and persistence of nontypeable Haemophilus influenzae in via modulation of lipooligosaccharides on the bacterial surface. 2009. Infection and Immuni77: 4081- 4091. Neelima Sukumar, Cheraton F. Love , Matt Conover, Nancy Kock, Purnima Dubey, Rajendar Deora. Active and Passive Immunization with Bordetella Colonization Factor A(BcfA) Protects Mice Against Respiratory Challenge with Bordetella bronchiseptica. 20Infection and Immunity, 77: 885- 895.
Gina Parise Sloan, Cheraton F. Love, Neelima Sukumar, Meenu Mishra, and Rajendar Deora. The Bordetella Bps Polysaccharide Is Critical for Biofilm Development in the Mouse Respiratory Tract. 2007. Journal of Bacteriology, 189: 8270-8276. Ann-Marie Turner, Cheraton Love, Rebecca Alexander, Pamela Jones. Mutational Analysis of Escherichia coli DEAD-box Protein CsdA. 2007. Journal of Bacteriology, 189: 2769-2776.
TEACHING SKILLS Taught General Biology and General Microbiology at Winston-Salem State
University, 2009-2010. Assisted in teaching Fundamentals of Bacteriology course, 2009.
PROFESSIONAL ACTIVITIES Vice president of Black Graduate Student Association (BGSA), 2008-2009. Assisted graduate school in recruiting at various professional school fairs, 2008. Helped organize Professional and Graduate School Forum, 2006. Member of BGSA, 2005- present.
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AWARDS Student Oral Presentation. FASEB Conference. Microbial polysaccharides of Medical/ Agricultural and Industrial Importance. June 2008.
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