Download ROLE OF SURFACE MOLECULES IN Campylobacter jejuni

ROLE OF SURFACE MOLECULES IN Campylobacter
jejuni COLONIZATION AND VIRULENCE IN
CHICKENS
By
Lisbeth E. Echevarría-Núñez
_____________________________
Copyright © Lisbeth E. Echevarría-Núñez 2012
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF VETERINARY SCIENCE AND MICROBIOLOGY
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
WITH A MAJOR IN MICROBIOLOGY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2012
THE UNIVERSITY OF ARIZONA
GRADUATE COLLEGE
As members of the Dissertation Committee, we certify that we have read the dissertation
prepared by Lisbeth E. Echevarría-Núñez entitled:
Role of surface molecules in Campylobacter jejuni colonization and virulence in
chickens
and recommend that it be accepted as fulfilling the dissertation requirement for the
Degree of Doctor of Philosophy
_________________________________________________________Date: 4/27/2012
Lynn A. Joens, Ph.D.
_________________________________________________________Date: 4/27/2012
V. K. Viswanathan, Ph.D.
_________________________________________________________Date: 4/27/2012
Carlos Reggiardo, Ph.D.
_________________________________________________________Date: 4/27/2012
Sadhana Ravishankar, Ph.D.
Final approval and acceptance of this dissertation is contingent upon the candidate’s
submission of the final copies of the dissertation to the Graduate College. I hereby certify
that I have read this dissertation prepared under my direction and recommend that it be
accepted as fulfilling the dissertation requirement.
_________________________________________________________Date: 4/27/2012
Dissertation Director: Lynn A. Joens
STATEMENT BY THE AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an
advanced degree at the University of Arizona and is deposited in the University Library
to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided
that accurate acknowledgment of source is made. Requests for permission for extended
quotation from or reproduction of this manuscript in whole or in part may be granted by
the head of the major department or the Dean of the Graduate College when in his or her
judgment the proposed use of the material is in the interests of scholarship. In all other
instances, however, permission must be obtained from the copyright holder.
SIGNED: Lisbeth E. Echevarría-Núñez
ACKNOWLEDGEMENTS
I would like to acknowledge my mentor Dr. Joens for given me the opportunity to
become a member of his laboratory and his unconditional advice. Special thanks to my
committee members: Dr. Gerba, Dr. Reggiardo, Dr. Ravishankar, and Dr. Viswanathan
from whom I have received great and unconditional advice.
Also I would like to acknowledge the person who believed in me and gave me a hand
when I most needed it. Dr. María Teresa Velez, you have dedicated years of hard work
and passion for education to this institution like no one else has, THANKS.
Thanks to all current and past members of Joens Lab that in one way or another were
involved during this process. Especially Dr. Kerry Cooper for all those months of
molecular training, which have not been in vain, THANKS.
Finally, I cannot thank enough my family and friends, especially Mom and Dad, for all
the support you guys have given me during all these years I have been away from home,
LOVE YOU both. Thanks to my grandparents for your unconditional love and support.
To Lynette and family thanks for all these years of your unconditional support. To Yesi’s
family (Angel, Derik and Lala) who joined me on this journey later on, but have been the
greatest support here, your presence here has been a blessing.
5 DEDICATION
To my role model, my Mom (Dr. Sonia E. Núñez), who has dedicated her life to educate
others and does it with passion and love. You have shown me the importance of a good
education. I know you had to make a stop in your life when you where in graduate
school, but this year we both have finished one of our greatest accomplishments in life
and this one I dedicate to you.
6 TABLE OF CONTENTS
LIST OF TABLES……………………………………………………………………...11
LIST OF FIGURES…………………………………………………………………….12
DISSERTATION FORMAT…………………………………………………………...15
CHAPTER I: LITERATURE REVIEW……………………………………………...16
I. Introduction……………………………………………………………………..…16
II. Epidemiology of Campylobacter infections……………………..………...……. 17
III. Campylobacter spp. in the Poultry System…………………………..………....18
IV. Animal model……………………………………………………………………20
V. Pathogenesis of Campylobacter infections……………………………………...21
VI. Campylobacter Virulence factors………………………………….……………24
A. Cytolethal distending toxin (CDT)…………………………………………...24
B. Flagella and Motility………………………………………………………….25
C. Adhesion and Invasion………………………………………………………..26
VII. Pili in bacteria………………………………………………………………….28
CHAPTER II: MUTAGENESIS OF PUTATIVE pilA GENE IN C. JEJUNI
(Cj1343c) AND ITS ROLE IN POULTRY COLONIZATION………………..……31
I. Abstract…………………………………………………………………………...31
II. Materials and Methods………………………………………………………….32
A. Culture of Bacterial Strains…………………………………………………...32
B. Biofilm formation……………………………………………………………..33
C. Scanning Electron Micrograph………………………………………..............33
7 TABLE OF CONTENTS – Continued
D. Generation of C. jejuni Cj1343c suicide vector…………..…………………..34
E. Generation of C. jejuni Cj1343c mutant………………….…………………...35
F. Poultry colonization assays………….…………………….…………………..35
III. Results…………………………………………………………………………..36
A. Detection of pilus-like appendages via scanning electron microscopy………36
B. Cj1343c is not involved in production of pilus-like appendages in C.
jejuni……………………………………………………………………………..36
C. Cj1343c is not required for poultry colonization…………………………….37
IV. Discussion………………………………………………………………………37
V. Chapter II Tables and Figures......................………………………………….41
CHAPTER III: CHAPTER III: PURIFICATION OF SURFACE-ASSOCIATED
STRUCTURES FROM C. JEJUNI……………………………………………..….….45
I. Abstract………………………………………………………...............................45
II. Materials and Methods…………………………………………........................46
A. Culture of Bacterial Strains……………………..............................................46
B. Purification of fibers reminiscent of pili……………………………………...46
C. Transmission Electron Micrograph (TEM)…………….……………………..47
D. French pressure cell disruption……………………………………………….47
E. Tricine-SDS-PAGE…………………………………………………………...48
F. Trypsin Digestion……………………………………………………………..48
8 TABLE OF CONTENTS – Continued
G. Mass spectrometry……………………………………………………………49
III. Results…………………………………………………………………………..49
A. TEM analysis of C. jejuni cells……………………………………………….49
B. SDS-PAGE of isolated pilus-like appendages in C. jejuni...............................49
C. Mass spectrometry analysis…………………………………………………...50
IV. Discussion……………………………………………………………………….50
V. Chapter III Tables and Figures......………………………………………..…..53
CHAPTER IV: MUTAGENESIS OF PUTATIVE GENES EXPRESSING
SURFACE-ASSOCIATED STRUCTURES ISOLATED FROM C. JEJUNI AND
THEIR ROLE IN C. JEJUNI PATHOGENESIS…………………………………….57
I. Abstract………………………………………………………...............................57
II. Materials and Methods………………………………………….........................58
A. Culture of Bacterial Strains……………………...............................................58
B. Generation of isogenic mutants……………….………………………………59
C. SEM of biofilms………………………………………………………………60
D. Poultry colonization assays…………………………………………………...61
E. Biofilm assay………………………………………………………………….62
F. Agglutination assays……………………………………………………….….62
G. Adhesion and invasion assays…………………………………………….…..63
H. Macrophage survival assays…………………………………………….…….64
9 TABLE OF CONTENTS – Continued
III. Results…………………………………………………………………………..65
A. Mutation strategy……………………………………………………………..65
B. Scanning electron micrograph of C. jejuni ΔCj0248 and Cj1475c C.
jejuni......................................................................................................................65
C.
ΔCj0248
and
ΔCj1475c
mutants
show
reduced
poultry
colonization………………………………………………………………………65
D. Reduced biofilm formation was observed for ΔCj0248 but not for ΔCj1475c.
Increased biofilm formation was observed for ΔCj1343c………...……………..66
E.
ΔCj0248,
ΔCj1475c
and
ΔCj1343c
have
no
adverse
effects
in
autoagglutination assays…………………………………………………………67
F. ΔCj0248 adheres better but invades less efficiently than wild type strain
ΔCj1475c and ΔCj1343c have no adverse effects in adhesion and invasion
assays…………………………………………………………………………….67
G ΔCj0248, ΔCj1475c and ΔCj1343c have no adverse effects in their ability to
survive intracellularly within macrophages.……………………..........................67
IV. Discussion……………………………………………………………………….68
V. Chapter IV Tables and Figures.……………………….……………...………..76
CHAPTER V: ATTENUATED SALMONELLA AS VECTOR TO DELIVER
VACCINE (Se:0248) AGAINST C. JEJUNI INFECTION……………………….....86
I. Abstract…………………………………………………………………………...86
10 TABLE OF CONTENTS – Continued
II. Material and Methods..........................................................................................88
A. Culture of Bacterial Strains…………………………………………………..88
B. Construction of an S. enterica vaccine strain………………………………....88
C. Expression of Cj0248…………...…………………………………………….89
D. Challenge of Birds………...………...………………………………………..89
E. Vaccination using Salmonella enterica……………………………………….89
III. Results..................................................................................................................90
A. Construction of an S. enterica vaccine strain…………………………………90
B. Expression of C. jejuni 0248 gene in the Salmonella vaccine strain………....90
C. Oral Vaccination with Salmonella……………………………………………91
IV. Discussion………………………………………………….……………………92
V. Chapter V Tables and Figures..…………………..………...………………….94
REFERENCES…………………………………………………………………………99
11 LIST OF TABLES
Table 1A. Cj1373c nucleotide sequence similarity to the pilT gene present in several
bacterial genomes………………………………………………………………………...41
Table 2A. Strains and plasmids used in this study……………………………………….41
Table 3A. Oligonucleotides used in this study…………………………………………..41
Table 4B. Strains and plasmids used in this study………………………………………53
Table 5B. Identification of proteins involved in the production of surface-associated
molecules identified by MS……………………………………………………………...53
Table 6C. Strains and plasmids used in this study……………………………………….76
Table 7C. Oligonucleotides used in this study…………………………………………...76
Table 8D. Strains and plasmids used in this study……………………………………….94
Table 9D. Oligonucleotides used in this study…………………………………………..94
Table 10D. First Oral Vaccination with Se:0248………………………………………..94
Table 11D. Second Oral Vaccination with Se:0248…………………………………….95
Table 12D. Third Oral Vaccination with Se:0248………………………………………95
12 LIST OF FIGURES
Figure 1A. Sequence alingment of the conserved, highly hydrophobic amino-terminal
domain consensus sequence commonly found in PilA proteins against Cj1343c signal
peptide sequence…………………………………………………………………………42
Figure 2A. Scanning electron micrograph of putative pili disseminating from the cell of
the C. jejuni strain NCTC 11168 flaAB mutant in different growth conditions. A.) Biofilm
24-hour. B.) Broth culture 24-hour. Arrows indicates putative pili……………………...43
Figure 3A. Scanning electron micrograph of Cj1343c. A.) Biofilms of C. jejuni
ΔCj1343c. B). Biofilms of C. jejuniΔFlaAB……………………………………………43
Figure 4A. Effects of ΔCj1343c mutation on in vivo poultry colonization. Error bar
indicates standard deviation……………………………………………………………...44
Figure 5B. Transmission electron micrograph of purified molecules from C. jejuni, A.)
TEM showing long filamentous appendages extracted from C. jejuni FlaAB mutants. B.)
TEM showing fragmented appendages extracted from C. jejuni FlaAB mutants. C.) TEM
showing fragmented appendages extracted from C. jejuni FlaAB mutants and their sizes.
D.) TEM showing extracted C. jejuni NCTC flagella and their sizes…………………...53
Figure 6B. Coomassie-stained SDS-PAGE gel containing pellet and supernatant of
isolated pilus-like appendages isolated form C. jejuni FlaAB mutant. Arrows indicate
bands that were excised for MS analysis………………………………………………...56
Figure. 7C. Analysis of the location of gene Cj1475c in the NCTC 11168 genome…….77
Figure 8C. Scanning electron micrograph of Cj2048 and Cj1475c mutants. A.) Biofilms
of C. jejuni ΔCj0248. B). Biofilms of C. jejuni ΔCj1475c……………………………...78
Figure 9C. Effects of ΔCj0248 mutation on in vivo poultry colonization, first trial. Error
bar indicates standard deviation………………………………………………………….78
Figure 10C. Effects of ΔCj0248 mutation on in vivo poultry colonization, second trial.
Error bar indicates standard deviation…………………………………………………...79
Figure 11C. Effects of ΔCj0248 mutation on in vivo poultry colonization, third trial.
Error bar indicates standard deviation…………………………………………………...79
Figure 12C. Effects of ΔCj1475c mutation on in vivo poultry colonization, first trial.
Error bar indicates standard deviation…………………………………………………...80
13 Figure 13C. Effects of ΔCj1475c mutation on in vivo poultry colonization, second trial.
Error bar indicates standard deviation…………………………………………………...80
Figure 14C. Effects of ΔCj1475c mutation on in vivo poultry colonization. Error bar
indicates standard deviation, third trial…………………………………………………..81
Figure 15C. Effects of Cj0248, Cj1475c and Cj1343c mutation in biofilm formation, at
24 h. Error bar indicates standard deviation……………………………………………..81
Figure 16C. Effects of Cj0248, Cj1475c and Cj1343c mutation in biofilm formation, at
48 h. Error bar indicates standard deviation……………………………………………..82
Figure 17C. Effects of Cj0248, Cj1475c and Cj1343c mutation in biofilm formation, at
72 h. Error bar indicates standard deviation……………………………………………..82
Figure 18C. Effects of Cj0248, Cj1475c and Cj1343c mutation in agglutination assays.
Error bar indicates standard deviation…………………………………………………...83
Figure 19C. Adherence assays. Effects of Cj0248, Cj1475c and Cj1343c mutations in the
ability to adhere to INT 407 cells………………………………………………………..83
Figure 20C. Invasion assays. Effects of Cj0248, Cj1475c and Cj1343c mutations in the
ability to invade to INT 407 cells………………………………………………………..84
Figure 21C. Macrophage survival assays. Effects of Cj0248, Cj1475c and Cj1343c
mutations in their ability to survive intracellularly within macrophages (24 h)…………84
Figure 22C. Macrophage survival assays. Effects of Cj0248, Cj1475c and Cj1343c
mutations in their ability to survive intracellularly within macrophages (48 h)…………85
Figure 23C. Macrophage survival assays. Effects of Cj0248, Cj1475c and Cj1343c
mutations in their ability to survive intracellularly within macrophages (72 h)…………85
Figure 24D. SDS-PAGE. 1.) Attenuated Salmonella without insert. 2.) Attenuated
Salmonella vector expressing C. jejuni 0248 gene. 3.) Molecular weight. Arrow indicates
band of appropriate molecular weight that might correspond to C. jejuni hypothetical
protein encoded by Cj0248………………………………………………………………96
Figure 25D. First oral vaccination with S. enterica (pYA3494) expressing Cj0248
(Se:0248). Error bar indicates standard deviation, p > 0.05……………………………..97
Figure 26D. Second oral vaccination with S. enterica (pYA3494) expressing Cj0248
(Se:0248). Error bar indicates standard deviation. Error bar indicates standard deviation,
p > 0.05…………………………………………………………………………………..97
14 Figure 27D. Third oral vaccination with S. enterica (pYA3494) expressing Cj0248
(Se:0248). Error bar indicates standard deviation, p > 0.05……………………………..98
Figure 28D. Plasmid map of cloning vector used in this study………………………….98
15 DISSERTATION FORMAT
This dissertation contains five chapters: Chapter I contains a comprehensive literature
review, Chapters II through V contains an abstract in which the project and research
objectives are defined, description of material and methods, results and discussion. Each
chapter contains an Appendix in which tables and figures referred in each chapter are
included. The dissertation was written and prepared by the degree candidate, Lisbeth E.
Echevarría-Núñez.
16 CHAPTER I: LITERATURE REVIEW
I. Introduction
Campylobacter spp. belongs to the epsilon class of proteobacteria, in the order
Campylobacteriales; this order includes two other genera, Helicobacter and Wolinella.
The genus Campylobacter includes 17 species, of which C. jejuni and C. coli are the most
important human pathogens (Allos B. M., et al., 2001); however, most human illness is
caused by one species C. jejuni. C. jejuni and C. coli have a genome of approximately
1.6-1.9 megabases (Mb), which is small compared to Escherichia coli, which have a
genome of approximately 4.5 Mb (Chang N. and Taylor D. E., 1990., Nuijten, P. J. M., et
al., 1990). Campylobacter spp. are small, curved to spiral, microaerophilic, Gramnegative rods with a polar flagellum at one or both ends of the cell. The cells are highly
motile, with a characteristic corkscrew kind of movement. The range for growth of the
thermophilic Campylobacter spp. is 34 ± 44°C, with an optimal temperature of 42ºC.
Campylobacter spp., normally spiral-shaped, have been reported to change into coccoid
forms on exposure to atmospheric oxygen levels or other stresses. These coccoid forms
have been coined viable but non-culturable (VBNC), and this form has been suggested to
be a dormant state required for survival under conditions not supporting growth of the
bacteria, e.g. during transmission, transport or storage (Rollins D. M., and Colwell R. R.,
1986). C. jejuni and related species are important human pathogens, causing acute human
enterocolitis, and they are the most common cause of foodborne diarrhea in many
industrialized countries. In industrialized countries the infection is often seasonal, more
often during summer and winter times, and usually targets young adults and young
17 children. Despite all efforts and recognition of its importance as a human pathogen,
relatively little is understood the mechanisms of C. jejuni-associated disease.
II. Epidemiology of Campylobacter infections
In the U. S. Campylobacter infections became reportable illnesses in many states in
the early 1980s, when appropriate culture techniques for the isolation of Campylobacter
spp. were developed. Today Campylobacter infections are recognized as one of the
leading bacterial causes of gastroenteritis in humans. The greatest risk factor to the
consumer is improper food-handling, which contributes to 40-60% of cases of foodborne
illness (de Jong AE., et al, 2008). The Center for Disease Control and Prevention (CDC)
estimates that approximately 124 persons with Campylobacter infections die each year.
The FoodNet estimates 13 cases are diagnosed each year for each 100,000 persons in the
population. However, many more cases are undiagnosed or unreported. It is estimated
that Campylobacter diarrheal illness affects about 2.4 million persons every year with an
estimated cost of treatment and loss of productivity exceeding $1 billion annually ( Tauxe
R. V., 1992.). An estimated one case of Guillain-Barré syndrome (GBS) occurs for every
1,000 cases of campylobacteriosis. During 2007, the Foodborne Diseases Active
Surveillance Network (FoodNet) of CDC's Emerging Infections Program conducts
population-based surveillance in ten U.S. states for all laboratory-confirmed infections
with select enteric pathogens transmitted commonly through food; among those are
Campylobacter species. In 2007 the FoodNet surveillance program included 45.9 million
persons, or 15.2% of the U.S. population. FoodNet sites identified 18,039 laboratoryconfirmed infections caused by the pathogens under surveillance. Of 16,801 bacterial
18 foodborne infections, most were caused by Salmonella (41%), followed by
Campylobacter (35%). The number of infections reported varied by pathogen and month;
44% of Campylobacter infections occurred from June through August. The
hospitalization status was determined for 95% of FoodNet cases. The percentage of
persons hospitalized for Campylobacter was 14. Sixty-four persons with laboratoryconfirmed foodborne infections were reported to have died. Of those, 7 were infected
with Campylobacter (FoodNet 2007). These numbers did not change significantly during
surveillance in years 2008 and 2009 (FoodNet 2008 and 2009).
III. Campylobacter spp. in the Poultry System
Campylobacter spp. colonize the intestinal mucosa of most warm-blooded hosts,
including all food-producing animals and humans. C. jejuni is found commonly in cattle,
sheep, pigs, poultry, wild birds, rabbits, other wild mammals, household pets, and in
natural water sources. C. jejuni establishes persistent, benign infections in chickens, but
causes significant inflammation and enteritis in humans. In chickens, C. jejuni colonizes
the mucus overlying the epithelial cells primarily in the ceca and the small intestine but
may also be recovered from elsewhere in the gut and from the spleen and liver. Colonized
chickens usually show no observable clinical symptoms of infection even when young
chicks are exposed to high doses under experimental conditions.
Poultry meat is considered to be an important source of infection (Friedman C.R.,
2000). Factors surrounding commercial poultry production and infection with
Campylobacter spp. are very diverse and poultry houses differ in the number of birds
present as well as the overall design of the house and biosecurity measures. In addition,
19 different breeds of chickens have vastly different growth rates, immunological traits and
genetics, further complicating research. Broiler houses remain uninfected for short
periods of time, however, birds can become colonized sporadically and serve as a source
of infection, resulting in rapid colonization of all birds in the house.
Broiler chickens are often intestinal carriers of C. jejuni, as chickens are
coprophagic, fecal shedding is an important factor in the dissemination of organisms
around large broiler flocks once the first bird becomes colonized (Evans S., and A. R.
Sayers. 2000). Once infected, birds can continue to shed the organism for over 42 weeks,
well past the harvest age of 6-7 weeks for broilers and well into the productive life for
layers (Lindblom et al., 1986). A chicken colonized with C. jejuni usually excretes large
amounts of bacteria up to 108 CFU’s, leading to contamination of broiler carcasses during
slaughter and processing (Ono K. and Yamamoto K., 1999., Shreeve, J. E., 2002,
Uyttendaele M.R., 1999).
It is estimated that up to 70% of Campylobacter infections are due to the
compsumption of contaminated poultry meat (Harris NV, et al 1986, Adak G. K, et al
1995, Harris NV, et al., 1986). The duration of colonization and shedding in poultry has
not been fully determined, however is generally accepted that colonization in chickens
persists at least for the life span of a broiler; 47 days. A recently published study
demonstrated that in a commercial poultry facility of 20,000 birds, 95% of the flock was
colonized within four to seven days once a single bird on the farm became infected (van
Gerwe et al., 2009). The major sources of contamination during the farm and at
processing facilities are: broiler house cleansing and disinfection (Pattison, M. 2001),
20 particularly because litter is routinely left in the houses between crops. Carry of C. jejuni
into the houses from the external environment via boots, external clothes, and equipment
can occur. As well as from wild and domestic animals and insects particularly because
studies have indicated the presence of C. jejuni isolated from the feces (Fernie, D., 1977,
Pearson, A. D., 1996). One of many approaches to reduce Campylobacter infections in
humans is to prevent C. jejuni colonization of broiler chickens. Despite all efforts to
reduce the levels of bacteria during the farm and processing activities, the prevalence of
C. jejuni on raw chicken in retail markets is still as high as 90 to 100% (Ruiz-Palacios et
al 1983, Scherer K., et al., 2006, Stern, N. J., 2001).
IV. Animal model
Since poultry is considered the main source of campylobacteriosis in humans,
researchers have worked to eliminate Campylobacter in the poultry system. Thus, many
models of C. jejuni colonization of the chick intestinal tract have been described. These
models significantly differ in the route of infection, age of the bird at the time of
infection, and the breed of chicken used in the study. Generally, there are two routes of
infections, by oral gavage, and infection via introduction of a carrier bird (Nachamkin
and Blaser, 2000). Each model represents different aspects of the typical flock
colonization and thus has roles in characterizing the disease. The oral gavage method
mimics the fecal oral transmission of the organism as it establishes within a flock.
Generally, birds 1 to 14 days of age are inoculated with a defined amount of
Campylobacter, and then given several days to several weeks for the bacteria to establish
colonization (Biswas et al., 2007; Nachamkin and Blaser, 2000). The age of the birds
21 when the bacteria is administered will affect the minimum infectious dose required, with
1-2 day old birds requiring about one log less viable Campylobacter than birds 14 days of
age (Ringoir et al., 2007). However, the age of the bird at the time of infection does not
affect the total bacterial load in the ceca, or the level of shedding of Campylobacter. This
model provides a great tool for identifying factors involved in initial poultry colonization.
The horizontal transmission model represents the passage from bird to bird within the
flock. In this model, a small percentage of the total birds in a trial group (generally less
than 20%) are infected with C. jejuni, and housed with the remaining uninfected birds
(Biswas et al., 2006; Nachamkin and Blaser, 2000). This style of infection has the distinct
advantage of identifying factors involved in the bird-to-bird transmission of C. jejuni in
poultry houses. For both models outlined above, many other factors will affect the
infection models. The type of diet can affect the load of Campylobacter in the ceca.
Previous studies have shown that an all-plant diet can significantly reduce the bacterial
load of C. jejuni in the ceca of the chicken, as compared to birds receiving an animalbased diet (Udayamputhoor et al., 2003).
V. Pathogenesis of Campylobacter infections
Infections with Campylobacter spp. lead to enteritis, an acute diarrheal disease,
with clinical manifestations similar to those of other bacteria that infect the
gastrointestinal tract, such as Salmonella spp. and Shigella spp. Therefore it is difficult to
distinguish between Campylobacter infections from these caused by other enteric
bacteria. In terms of diagnosis, Campylobacter infections can present a wide range of
symptoms from general malaise, diarrhea, often accompanied by fever and severe
22 abdominal cramps; however, some individuals may present fever and abdominal pain
without diarrhea. In most patients, the diarrhea is either loose and watery or grossly
bloody with 8–10 bowel movements occurring per day at the peak of illness (Blaser M.
J., et al., 1983). After ingestion of contaminated food with Campylobacter spp. in order
for the infection to be established, Campylobacter spp. must survive the acidic
environment in the stomach, and then colonize the jejunum and the ileum. The ability of
the bacteria to colonize also relies on the rapid motility that Campylobacter spp. presents
thus facilitating the migration and attachment to the mucosal surfaces.
The infective dose of Campylobacter spp., is relatively low, doses as low as 500
microorganisms have been reported to establish an infection. However, infective doses
ranges from 500 to 106 organisms (Black M. J., et al 1992, Black, R. E., et al., 1988).
Variation in infectious dose is thought to be due to either individual susceptibility or to
the relative virulence of the organism. The incubation period ranges from eight to
eighteen days. However the disease is usually self-limiting, lasting from two to seven
days, which may account for the high numbers of cases that are undiagnosed or
unreported. However, it occasionally is fatal in infants and young adults (Skirrow, M., et
al 1992, Allos B. M., 2001). There are at least three mechanisms by which
Campylobacter may induce illness: intestinal adherence and production of toxins,
bacterial invasion and proliferation within the intestinal mucosa, inducing cell damage
and inflammatory responses, and extraintestinal translocation in which organisms can
cross the intestinal mucosa and migrate via the lymphatic system to various
23 extraintestinal sites causing meningitis, cholecystitis, urinary tract infections (RuizPalacios, 1993).
Upon infection, C. jejuni crosses the mucus layer covering the epithelial cells and
adheres to these cells, and a subpopulation subsequently invades the epithelial cells. The
invasion of epithelial cells can lead to the mucosal damage and inflammation. Electron
and light microscopy studies have demonstrated the organism within epithelial cells
lining the gut lumen as well as in granulocytes, parenchyma cells, and mononuclear cells
located within the lamina propria. This intracellular existence provides this fastidious,
slow-growing organism an unoccupied niche where microbial competition is reduced or
non-existent. Additionally, the intracellular niche is thought to shelter organisms from
immune surveillance. Gross lesions may include hemorrhage, edema, and catarrhal
inflammation and are found in the colon and small intestine. Microscopically, lesions
consist of villous erosion due to necrosis, and epithelial cell sloughing, with exudates
containing inflammatory cells and fibrin (Wassenaar, T. M., 1999).
Campylobacteriosis can cause life-threatening bloodstream infections in persons
with compromised immune systems. Also, campylobacteriosis has been associated with a
chronic sequela, Guillain-Barré Syndrome (GBS). GBS, is a demyelinating disorder
resulting in acute neuromuscular paralysis. Up to 40% of patients with the syndrome have
evidence of a recent Campylobacter infection. Approximately 20% of patients with GBS
are left with some disability, and approximately 5% die despite advances in respiratory
care. Campylobacteriosis is also associated with Reiter syndrome, a reactive arthropathy.
In approximately 1% of patients with campylobacteriosis, the sterile postinfection
24 process occurs 7 to 10 days after onset of diarrhea. Multiple joints can be affected,
particularly the knee joint. Pain and incapacitation can last for months or become chronic.
Both GBS and Reiter syndrome are thought to be autoimmune responses stimulated by
recurrent infections. However, the pathogenesis of GBS and Reiter syndrome is not
completely understood.
VI. Campylobacter Virulence factors
Since the completion of the genome sequence of C. jejuni, many studies suggest
that Campylobacter possess a substantial number of virulence factors. These genes have
been shown to be associated with C. jejuni adherence (CadF, JlpA, LOS, MOMP, PEB1),
invasion (CiaB), motility (flagella) (Wassenaar, T. M., et al., 1993), toxin production,
extracellular matrix binding, internalization into enterocytes, and a Type IV secretion
system. (Parkhill J, et al., 2000).
A. Cytolethal distending toxin (CDT)
CDT production has been described in several Gram-negative bacteria, including
Escherichia coli (Pickett, C. L., 1994, Scott, D. A., et al 1994, Slutsker LA, et al, 1997),
Haemophilus ducreyi (Cope, L. D., et al, 1997), Shigella dysenteriae (Okuda, J., et al
1995, Okuda, J., et al, 1997), and Helicobacter spp. (Young, V. et al, 2000). CDT
production by Campylobacter was first discovered in 1988. The role of CDT in human
campylobacteriosis is not well understood. Although, all C. jejuni strains tested to date
appear to possess the CDT genes (Eyigor A., et al, 1999, Pickett, C. L., et al, 2002,
Zeytin. 1996), the levels of toxin activity expressed are strain dependent. The C. jejuni
CDT is encoded by a three-gene operon (cdtABC), and isogenic C. jejuni cdt mutants
25 lose all CDT activity (Purdy, D., et al 2000, Whitehouse, C.A., et al, 1998). Cytolethal
distending toxin activity causes certain cell types (such as HeLa cells and Caco-2 cells) to
become slowly distended, which progresses into cell death. CDT involvement in diarrhea
is believed to be caused by distortion of crypt cells into functional villus epithelial cells,
which causes a temporary erosion of the villus and a subsequent loss of absorptive
functions (Whitehouse, C.A., et al, 1998).
B. Flagella and Motility
Several studies have indicated that intact flagella are an important colonization
factor of C. jejuni (Nachmkin I., et al, 1993). C. jejuni contains polar flagella at one or
both ends of the cell. The flagellar filament consists of multimers of the protein flagellin
and is attached by the hook protein to a basal structure, embedded in the cell membrane,
which serves as a motor for rotation. The flagellin locus contains two adjacent genes,
flaA, encoding the major flagellin and flaB, encoding a minor flagellin. Both genes are
independently transcribed, with the flaA gene regulated by a fliA gene (encoding sigma28) and the flaB gene regulated by an rpoN gene (encoding sigma-54) (Hendrixson DR.,
et al 2004, Nuijten PJ., et al, 1990). There are numerous conditions that affect flaA and
flaB promoter activity, including temperature, pH, osmolality, and inorganic nutrients.
Studies have shown that lower pH, bovine bile, deoxycholate, fructose, high osmolarity
and chemotactic effectors such as aspartate, glutamate, citrate, fumarate, ketoglutarate
and succinate all upregulate the flaA promoter (Alm RA, et al, 1993). The flaA gene
seems to be highly conserved among Campylobacter isolates and transcription is usually
higher in this gene than that of the flaB gene (Guerry P., et al, 1900). Transcription of
26 sigma-54 dependent genes, necessary for assembly of the hook-basal body filament
structure, is regulated by a two-component system composed of the sensor kinase FlgS
and the response regulator FlgR (Wosten MM., et al, 2004). Experiments with mutants
have shown that flaA, but not flaB is essential for colonization of chickens, although
probably both are needed for full motility. Other studies have shown differential
expression patterns of flagella genes between strains of C. jejuni that colonized chickens.
Flagella genes that were differentially expressed contained a modification in the
hypervariable region of the C. jejuni genome (Hiett K.L., 2008). These findings further
support the fact that flagella are an important colonization factor for C. jejuni. The
flagellar apparatus also functions as a type III secretion apparatus for the Campylobacter
invasion antigens (Cia proteins) (Konkel M., et al, 2004). These proteins are important
for in vitro cell invasion and chick colonization, and their secretion is enhanced upon
exposure to chicken mucus. Despite numerous studies, the role of motility in the
colonization GI tract by C. jejuni is not fully understood. More likely motility is needed
for initial colonization of its ideal niche, the mucus, but it is not known if motility is
important for the persistence of C. jejuni in the intestinal tract, leading to long-term
colonization (Beery JT., et al 1988, Hartley-Tassell LE., et al, 2010). Therefore, C. jejuni
flagellum serves multiple functions in the adaptation of C. jejuni to the chicken GI tract.
C. Adhesion and Invasion
Successful establishment of infection by bacterial pathogens requires adhesion to
host cells, colonization of tissues, and in certain cases, cellular invasion followed by
intracellular multiplication, dissemination to other tissues, or persistence (Pizarro-Cerda
27 J., et al, 2006). The ability of C. jejuni to enter, invade and survive within mammalian
cells has been studied extensively; however, it appears to be strain dependent. Other
adhesion factors beside flagella may also play an important role in host colonization by
C. jejuni. Despite the lack of identifiable adherence organelles such as pili, several
proteins contribute to C. jejuni adherence to eukaryotic cells.
The most studied adherence factors in C. jejuni are CadF and JlpA. CadF binding
to fibronectin, which is localized basolaterally on epithelial cells, mediates adherence in
C. jejuni, promotes bacteria–host cell interactions, and facilitates the colonization of
chickens (Konkel ME., et al., 1997). Fibronectin is a 220 kDa glycoprotein that is present
at regions of cell-to-cell contact in the gastrointestinal epithelium, thus providing a
potential binding site for pathogens (Quaroni et al., 1978). The fibronectin-binding
domain of CadF consists of amino acids 134–137 (FRLS), which represents a novel
fibronectin-binding motif (Konkel, M. E., et al., 2005). CadF is required for maximal
binding and invasion by C. jejuni in vitro, and cadF mutants are greatly reduced in chick
colonization when compared to the wild type (Monteville, M. R., et al., 2003). CadF is
similar to E. coli OmpA and forms membrane channels but its actual function have not
been elucidated (Mamelli, L., et al., 2006).
Another characterized adhesin, a surface lipoprotein, that has been shown to be
essential in the binding to human epithelial cells, is JlpA. JlpA mediates adherence of the
bacterium to epithelial cells by the interaction with heat shock protein 90∝ (Hsp90∝).
Hsp are typically involved in signal transduction, cell cycle control and transcriptional
regulation. Hsp90α interaction with JlpA initiates signaling pathways that lead to
28 activation of NF-κB and p38 MAP kinase, two central components in host
proinflammatory responses. This indicates that some of the inflammation that is observed
during C. jejuni pathogenesis might be related to JlpA-dependent adherence (Jin, S. et al
2001, Jin, S., et al., 2003). CapA, another lipoprotein, was implicated as a possible
adhesin for C. jejuni. CapA is an autotransporter that is homologous to an autotransporter
adhesin, and CapA-deficient mutants have decreased adherence to Caco‑2 cells and
decreased colonization and persistence in the chick model (Ashgar, S. S. et al., 2007).
Another putative adhesin is Peb1; which is located in the periplasm and shares
homology to the periplasmic-binding proteins of amino acid ATP-binding cassette (ABC)
transporters. Peb1 binds to both aspartate and glutamate with high affinity, and peb1deficient mutants cannot grow if these amino acids are the major carbon source (LeonKempis M., et al, 2006). Peb1 contains a predicted signal peptidase II recognition site, a
common motif in surface-localized lipoproteins, and so there is a possibility that Peb1 is
surface accessible. Mutants that lack peb1 colonize mice poorly, but this could be
attributed to the loss of either the adhesion or the amino-acid-transport functions, or both
(Pei, Z. et al., 1998). Another periplasmic protein, the glycoprotein Cj1496c, which has
homology to a magnesium transporter, is also required for wild-type levels of adherence.
The mechanism by which these periplasmic proteins contribute to host-cell adherence by
C. jejuni is unclear (Kakuda, T. and DiRita 2006).
VII. Pili in bacteria
Many bacterial species possess long filamentous structures known as pili or
fimbriae extending from their surfaces (Shreeve, J. E., et al, 2002). Pili are important
29 virulence factors for several diseases, in particular infections of the urinary, genital and
gastrointestinal tract. Bacterial pili are defined as non-flagellar, proteinaceous, multisubunit surface appendages involved in adhesion to other bacteria, host cells or
environmental surfaces (Fronzes, R., 2008, Ottow J. C., 1975). Pili have been implicated
in crucial host–pathogen interactions such as colonization, biofilm formation, invasion
and attachment, signaling events, phage transduction and DNA uptake. Pili in Gramnegative bacteria are typically formed by non-covalent homopolymerization of major
pilus subunit proteins called pilins, which generates the pilus shaft. The pilus shaft is
composed of several hundred (probably thousands) of small 15 – 25 kDa subunits or
pilins.
There are four distinct groups of pili in Gram-negative bacteria based on their
assembly pathway: pili assembled by the chaperone-usher pathway, the Type IV pili, pili
assembled by the extracellular nucleation/precipitation pathway (curli pili) and pili
assembled by the alternative chaperone-usher pathway (CS1 pilus family). The most
widespread class known in Gram-negative bacteria is the Type IV pili (Craig, L. and Li,
J. 2008, Craig, L., et al., 2004). Type IV pili are present in a variety of pathogens such as
E. coli, Salmonella enterica, Pseudomonas aeruginosa, Legionella pneumophila,
Neisseria gonorrhoeae, and Vibrio cholerae. Type IV pili are thin usually 6-8 nm wide,
flexible, several micrometers long, and aggregates the forming of bundles. The
biogenesis of Type IV pili requires specific proteins, including: the major pilin subunit
(PilA); a specific inner-membrane prepilin peptidase that cleavages the signal peptide
(PilD); an ATPase that powers the assembly of the pilus (PilB); an inner-membrane
30 protein that recruits the ATPase from the cytoplasm (PilC); and an outer-membrane
secretin that is needed for the pili to emerge on the surface of the bacterium (PilQ)
(Mattick J. S., 2002., Proft T., et al., 2009, Strom, M. S. and Lory, S., 1993., Wolfgang,
M., et al., 2000). Since it is commonly found on pathogenic bacteria, pili are important
targets for vaccine development. Many pili vaccines have been developed and tested
against various Gram-negative microorganisms and have been shown to be highly
effective in reducing infection and disease ( Ellemant C. & Stewartd J. 1988).
31 CHAPTER II: MUTAGENESIS OF C. JEJUNI 1343C GENE AND ITS ROLE IN
POULTRY COLONIZATION
I. Abstract
Campylobacter spp. is one of the two major causes of foodborne illness
throughout the world. Campylobacter accounts for the most common causes of diarrheal
illness caused by bacterial pathogens worldwide and in the United States. The FoodNet
indicates that approximately 13 cases are diagnosed each year for each 100,000 persons
in the population. However, many more cases are undiagnosed or unreported. It is
estimated that Campylobacter diarrheal illness affects about 2.4 million persons every
year with an estimated cost of treatment and loss of productivity exceeding $1 billion
annually. Previous work in our laboratory on biofilms has demonstrated the presence of
fibers (pili) disseminating from the cell wall of C. jejuni isolates not expressing flagella
(flaAB mutants). To further investigate this finding, annotation analysis of C. jejuni strain
was initiated and a gene (Cj1473c) with nucleotide sequence homology to the pilT gene
present in several bacterial genomes was identified (Table 1A). In the structure of a type
IV pilus, PilT is the retraction ATPase that aids removal of pilin subunits from the base
and with the pilin subunits being transferred to the inner membrane. In addition another
gene (Cj1343c) was identified having sequence homology to pilA gene in several
bacterial genomes. These searches revealed that putative periplasmic protein encoded by
the Cj1343c gene is homologous to several proteins involved in type II secretion systems,
biogenesis of type IV pili, and competence for natural transformation in Gram-positive
and Gram-negative species. Moreover Cj1343c contains homology to a conserved
32 consensus sequence found in PilA proteins, which forms the core of the pilus fiber in
bacteria. Based on these findings we hypothesized that C. jejuni produces pili and that
Cj1343c is involved in the production of pilus-like appendages of pili in C. jejuni. This
chapter will cover the following aims:
1. Further analyze the production of pilus-like appendages in C. jejuni in
various culture conditions (broth, and biofilms) through scanning electron
microscopy (SEM)
2. Determine the role of Cj1343c gene in expression of pilus fibers
3. Characterize the role of Cj1343c in poultry colonization
Results indicated that C. jejuni pilus-like appendages disseminating from the cell wall of
the bacterium in various culture conditions. Upon mutation of Cj1343c, biofilms were
analyzed through SEM, micrographs revealed the presence of pilus-like appendages
disseminating from the cell wall of the mutant strain. Lastly, our results indicated that
Cj1343c is not involved in poultry colonization. Further analysis needs to be performed
in order to purified and identified putative genes involved in the production of the
surface-associated structures seen in C. jejuni.
II. Materials and Methods
A. Culture of Bacterial Strains
All C. jejuni strains used in this study were routinely grown on Mueller Hilton
agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Plates were
supplemented with 5% citrated bovine blood (MHB; Quad Five, Ryegate, MT, USA) at
37°C in an environment supplemented with 10% CO2. When needed media was
33 supplemented with antibiotics at the following concentrations: chloramphenicol 15
μg/ml, and kanamycin 50 μg/ml. All E. coli strains used in this study were grown on
Luria-Bertani (LB) agar (BD, Sparks, MD) at 37°C; when needed, media was
supplemented with antibiotics at the following concentrations: chloramphenicol 15
μg/ml, kanamycin 50μg/ml and ampicillin 100 μg/ml. All strains and plasmids as well as
primers used in this study are listed in Table 2A and Table 3A respectively.
B. Biofilm formation
The flaAB mutant was grown overnight in Mueller-Hinton broth (MHB) with
shaking at 37oC with 5% CO2 to an OD600 of 0.8-1.0. Sterile glass cover slips were
mounted in a 12-well polystyrene plate in which each well contained 1 ml sterile MHB,
which was inoculated with the overnight flaAB mutant culture with a starting OD600 of
0.05. The plate was incubated at 37oC with 10% CO2 for 24 hours. Cover slips coated
with biofilm were removed and prepared for SEM by rinsing with water, fixing in
phosphate buffered 2.5% glutaraldehyde for 3 hours and finally rinsing with phosphate
buffer.
The
cover
slips
were
reacted
to
an
ethanol
series
followed
by
hexamethyldisilazane in order to dehydrate the sample. Then, the biofilms were coated
with chromium, and observed using an Ultra-High Resolution Field Emission-SEM S4800 (University Spectroscopy and Imaging Facilities, University of Arizona).
C. Scanning Electron Micrograph
Cover slips coated with biofilm were removed and prepared for SEM by rinsing
with water, fixing in phosphate buffered 2.5% glutaraldehyde for 3 hours and finally
rinsing with phosphate buffer. The cover slips were reacted to an ethanol series followed
34 by hexamethyldisilazane in order to dehydrate the sample. Then, the biofilms were coated
with chromium, and observed using an Ultra-High Resolution Field Emission-SEM S4800 (University Spectroscopy and Imaging Facilities, University of Arizona).
D. Generation of C. jejuni Cj1343c suicide vector
C. jejuni total DNA was extracted using the DNeasy Blood and Tissue (Qiagen,
Valencia, CA). For the inactivation of the Cj1343c gene primers were designed to
amplify approximately 1000-bp upstream and downstream regions of the target gene,
such that the whole open reading frame was omitted. The target DNA was amplified via
PCR, and the products were visualized by agarose gel electrophoresis. Bands of the
correct size were excised from the gel and purified using a gel extraction kit (Qiagen,
Valencia, CA) and confirmed by sequence analysis (University of Arizona Genetics
Core). The PCR products were inserted separately into a pCR2.1-TOPO vector following
the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Thereafter, one fragment was
cloned into the pCR2.1 vector harboring the other fragment, and a chloramphenicol
resistance cassette was inserted between the two flanking regions at the primer built NheI
site. The resulting plasmid contained upstream and downstream flanking regions of target
gene. The presence and correct orientation of the inserts were confirmed by submitting
the plasmid for sequencing analysis (University of Arizona Genetics Core). The
chloramphenicol acetyltransferase gene (cat; chloramphenicol resistance, CmR) was
amplified by PCR from the plasmid pUOA23 yielding a fragment of approximately 800bp. To confirm that the chloramphenicol cassette was inserted between the upstream and
downstream regions of the target gene, a PCR assay was performed on the
35 chloramphenicol-resistant colonies. The insertion of the cat gene yielded a fragment that
was 800-bp larger than that of the plasmid containing both upstream and downstream
regions of the target gene alone. Primers used in this study are listed in Table 4.
E. Generation of C. jejuni Cj1343c mutant
Plasmid DNA containing flanking DNA and cat resistance cassette was
electroporated into C. jejuni NCTC 11168. Electrocompetent cells and electroporation of
C. jejuni NCTC 11168 were made as previously described (DiRita et al., 2008).
F. Poultry colonization assays
Chicken studies were performed in accordance with the Institutional Animal Care
and Use Committee of the University of Arizona (IACUC). One-day old chicks were
obtained from a commercial hatchery (Murray McMurray, Webster City, Iowa). To
determine if the birds were free of C. jejuni, at arrival the chicks were swabbed and direct
plated onto a MHB plates. The birds were housed in individual round ½” pegboard
brooders; each brooder with enough space to provide ~1 ft2 per bird. Birds were housed
on fine wood shaving litter, which were changed on a weekly basis or more frequently, if
required. The studies were performed under controlled temperatures. All chickens were
fed a commercial chick starter mash for the entire study; feed and water were placed in
large automatic feeders allowing birds from each group to feed and drink ad libitum.
Birds had the automatic feeders filled or topped off on a daily basis, and waterers were
cleaned and filled on a daily basis. On the day of challenge, eight hours prior to
challenging the chickens, feed was removed, and the feed was returned to the birds one
hour after challenge. Chickens were orally challenged with C. jejuni mutant or NCTC
36 11168 wild-type strain (positive control) at 14 days of age; birds were challenged by
gavage with 1 ml of a C. jejuni suspension (105 cfu/ml) in phosphate buffered saline
(PBS). A negative control group was challenged with PBS only. Chickens were
euthanized by carbon dioxide asphyxiation and necropsied at 25 days of age. Each cecum
were collected, processed, the feces serially diluted, and direct plated to quantify the
presence of C. jejuni in each bird. There were 15 chickens per group (n=10) and
experiments were performed in triplicate to confirm the statistical significance of each
designed study.
III. Results:
A. Detection of pilus-like appendages via scanning electron microscopy (SEM).
The production of pilus-like appendages in C. jejuni in various culture conditions
(broth, and biofilms) was investigated using scanning electron microscopy (SEM). By
culturing C. jejuni on glass coverslips contained within a 24-well tissue culture plate it
was possible to recover the biofilm growth for high-resolution SEM analysis. Using SEM
it was found that the bacteria within the static culture (Figure 2A) produced numerous
pilus-like appendages. To determine that the fibers disseminating from the cell wall of C.
jejuni were not flagella, scanning electron microscopy studies were performed on
biofilms and broth cultures from C. jejuni NCTC 11168 flaAB mutants.
B. Cj1343c is not involved in production pilus-like of appendages in C. jejuni.
Based on bioinformatics analysis we hypothesized that gene Cj1343c was involved
in the production of the pilus-like appendages previously described. However, after
generating a Cj1343c isogenic mutant, scanning electron microscopy still showed the
37 presence of pilus-like appendages disseminating from the cell wall of the mutant Figure
3A.
C. Cj1343c is not required for poultry colonization.
In this study we evaluated the role of Cj1343c gene in C. jejuni poultry
colonization and found that the deletion of Cj1343c had no negative effect on the ability
of C. jejuni to colonize poultry. Birds were divided in to groups of 10. At day 14 the birds
were orally inoculated with C. jejuni NCTC 11168 and C. jejuni ΔCj1343c. The ability
of both strains to colonize the chicks was assessed by enumeration of bacteria in the ceca
10 days post-inoculation. The NCTC 11168 parent strain used in our study was able to
colonize chicks at a level of 1 x 108 CFU per g of cecal content with an inoculum of 105
CFU. The C. jejuni Cj1343c mutant exhibited no significant difference in colonization
numbers when compared to the wild-type strain Figure 4A (p > 0.05).
IV. Discussion
Adhesion by bacterial pathogens is often mediated by fimbrial or pili structures.
In this study we have found that C. jejuni flagella deficient strains express pilus-like
appendages when grown in different culture conditions. C. jejuni was long thought to not
produce fimbriae or pili, but one unconfirmed report described the production of
fimbrial-like appendages by C. jejuni and C. coli when the bacteria were grown in the
presence of bile salts (Doig, P., 1996). However, five years later it was reported that those
appendages were not pili, but instead were a bacteria-independent morphological artifact
of the growth medium (Gaynor E. C., 2001). As of today no data exists to support that C.
38 jejuni produces pili; here we provided evidence that C. jejuni produces pilus-like
appendages disseminating from the cell wall of C. jejuni in various culture conditions.
Subsequent BLAST searches found that a putative periplasmic protein encoded by
the Cj1343c gene was homologous to several proteins involved in type II secretion
systems, biogenesis of type IV pili, and competence for natural transformation in Grampositive and Gram-negative species. C. jejuni 1343c gene (Cj1343c) encodes a protein
that is 171 amino acids in length; with a its predicted size of 18-kDa. BLAST searches
indicate that Cj1343c appears to be conserved among different C. jejuni strains, including
a C. jejuni RM1221 poultry isolate, a C. jejuni NCTC 11168 human isolate, and a C.
jejuni S3 poultry isolate. Analysis of the location of this gene in the Campylobacter
genome using the NCBI genome sequence tool revealed that Cj1343c is in a putative
operon Cj1340c-Cj1347c. All of these genes are located in the negative strand. There is a
9bp gap between Cj1347c and Cj1348c.
The SignalP online tool was used to predict the presence and location of putative
signal peptide cleavage sites. A signal peptide was found by the Cj1343c sequence with a
signal peptide cleavage site predicted to be located between amino acid positions 21-25
(LAAIAL). Based on this finding this particular gene was predicted to encode a putative
periplasmic protein and was known to have some similarity to the N-terminus of
secretion proteins, such as the E. coli putative general secretion pathway proteins. The
Conserved Domain Database (CDD) was used to search for conserved domains within
the putative periplasmic protein encoded by the Cj1343c gene; however, no conserved
domains were found.
39 Further analysis of the Cj1343c signal peptide sequence revealed that it was
homologous to signal peptides found in the PilA protein of various bacteria. These signal
peptides are primarily composed of a single small protein subunit, usually termed PilA or
pilin, which are arranged in a helical conformation with 5 subunits per turn and which
may be glycosylated, and/or phosphorylated in different species. Pilins from different
species are usually 145-160 amino acids in length and have a short positively charged
leader sequence, and a conserved and hydrophobic amino-terminal domain with a
consensus sequence (FTLIELMIVVAIIGILAAIALPAYQDYTARSQ), which forms the
core of the pilus fiber (Mattick J. S., 2002). Sequence alignment revealed that Cj1343c
signal peptide sequence has a 68% identity to the consensus sequence found in pilA. A
diagram of the sequence alignment can be found in Figure 1A.
Pili have been implicated in crucial host–pathogen interactions such as
colonization, biofilm formation, invasion and attachment, signaling events, phage
transduction and DNA uptake. These finding were very exciting because in a previous
study, transposon-generated mutants were analyzed for identification of genes involved
in natural transformation in C. jejuni (Wiesner R. S., et al., 2003). Briefly, a solo
transposon mutant library of C. jejuni 81-176 was created. Eleven mutants were
identified as having a reduction in the ability of C. jejuni to be transformed by 1,000-fold.
Among those genes were Cj1343c and Cj1475c, including those in the putative operon
(Cj1470c, Cj1471c, Cj1472c, Cj1473c, and Cj1474c) in which Cj1475 is located. Based
on these findings, we hypothesized that Cj1343c was involved in the production of piluslike appendages seen in C. jejuni.
40 Despite the involvement in transformation, Cj1343c having homology to the pilA
consensus sequence commonly found in bacteria that produces pili and been identified
from purified fibers been produced by C. jejuni, Cj1343c is not involved in the
production of those appendages observed previously as demonstrated by the presence of
pilus-like appendages in the mutant strain. Many Gram-negative organisms have an
absolute requirement for type IV pili and the type II secretion system, the fact that no
type II secretion system have been describe in C. jejuni could explain the lack of a type
IV pilus in the bacterium.
In addition, we investigated whether the mutation of Cj1343c had a role in
commensal colonization of broiler chickens. Cj1343c was found not to be involved in
commensal chicken colonization, as demonstrated by the ability of the mutant strain to
colonize broilers chickens to that of wild type levels. Further analysis of this mutant
needs to be performed in order to determine what role it might have in other virulence
assays such as biofilm, agglutination, attachment and invasion. To further investigate and
identify which elements are involved in the production of pilus-like appendages in C.
jejuni, we continued studies in order to find alternated approaches that will facilitate the
purification of these appendages. These are described in the next chapter.
41 V. Chapter II Tables and Figures
Table 1A. Cj1373c nucleotide sequence similarity to the pilT gene present in several
bacterial genomes
Bacterium
Pseudomonas fluorescens
Protein encoded by homologous gene
Pilin biogenesis protein PilT
Methylococcus capsulatus strain Bath
Twitching motility protein PilT
Synechoccus sp. RCC307
Type IV pili assembly protein, pilus retraction ATPase PilT
Rubrobacter xylanophilus DSM 9941
Pilus retraction ATPase PilT
Desulfovibrio vulgaris strain ‘Miyazaki F’
Twitching motility protein
Table 2A. Strains and plasmids used in this study
Strain or plasmid
Bacteria
C. jejuni NCTC 11168
TOP10
Plasmids
pCR2.1-TOPO
pCR1343c
pUOA23
Characteristic
Source
Clinical isolate
F- mcrA Δ(mrr-hsdRMSmcrBC) Φ80lacZΔM15
ΔlacΧ74 recA1 araD139
Δ(araleu) 7697 galU galK rpsL
(StrR) endA1 nupG
This study
Invitrogen
Cloning vector
Contains both flanking regions
and CmR marker
Contains C. coli plasmid C-589
chloramphenicol
acetyltransferase (cat) gene
Invitrogen
This study
Dr. Van der Walls
Table 3A. Oligonucleotides used in this study
Primer Sequence
Sequence (5’ > 3’)
Restriction site
Cj1343c-F1
Cj1343c-R1
Cj1343c-F2
Cj1343c-R2
Cat-F1
Cat-R
acactgGAGCTCataaaaaaatttcccaagaa
aaggatatagatgaaaaaagGCTAGCgaagga
gttgtgGCTAGCggaatgtaatgatagtgagt
cttgagaaacttggttttgaGTCGACattcct
atGCTAGCtaacccattgctcggcggtgttcctttcca
tcgcactgataaaaaccctttaggaaGCTAGCaa
SacI
NheI
NheI
SalI
NheI
NheI
42 Figure 1A. Sequence alingment of the conserved, highly hydrophobic amino-terminal
domain consensus sequence commonly found in PilA proteins against Cj1343c signal
peptide sequence. The asterisks represents the exact match between two amino acids (the
identity).
43 Figure 2A. Scanning electron micrograph of putative pili disseminating from the cell of
the C. jejuni strain NCTC 11168 flaAB mutant in different growth conditions. A.) Biofilm
24-hour. B.) Broth culture 24-hour. Arrows indicates putative pili.
Figure 3A. Scanning electron micrograph of Cj1343c mutant. A.) Biofilms of C. jejuni
ΔCj1343c. B). Biofilms of C. jejuniΔflaAB.
44 2.56E+09 1.71E+08 Log 10 CFU/g 1.14E+07 7.59E+05 5.06E+04 3.38E+03 2.25E+02 1.50E+01 1.00E+00 NCTC WT NCTC::Cj1343c Figure 4A. Effects of ΔCj1343c mutation on in vivo poultry colonization. Error bar
indicates standard deviation, p > 0.05, n = 10 chickens per strain.
45 CHAPTER III: PURIFICATION OF SURFACE-ASSOCIATED MOLECULES
FROM C. JEJUNI
I. Abstract
It is well known that bacteria first need to attach and later be internalized in order
to establish an infection. C. jejuni’s ability to attach, and survive intracellularly has been
studied. Campylobacter possess several virulence factors; however, only a few genes
have been associated with C. jejuni virulence, such as those involved in adherence,
invasion, and motility. We have previously shown pilus-like appendages disseminating
from the cell wall of C. jejuni. Based on those results we initiated a bioinformatics
analysis of the Campylobacter genome. We identified a gene, Cj1343c, with homology to
pilA genes from several bacterial genomes. Upon mutation of the Cj1343c gene, we
determined that it was not involved in the production of a pilus-like appendage
previously shown by scanning electron micrograph of flagella deficient strains of C.
jejuni growing on biofilms. This study was initiated in order to find alternate approaches
that will allow us to purify and identified proteins involved in the production of surfaceassociated structures. We used transmission electron microscopy to examine the fine
structure of purified cells grown in biofilms. Since pili range in diameter from 2-10 nm,
transmission electron microscopy is the best approach to directly view theses structures.
Negative staining and TEM is widely used to identify surface structures on bacterial cells,
and allows physical characteristics such as size, and shape to be determined. This chapter
will address the following aims:
1. Purification of fibers reminiscent of pili
46 2. Identify genes involved in the expression fibers reminiscent of pili
We were able to isolate C. jejuni surface-associated structures from C. jejuni by using
mechanical shearing and differential centrifugation and we were able to visualize them
through TEM. Mass spectrometry assays resulted in the identification of two genes
involved in the expression of surface-associated structures genes in C. jejuni. Here we
provide data that supports the idea that C. jejuni produces pili, and this might have a
significant role in understanding the pathogenesis of C. jejuni. Moreover, it will provide a
new target for potential vaccine development against C. jejuni infection in poultry
production system. Thus reduction of C. jejuni in chickens will directly impact the
number of C. jejuni infection in humans.
II. Materials and Methods:
A. Culture of Bacterial Strains
All C. jejuni strains used in this study were routinely grown on Mueller Hilton
agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Plates were
supplemented with 5% citrated bovine blood (MHB; Quad Five, Ryegate, MT, USA) at
37°C in an environment supplemented with 10% CO2. Selective media was supplemented
with antibiotics at the following concentrations: kanamycin 50 μg/ml. All strains and
plasmids used in this study are listed in Table. 4B.
B. Purification of fibers reminiscent of pili
Surface-associated molecules were purified as follows: in brief, C. jejuni strain
NCTC 11168 flaAB mutant was grown overnight in one liter of Mueller-Hinton broth
(MHB) with shaking at 42oC with 10% CO2. Surface structures were mechanically
47 sheared from the bacterial surface by vigorous vortexing for 5 min. After shearing, the
bacterial suspensions were centrifuged at 10, 000 x g for 10 min, and both the supernatant
and pellets were collected. Bacterial pellets were washed with 10ml of 10mM Hepes.
Cells were lysed by French pressure cell disruption (as described below). Surface
structures were collected by ultracentrifugation at 100, 000 x g for 1 h at 4°C (Beckman,
SW40 rotor). The pellet and the supernatant were re-suspended in 2 ml of 10 mM
HEPES, pH 7.4, using an 18- gauge needle. The pellet was washed in a total volume of
10 ml of 10 mM HEPES, pH 7.4, and spun again in the ultracentrifuge, using the
conditions described above. The pellet and the supernatant were re-suspended in 5 ml 1
% (w/v) N- lauroylsarcosine (Sarkosyl) (Sigma) in 10 mM HEPES, pH 7.4, and
incubated at 37°C for 30 min with shaking. The Sarkosyl-treated samples were spun at
100, 000 x g for 1 h at 4°C (Beckman, SW40 rotor) and the pellet washed with 10 ml of
10 mM HEPES, pH 7.4. Following the final ultracentrifugation, the pellet was resuspended in 500 μl 10 mM HEPES, pH 7.4. Samples were treated with a cocktail of
proteases inhibitor to prevent protein degradation.
C. Transmission Electron Micrograph (TEM)
10 μl of the resulting pellet containing isolated fibers reminiscent of pili were
pippetted onto Formvar coated 200 mesh copper grids for 1 minute. The grids were then
negatively stained using 1% phosphotungstic acid. Photographic images were made at
various instrumental magnifications to resolve the details of filaments and relative
diameter of fibers reminiscent of pili were measured for comparison.
D. French pressure cell disruption
48 C. jejuni strain NCTC 11168 was lysed by passing the culture twice through a
French press (Thermo Electron Corporation) at 1000 p.s.i. (6.9 MPa; 40K cell). The lysed
cell preparation was centrifuged at 10, 000 x g for 10 min at 4°C to remove cell debris
and unlysed cells (Hobb R. I. et al. 2009).
E. Tricine-SDS-PAGE:
Sample concentrations were adjusted so that a suitable amount of protein could be
loaded onto the gel and meet the minimal staining concentration to view the proteins
(Syrovy, L. and Hodny, Z., 1991). Pre-casted 10–20% resolving Tricine-SDS-PAGE gels
were used for this analysis (Bio-Rad, Hercules, CA). Samples were mixed with sample
buffer and boiled for 10 min. The gel was mounted in a vertical electrophoresis apparatus
(Mini-Protean 3, Bio-Rad), and 1X running buffer was added. The running conditions
were set as follows: initial voltage of 30V was used until the sample had completely
entered into the stacking gel. Then the voltage was increased to 190V until the sample
was at the end of the gel. The gel was then fixed for 15 minutes (50% Methyl Alcohol,
10% Acetic Acid, 100mM Ammonium acetate) and the proteins visualized by staining
with Coomassie dye in 10% acetic acid for 30 minutes. The gel was then de-stained twice
in 10% acetic acid for 1 hour. Finally, the gel was washed several times in D2H2O
(Schagger H., 2006).
F. Trypsin Digestion
After washing the gel in double sterile water (dsH2O) for 15 minutes bands of
interest were excised and placed in acetonitrile (ACN): dsH2O (50/50) for 15 minutes.
After treatment with the ACN solution, 100mM of Ambic was added to the gel for 5
49 minutes, which was followed by a 1:1 solution of ACN for another 15 min. The solution
was removed and samples were dried in a speed-vacuum for 15 min. Trypsin digestion
was then initiated by adding trypsin (400 ng of trypsin per band) followed by an
incubation step for 45 min on ice. The trypsin solution was removed and discarded and
digestion buffer without trypsin was added to the samples and the band were added and
incubated overnight.
G. Mass spectrometry
The mass spectrometry work was performed by the University of Arizona core
facility (Arizona Proteomics Consortium, University of Arizona).
III. Results
A. TEM analysis of C. jejuni cells
To ensure that structures observed through transmission electron microscopy were
not flagella, the C. jejuni NCTC flaAB mutant was used for TEM analysis. First, flaAB
mutants were grown in MH broth at 42°C with shaking for 24 h and subjected to
mechanical shearing, pelled by centrifugation and later analyzed by TEM, Figure 5B.
Experiments were repeated twice.
B. SDS-PAGE of isolated surface molecules in C. jejuni
In order to identify putative proteins that might be involved in the production of
surface-associated molecules in C. jejuni, mechanically isolated fibers were separated by
SDS-PAGE and stained. These experiments demonstrated the presence of proteins that
ranged in size between 25-10 kDa in both the pellet and supernatant fractions, Figure 6B.
The proteins of interest were excised from the gel and treated with a trypsin digest and
50 later analyzed using mass spectrometry. Experiments were repeated twice.
C. Mass spectrometry analysis
Bands of interest were excised from the gel and then trypsin digested for the
generation of tryptic peptides. Samples were then analyzed by mass spectrometry.
Generated peptides were analyzed by Scaffold 3 software, which identifies proteins by
validating data from multiple search engines. Experiments were repeated twice. Proteins
that were then identified through MS and their gene products, molecular weight,
subcellular location, and predicted function summarized in Table 5B.
IV. Discussion
This study was initiated based on the previous findings in which C. jejuni flaAB
mutants showed pilus-like surface-associated structures, when grown in both broth and
biofilms. We decided to concentrate on methods that would allow us to purify and
visualize pilus-like surface-associated structures observed in C. jejuni. Bacteria have to
attach to specific host cells as a crucial step in establishing an infection. This process is
necessary for colonization of host tissue and is mediated by surface-exposed adhesins.
Basically bacteria face net repulsive forces during colonization caused by the negative
charges of both the bacterium and the host cell. This can be overcome by a cell surface
structures in which the adhesin is located at the tip of hair-like, peritrichous, nonflagellar, or filamentous surface appendages known as pili. Pili are important virulence
factors for several diseases, in particular infections of the urinary, genital and
gastrointestinal tracts (Proftac T. and Bakerb E., 2009). Pili are polymeric, hydrophobic,
filamentous appendages, which extend several micrometers from the bacterial surface.
51 Pili are generally composed of a major repeating subunit called pilin and, in some cases,
a minor tip-associated adhesin subunit.
We isolated C. jejuni surface-associated structures by using mechanical shearing
and differential centrifugation techniques commonly used to prepare pili from other
microorganisms. Over the past five decades, several distinct pilus types have been
identified, most of which were described and characterized in Gram-negative bacteria.
The best characterized pilus structures include; type I pili (Olsen, A., 1989, Kikuchi, T.,
2005) (expressed by enteropathogenic Escherichia coli), type IV pili (Hahn, E., et al.,
2002, Craig, L., 2004) (expressed by E. coli, and Pseudomonas and Neisseria species)
and curli pili (Craig, L., et al 2004, de Jong A. E., et al., 2008). Type I pili appear as
peritrichous, rigid, rod-like structures of 1–2 μm in length, and they have a visible
flexible tip that is known to be involved in bacterial interaction with receptors on the
host-cell surface. Type IV pili are a similar length, but they appear to be more flexible
and often form bundles at polar locations when compared to Type I pili. Curli pili are, as
their name suggests, coiled structures. All three pilus types are formed through the noncovalent association of pilin subunits into regular, polymeric structures.
The pilus-like surface-associated structures produced by C. jejuni range from 2-3
nm in diameter and appear to be flexible, long filaments that extend from the cell surface
several micrometers. We included a C. jejuni strain expressing flagella and measurements
revealed that flagella were about 10-12 nm wide, thus facilitating the identification of
pilus-like surface-associated structures. Based on diameter, length, and morphology we
hypothesized that indeed C. jejuni produces a type of pili. To further analyze these
52 structures, mechanically sheared fibers were subjected to SDS-PAGE analysis. Pili in
Gram-negative bacteria are typically formed by non-covalent homopolymerization of
major pilus subunit proteins called pilins, which generates the pilus shaft. The pilus shaft
is composed of several hundred (probably thousands) of small 15 – 25 kDa subunits or
pilins. After analysis by SDS-PAGE and Coomassie brilliant blue staining procedures,
bands that were in the range typical for pilins, 10–25 kDa were observed. Bands were
excised from the gel and analyzed by mass spectrometry. Four proteins were identified
twice by MS analysis and are listed in Table 1. Bioinformatics analysis showed no
sequence homology of proteins identified by MS to any known pili proteins. However, it
is possible that C. jejuni produces a unique type of pili that have not have been previously
described. Here we provide data that supports the idea that C. jejuni produces pili, and
this might have a significant role in understanding the pathogenesis of C. jejuni.
53 V. Chapter III Tables and Figures
Table 4B. Strains used in this study
Strains
Bacteria
C. jejuni NCTC 11168
C. jejuni ΔflaAB
Characteristic
Source
Clinical isolate
C. jejuni strain not expressing
genes flaA and flaB
This study
Joens
Table 5B. Identification of proteins involved in the production of surface-associated
molecules identified by MS
Gene
Cj0113
Predicted
Function
peptidoglycan
associated
lipoprotein
(omp18)
Cj0998c
putative
periplasmic
protein
Cj0091
putative
lipoprotein
Cj0248
hypothetical
protein
MW
(kDa)
15
21
22
32
Signal
Sequence
Yes
Lipoprotein
signal
peptide
Yes
Yes
No
Predicted
cleavage
site
Predicted
N-terminal
sequence
Fingerprint:
Motif
Helix
Subcellular
location
MKKILFT
SIAALAV
VISGC
NKVYFDFDK
FNIRPDMQN
VVST
ADRIAVKSY
GETNPVCTE
KTKACD
one transmembrane
helix
Outer
Membrane
LLA-SA
MKKILVS
VLSSCLL
ASALS
LASYLKGAK
ATIKPSNAF
MG
ISAKITMDK
KSTIVP
one transmembrane
helix
Unknown
AQT-AY
MKKTKIL
GTALIGA
LLFSGC
one transmembrane
helix
Unknown
three
transmembrane
helices
Unknown
ALA-VV
N/A
MIGDMN
ELLLKSV
EVLPPLP
N/A
VSKLRKYVS
EANSNIETM
KVAEII
LLGVGNIINI
VMADSIRDN
FKIDV
A.)
B).
54 55 C.)
D.)
Figure 5B. Transmission electron micrograph of purified surface molecules from C.
56 jejuni A.) TEM showing long filamentous appendages extracted from C. jejuni flaAB
mutants. B.) TEM showing fragmented appendages extracted from C. jejuni flaAB
mutants. C.) TEM showing fragmented appendages extracted from C. jejuni flaAB
mutants and their sizes. D.) TEM showing extracted C. jejuni NCTC flagella and their
sizes.
Figure 6B. Coomassie-stained SDS-PAGE gel containing pellet and supernatant of
isolated surface molecules isolated form C. jejuni flaAB mutant. Arrows indicate bands
that were excised for MS analysis.
57 CHAPTER IV: MUTAGENESIS OF PUTATIVE GENES EXPRESSING
SURFACE-ASSOCIATED STUCTURES ISOLATED FROM C. JEJUNI AND
THEIR ROLE IN C. JEJUNI PATHOGENESIS
I. Abstract
Previously we have identified genes that might be involved in the production of
pilus-like surface-associated structures previously shown in C. jejuni. TEM analysis
revealed surface-associated structures appearing to be long and flexible filaments that
extend from the cell surface forming a mesh-like structure. Fibers reminiscent of pili
produced by C. jejuni range from 2-3 nm in diameter and several micrometers in length
consistent with known pili that have previously described in the literature. Pili, as
adhesive organelles, have been implicated in other functions, such as phage binding,
DNA transfer, biofilm formation, cell aggregation, host cell invasion and twitching
motility. This chapter addressed the following aims:
1.
Determine the role of putative genes (Cj0248, Cj1475c and Cj0998c) in
expression of fibers reminiscent of pili. Isogenic mutants were made and
examined by scanning electron microscopy (SEM) to confirm the loss of pili.
2.
Determine the role of isogenic mutants in the intestinal poultry colonization.
3.
Determine the role of isogenic mutants in biofilm production, attachment and
invasion assays, agglutination assays, and macrophage survival.
Results indicated that genes Cj0248 and Cj1475c were not involved in the expression of
surface-associated structures seen in C. jejuni. Poultry colonization studies revealed that
Cj0248 and Cj1475c are important colonization factors of C. jejuni. Biofilms studies
58 revealed that Cj0248 is involved in initial biofilm formation; Cj1475c is not involved C.
jejuni biofilm formation. Cj1343c showed an increased biofilm formation, thus Cj1343c
either represent a global regulator of transcription of genes involved in biofilm formation
or either represents the major constituent of the biofilm in C. jejuni. Attachment and
invasion assays revealed that Cj0248 not require for attachment to INT407 cells but is
require for internalization into cells. Here we have identified two genes required for cecal
colonization, leading to commensalism in the chick gastrointestinal tract. Further analysis
is needed in order to elucidate the exact role of Cj1343c in biofilm formation. These
studies provide insight new colonization factors in pathogenesis of C. jejuni. Moreover, it
provides new target for potential vaccine development against C. jejuni infection. Thus
reduction of C. jejuni in chickens will directly impact the number of C. jejuni infection in
humans.
II. Materials and Methods
A. Culture of Bacterial Strains
All C. jejuni strains used in this study were routinely grown on Mueller Hilton
agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Plates were
supplemented with 5% citrated bovine blood (MHB; Quad Five, Ryegate, MT, USA) at
37°C in an environment supplemented with 10% CO2. Selective media was supplemented
with antibiotics at the following concentrations: chloramphenicol 15 μg/ml, and
kanamycin 50 μg/ml. All E. coli strains used in this study were grown on Luria-Bertani
(LB) agar (BD, Sparks, MD) at 37°C; selective media was supplemented with antibiotics
at the following concentrations: chloramphenicol 15 μg/ml, kanamycin 50 μg/ml and
59 ampicillin 100 μg/ml. All strain and plasmids used in this study are listed in Table 6C.
Primers used in this study are listed in Table 7C.
B. Generation of isogenic mutants
C. jejuni total DNA was extracted using the DNeasy Blood and Tissue extraction
kit (Qiagen, Valencia, CA). For the inactivation of the genes, primers were designed to
amplify approximately 1000-bp upstream and downstream regions of the target gene,
such that the whole open reading frame was omitted. The target DNA was amplified via
PCR, and the products were visualized by agarose gel electrophoresis. Bands of the
correct size were excised from the gel and purified using a gel extraction kit (Qiagen,
Valencia, CA) and confirmed by sequence analysis (University of Arizona Genetics
Core). The PCR products were inserted separately into pCR2.1-TOPO vector following
the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Cells were screened using
pre-warmed selective ampicillin plates (50 μg/ml), supplemented with X-gal for
blue/white screening. In order to build a pCR2.1 plasmid that contains both upstream and
downstream sequences of the target gene we digested each plasmid separately. The
pCR2.1 vector containing each separate gene fragment was digested with the same set of
enzymes. Digests were separated by electrophoresis and the proper sized bands were
excised from the gel and purified as previously describe. The DNA were then ligated and
electroporated into E. coli DH5α (0.1-cm electroporation cuvette and pulsed 1. 8 kV, 50
μF, 200Ω) with a Gene Pulser (Bio-Rad). After recovery in 1mL of SOC media (1 h,
37°C, with shaking), the bacteria were harvested and plated onto pre-warmed selective
LB agar plates containing ampicillin (50 μg/ml). The presence and correct orientation of
60 the gene inserts were confirmed by submitting the plasmid for sequencing analysis
(University of Arizona Genetics Core). The chloramphenicol acetyltransferase gene (cat;
chloramphenicol resistance, CmR) was amplified by PCR from the plasmid pUOA23
yielding a fragment of approximately 800-bp. For the inactivation of the target gene, the
cat resistance cassette was cloned into the NheI restriction site of the plasmid containing
the flanking regions of the target gene. After ligation, the plasmid was electroporated into
electrocompetent E. coli INVαF’ as described above. Chloramphenicol-resistant colonies
were screened on chloramphenicol LB agar plates (15 μg/ml). To confirm that the
chloramphenicol cassette was inserted between the upstream and downstream regions of
the target gene, a PCR assay was performed on the chloramphenicol-resistant colonies.
The insertion of the cat gene yielded a fragment that was 800-bp larger than the plasmid
containing both the upstream and downstream regions of the target gene alone. Plasmid
DNA containing flanking DNA and cat resistance cassette was electroporated into C.
jejuni NCTC 11168. Electrocompetent cells and electroporation of C. jejuni NCTC
11168 were made as previously described (DiRita et al., 2008).
C. SEM of biofilms
The Cj0248 and Cj1475c mutants were grown overnight in Mueller-Hinton broth
(MHB) with shaking at 37oC with 5% CO2 to an OD600 of 0.8-1.0. Sterile glass cover
slips were placed into a 12-well polystyrene plate in which each well-contained 1 ml
sterile MHB, inoculated with an overnight C. jejuni flaAB mutant culture having an OD600
of 0.05. The plate was incubated at 37oC with 10% CO2 for 24 hours. Cover slips coated
with biofilm were removed and prepared for SEM by rinsing with water, fixing in
61 phosphate buffered 2.5% glutaraldehyde for 3 hours and finally rinsing with phosphate
buffer.
The
cover
slips
were
reacted
to
an
ethanol
series
followed
by
hexamethyldisilazane in order to dehydrate the sample. Then, the biofilms were coated
with chromium, and observed using an Ultra-High Resolution Field Emission-SEM S4800 (University Spectroscopy and Imaging Facilities, University of Arizona).
D. Poultry colonization assays
Chicken studies were performed in accordance with the Institutional Animal Care
and Use Committee of the University of Arizona (IACUC). One-day old chicks were
obtained from a commercial hatchery (Murray McMurray, Webster City, Iowa). To
determine if the birds were free of C. jejuni at arrival, the chicks were swabbed and the
fecal sample direct plated onto a Cefex plate. The birds were housed in individual round
½” pegboard brooders; each brooder containing enough space to provide ~1 ft2 per bird.
Birds were housed on fine wood shaving litter, which were changed on a weekly basis or
more frequently if required. All chickens were fed a commercial chick starter mash for
the entire study; feed and water were placed in large automatic feeders allowing birds
from each group to feed and drink ad libitum. On the day of challenge, eight hours prior
to challenging the chickens, feed was removed, and the feed returned to the birds one
hour after challenge. Chickens were challenged at 15 days of age by gavage with 1-ml
(105 CFU) if the C. jejuni mutant or wild-type strain. A negative control group received
PBS only. Chickens were euthanized by carbon dioxide asphyxiation and necropsied at
25 days of age. The ceca for each bird was collected, processed, and the feces serially
diluted for bacteriological culturing to quantify the presence of C. jejuni in each bird.
62 Experiments were performed in triplicate to confirm the statistical significance of each
designed study.
E. Biofilm assay
The biofilm assays were performed as previously described (Reeser et al., 2007)
with modifications. Briefly, Mueller Hilton broth in a 24-well polystyrene plates were
inoculated with C. jejuni to a final concentration of OD600 of 0.05. Samples were
incubated statically at 37°C under an atmosphere of 10% CO2. At desired time points (24,
48 and 72 hrs), planktonic cells and media were removed carefully without disturbing the
biofilm. Each well was carefully washed twice with PBS and dried for 5 min at 55°C.
Dried wells were stained with 0.1% crystal violet for 5 min, then rinsed three times with
D2H2O. Wells were de-stained with 80% ethanol and 20% acetone for 5 min and a 100 μl
of de-stained sample was collected and placed in a 96-well plate. The OD600 of the freefloating cells and the A570 of the de-stained sample were measured. Background values
due to non-specific staining of the 24-well plate by crystal violet was accounted for by
subtracting the values obtained from wells of the negative controls (no bacteria).
F. Agglutination assays
Autoagglutination assays were performed as described previously by (Misawa
and Blaser 2000) with the modifications detailed below. Bacterial strains from glycerol
stocks were grown on MHB plates supplemented with the appropriate antibiotics at 37°C
for 48 h and then re-streaked onto fresh plates and grown overnight. Growth from these
plates was re-suspended in phosphate-buffered saline (PBS) (Sigma-Aldrich, United
Kingdom), and the optical density at 600nm (OD600) was measured and adjusted to 1.0 ±
63 0.2. Two milliliters of each bacterial suspension was then dispensed into a sterile glass
tube (13 x 100 mm), and the tubes incubated at 37°C for 24 h. After incubation, 1 ml of
the bacterial suspension from each glass tube was carefully removed and the OD600 was
measured. Each strain was tested in triplicate. To account for the slight variations
between the OD600s of the strains tested, the data were normalized by subtracting the
OD600 measured after 24 h from the starting OD600, dividing by the starting OD600, and
finally multiplying by 100 to give the percent autoagglutination. Results were considered
to be statistically significant if P was <0.05.
G. Adhesion and invasion assays
The attachment assays were performed as previously described (Malik-Kale et al.
2007). In brief, a 24-well tissue culture tray were seeded with 1.0 x 105 INT 407 human
intestinal cells per well, and cells were incubated for 18 hours at 37°C in a humidified,
5% CO2 incubator (MOI of 0.01). The cells were rinsed with Minimal Essential Medium
(MEM)-10% fetal bovine serum (FBS) and inoculated with approximately 1x107 CFU of
the mutant or wild-type strain.
Tissue culture trays were incubated at 37°C in a
humidified, 5% CO2 incubator. For bacterial attachment, the infected monolayers were
incubated for 3 hours, rinsed three times with MEM-10% FBS, and re-incubated for an
additional 3 hours. Then, the cells were rinsed three times with phosphate buffered saline
(PBS), and the epithelial cells lysed with a solution of 0.1% TritonX-100. The
suspensions were serially diluted and the number of viable, adherent bacteria was
determined by counting the resultant colonies on MH blood plates. Experiments were
completed in triplicates. To measure bacterial invasion cell monolayers were generated as
64 described above. The infected monolayers were incubated for 3 hours, rinsed three times
with MEM-10% FBS, and incubated for an additional 3 hours in MEM-10% FBS
containing gentamicin to a final concentration of 250 μg/ml. Then, the cells were rinsed
three times with phosphate buffered saline (PBS), and cells were lysed with a solution of
0.1% TritonX-100. Then suspensions were serially diluted and the number of viable
intracellular bacteria was determined by counting the resultant colonies on MH blood
plates. Experiments were completed in triplicates.
H. Macrophage survival assays
Murine macrophage cells (J774A.1, ATCC) were routinely maintained in
Dulbecco’s minimal essential media (DMEM) supplemented with 10% fetal bovine
serum (FBS) in 75cm2 flasks at 37°C in a humidified, 5% CO2 incubator (2 x 105 cells per
well). Each well of a 24-well tissue culture tray was seeded with approximately 1.0 x 107
CFU/ml of mutant and wild-type strains (MOI of 0.02). The monolayers were washed 3
times after a 3 hours uptake period and further incubated in DMEM-10%FBS. Three
hours prior to each endpoint of 24 h, 48 h, and 72 h, wells were washed twice. To kill
extracellular bacteria cells were incubated for an additional 3 hours in DMEM-10%FBS
containing bactericidal gentamycin to a final concentration of 250 μg/ml. At each
endpoint, cells were washed three times with sterile PBS, and the number of internalized
C. jejuni were determined by lysis of the macrophage cells with 200 μg of deoxycholate
(0.5%w/v) standardized to 1ml sterile PBS. The suspensions were serially diluted and the
number of viable bacteria determined by counting the resultant colonies on MHB plates,
as colony forming units (CFU). All assays were performed in duplicates.
65 III. Results:
A. Mutational strategy
For the inactivation of the genes, primers were designed to amplify upstream and
downstream regions of the target gene, such that whole open reading frame was omitted.
Genes Cj0248 and Cj1475c were successfully deleted from the C. jejuni genome.
However, after unsuccessful multiple attempts to mutate Cj0998c, we hypothesized that
Cj0998c might be an essential gene of C. jejuni.
B. Scanning electron micrograph of C. jejuni ΔCj0248 and Cj1475c
After mutation of genes Cj0248 and Cj1475c, mutants were grown on sterile glass
cover slips to form biofilm and submitted for SEM analysis. After examining the
scanning electron micrographs, long and flexible filaments about 2-4 μm in length were
observed in the mutant strains, Figure 8C.
C. C. jejuni ΔCj0248 and ΔCj1475c mutants show reduced poultry colonization
To evaluate the role of Cj0248 and Cj1475c in host colonization, isogenic mutants
were generated and the mutants assayed in their ability to colonize poultry. In brief, birds
were divided into groups of 15, and on day 14, orally inoculated with either C. jejuni
ΔCj0248, ΔCj1475c or C. jejuni NCTC 11168. The ability of both strains to colonize the
chicks was assessed by enumeration of bacteria in the ceca 10 days post-inoculation. The
NCTC 11168 strain used in our study was able to colonize chicks at a level of 1 x 108
CFU per g of cecal content with an initial inoculum of 105 CFU/ml. The C. jejuni Cj0248
mutant strain exhibited a significant reduction in chick colonization as compared to the
wild-type strain. In the first trial, C. jejuni ΔCj0248 either, exhibited a complete
66 reduction of poultry colonization or bacterial levels were below the detection limit (102)
of the assay (Figure 9C; p = 0.01). In the second trial C. jejuni ΔCj0248 exhibited a
significant reduction in colonization when compared to the wild type strain. (Figure 10C;
p = 0.01). In the last trial, C. jejuni ΔCj0248 exhibited a 4-log reduction in the chick
colonization of chicks when compared to the wild type strain (Figure 11C). The C. jejuni
Cj1475c mutant exhibited a difference in chick colonization when compared to the wildtype strain. In the first trial C. jejuni ΔCj1475c showed a 3-log reduction in chick
colonization when compared to the wild type strain (Figure 12C; p = 0.05). In the second
trial, the C. jejuni ΔCj1475c mutant strain exhibited a 3-log reduction in chick
colonization when compared to the wild type strain (Figure 13C; p = 0.05). In the last
trial, C. jejuni ΔCj1475c exhibited no significant difference in chick colonization when
compared to the wild type strain (Figure 14C; p > 0.05).
D. Reduced biofilm formation was observed for ΔCj0248 but not for ΔCj1475c.
Increased biofilm formation was observed for ΔCj1343c
The ability of mutant strains to form a biofilm was investigated using a crystal
violet staining procedure. At 24 h C. jejuni ΔCj0248 showed decreased biofilm formation
when compared to the wild type strain (Figure 15C; p = 0.04). In contrast, C. jejuni
ΔCj1343c shows a dramatically increased ability to form a biofilm at 24, 48 and 72 h
when compared to the wild type strain (Figure 136C; p = 0.01). At 48 h no significant
difference is observed in the ability of the mutants to form a biofilm when compared to
wild type strain, Figure 17C. No significant difference was observed in biofilm formation
of ΔCj1475c when compared to the control.
67 E. ΔCj0248, ΔCj1475c and ΔCj1343c have no adverse effects in autoagglutination
assays
The role of auto agglutination in C. jejuni pathogenesis has not been determined.
However, the importance of auto agglutination in virulence has been described for other
pathogenic bacteria, including enteropathogenic Escherichia coli, and Vibrio cholerae
(Chiang, S. L., et al., 1995, Knutton, S., et al., 1999, Roggenkamp, A., 1995). This
activity has been associated with pilins and/or outer membrane proteins that have been
demonstrated to be critical for pathogenesis. Thus we investigated the ability of C. jejuni
Cj0248, Cj1475c and Cj1343c mutant strains to agglutinate. Our study indicates that the
C. jejuni Cj0248, Cj1475c and Cj1343c agglutinated at the same levels as wild type
strains (Figure 18C). Interestingly, Cj1343c mutant showed a significant difference in
the ability to autoagglutinate (Figure 18C; p < 0.05).
F. ΔCj0248 adheres better but invades less efficiently than wild type strain. ΔCj1475c
and ΔCj1343c have no adverse effects in adhesion and invasion assays
The ability of C. jejuni ΔCj0248, ΔCj1475c and ΔCj1343c to attach and to
invade epithelial cells was examined using gentamicin protection assays. The ability to
attach was significantly higher in ΔCj0248 when compared to the wild type strain, but
not its ability to invade. ΔCj1475c and ΔCj1343c attached and invaded at levels
comparable to the wild type strain (Figure 19C and Figure 20C).
G. ΔCj0248, ΔCj1475c and ΔCj1343c have no adverse effects in their ability to survive
intracellularly within macrophages
68 The ability of C. jejuni ΔCj0248, ΔCj1475c and ΔCj1343c to survive
intracellularly within macrophages was examined using the gentamicin protection assays.
C. jejuni ΔCj0248, Δ1475c and Δ1343c became internalized by macrophage in a less
efficient manner than the wild type strain, however by 72 h, almost all bacteria were
killed by macrophages (Figures 21C, 22C and 23C).
IV. Discussion:
Here we describe the findings of our mutational work and the role of Cj0248,
Cj1474c and Cj1343c in C. jejuni pathogenesis, including commensal colonization,
biofilm formation, agglutination, adherence and invasion, as well as intracellular survival
within macrophages. Upon mutation of these genes (Cj0248, Cj1474c and Cj1343c),
examination of the scanning electron micrographs shows that the ability of mutant to
produce pilus-like appendages disseminating from the cell was not affected. Despite the
involvement of Cj1343c and Cj1475c in transformation, Cj1343c having a 68% identity
to the pilA consensus sequence commonly found in bacteria that produces pili and
Cj1475c been in the same operon of a homolog to the PilQ protein (Cj1473c); Cj1343c
and Cj1475c are not involved in the production of C. jejuni pilus-like surface-associated
structures previously observed.
C. jejuni 1475c gene (Cj1475c) encodes a protein that is 105 amino acids in
length; its predicted size is 12-kDa. It is listed as a hypothetical protein with putative
uncharacterized protein CtsR function. Using the online SignalP tool, no signal peptide
was found on the Cj1475c sequence. Also, Cj1475c appears to be conserved among
different C. jejuni strains, including C. jejuni RM1221 poultry, C. jejuni NCTC 11168
69 human isolate, C. jejuni S3 poultry isolate, and C. coli isolates. Cj1475c has been
identified as being part of a Campylobacter transformation system (cts). In a previous
study, a solo transposon mutant library of C. jejuni 81-176 was created (Wiesner R. S., et
al 2003). Mutants had a reduction in the ability of C. jejuni to be transformed. Among
those genes were Cj1475c including those in the putative operon in which Cj1475 is
located (Cj1470c, Cj1471c, Cj1472c, Cj1473c, Cj1474c, and Cj1476) (Figure 7C). The
type II cts homologues that were identified through transposon mutagenesis were termed
as follows: CtsD (Cj1474c), CtsE (Cj1471c), and CtsF (Cj1470c). Cj1470c, Cj1471c, and
Cj1474c genes were among the genes with homology to genes required for diverse
macromolecular transport processes in a range of bacterial species. CtsD is homologous
N. gonorrhoeae PilQ, which is thought to be an outer membrane pore involved in natural
transformation and type IV pilus biogenesis. BLAST searches reveal CtsR are
homologous to post-translational modification proteins found in Nitrosomonas sp., and
phage proteins in Listeria monocytogenes. However, no significant homology to any
proteins with known protein function in other bacterial species was found for CtsR.
Commensal colonization studies revealed that the mutation of Cj1475c
significantly decreased the commensal colonization of chicks in two of three trials. No
differences were observed between the wild type and mutant strains in regards to biofilm
and this particular mutant strain ability to agglutinate, adhere and invade as well as
intracellular survival within macrophages.
The ability of ΔCj1475c to attach and to become internalized was similar to the
wild type strain, indicating that the reduced colonization we observed is not due to the
70 inability of the mutant to attach and invade epithelial cells. Based on this results we could
speculate whether Cj1475c play a role in other steps during colonization prior to or after
to attachment and invasion. Upon ingestion, the organism is exposed to numerous insults
in the gastrointestinal tract, such as low stomach pH, and exposure to bile salts. The
ability to survive these insults is essential for infection. Some C. jejuni strains are more
sensitive to low pH than others, thus acid tolerance is strain dependent. In the small
intestine, C. jejuni is exposed to bile salts, low oxygen levels, and iron availability is
limited. We can hypothesize that Cj1475c is involved in bile tolerance in C. jejuni. Bile is
composed of a mixture of bactericidal detergents, thus resistance to bile is an important
determinant during infection. In a variety of enteric pathogens a correlation between
reduced ability to tolerate bile and reduce colonization have been observed (Lacroix et al
1996, Bina and Mekalonaos 2001). Although a number of studies have highlighted the
mechanism of resistance of Campylobacter to bile, little is known about the effect of bile
on Campylobacter virulence determinants.
Further review of the literature we found a study in which Cj1472c, Cj1473c and
Cj1475c have been identified to be up regulated in the presence of bile (Malik-Kale P., et
al., 2008). Bile has been shown to regulate virulence gene expression in several
gastrointestinal pathogens; the ability of bile acid deoxycholate (DOC) was assessed in
virulence gene expression in C. jejuni. Microarray analyses were developed with RNA
extracted from C. jejuni cultured in the presence and absence of DOC. A total of 156
genes, including Cj1472c, Cj1473c and Cj1475c were up-regulated 2.3-fold, 1.7-fold and
2.2-fold, respectively in the presence of DOC. It is important to mention that in the
71 studies described above regarding Cj1475c mutation, results observed cannot exclusively
be linked to the mutation of this particular gene, as Cj1474c is located in an operon and
polar effects in downstream genes are not excluded.
C. jejuni Cj0248 gene (Cj0248) encodes a protein that is 285 amino acids in
length; its predicted size is 32-kDa. Using the online SignalP tool, no signal peptide was
found on Cj0248 sequence. Analyses revealed that Campylobacter jejuni 0248 gene
(Cj0248) does not appear to be in an operon; however there is a small gap between
Cj0248 and Cj0249. Cj0248 is encoded in the positive strand. Cj0248 is also conserved
among different C. jejuni strains including C. jejuni RM1221, C. jejuni NCTC 11168
human isolate, and C. jejuni S3 poultry isolate. The crystal structure of Cj0248 has been
described (Xu Q. et. al. 2006). Cj0248 belongs to a large protein family that includes
members such as hydrolases and signal-transduction proteins.
The mutation of Cj0248 severely affected the ability of C. jejuni to colonize
chicks. Mutation of Cj0248 also decreased biofilm formation for 24 h. This is consistent
with our results in which mutation of Cj0248 affected its ability to colonize broilers
chickens. The polar flagellum is the best-characterized virulence factor in C. jejuni;
flagellar motility is required for efficient colonization of chickens and humans. In 2004, a
transposon mutant library was generated to identify genes required for a virulence
determinant, 28 mutants were identified to have reduced motility and Cj0248 was one
among them (Hendrixson D. R., et al., 2001). Motility has been found to be involved in
the early stages of biofilm formation. This finding might explain the reduced biofilm
formation we observed in our study by this particular mutant.
72 In a different study a transposon mutant library was generated to identify genes
involved in colonization of the chick gastrointestinal track (Hendrixson D. R., and DiRita
V. J., 2004). Consistent with the previous study mutants showed reduce motility
(Hendrixson D. R., et al., 2001). The transposon inserted itself in genes required for
transcription, secretion or structural components of flagellar, chemotaxis proteins with no
homology to proteins with known functions. Mutation of Cj0248 resulted in reduction in
the bacterial loads in the chick ceca when compared to wild-type strain. Cj0248 represent
a new potential virulence factor important in the poultry colonization.
Following passage through the stomach, C. jejuni must interact with host mucosa
in order to cause disease. Some of these interactions include attachment, invasion, and
signal transduction events. Studies have shown that C. jejuni interacts very closely with
host cells during an infection. Upon establishing an association with the human intestinal
epithelial cells, Salmonella as well as E. coli secrete effector proteins into the host cell
that initiates host signal transduction events that can lead to re-arrangements and/or
internalization of the bacteria. In fact, invasion of epithelial cells is considered a
virulence factor in many pathogenic bacteria including Salmonella, Shigella, E. coli and
Listeria (Falkow et al 1992, Takeuchi et al 1967, Sansonetti 1991).
Previous studies have showed that mutations in the major flagellum subunit gene,
flaA, resulting in bacteria with truncated flagella, produce a mutant that has diminished
motility and is unable to invade intestinal epithelial cells in vitro (Guerry, P., et al., 1991,
Wassenaar, T. M., et al 1991). Furthermore, a mutation in pflA resulted in bacteria with
paralyzed flagella; this mutant was still able to adhere but is not capable of invasion in
73 vitro (Yao, R., et al., 1994). We found that Cj0248 mutant adhered to INT407 cells to a
greater degree when compared to the wild type strain, however, the level of invasion was
reduced. Previous studies have showed that only 66% of intestinal epithelial monolayers
are invaded (Hu, L., et al., 1999) suggesting that there are other factors besides adherence
that influences the ability of the bacteria to invade. This indicates that reduce colonization
observed upon mutation of Cj0248 gene is not due to the reduce motility or its inability to
attach and but its inability to invade epithelial cells.
Bacteria use the intestinal barrier as an entry site, exploiting the target host cell
cytoskeletal machinery to trigger their uptake by non-phagocytic cells, thereby gaining
access to the host. This might explain why no difference was observed between ΔCj0248
and wild type strain in their ability to survive intracellularly within macrophages. In fact
only a small percentage of C. jejuni survives within professional phagocytic cells.
Suggesting that C. jejuni is internalized in a similar manner as other pathogenic bacteria,
by non-phagocytic cells.
Lastly we also investigated the role of Cj1343c biofilm formation, agglutination,
adherence and invasion as well as intracellular survival within macrophages. As
previously describe in Chapter II, mutation of Cj1343c had no effect in the ability of the
bacterium to commensally colonized chicks when compared to wild type strain. Our
result indicates that ΔCj1343c can persist and multiply within epithelial cells in vitro and
survive intracellularly within macrophages at levels similar to the wild type strain.
However, biofilm formation was increased upon mutation of Cj1343c gene. We can
74 speculate whether Cj1343c is a regulator of transcription of genes involved in biofilm
formation, thus when mutated uncontrolled biofilm growth is observed.
Biofilms are composed of an extracellular material, which it is mostly produced
by the bacteria, and consists of an accumulation of different types of biological molecules
known as extracellular polymeric substances (EPS). The EPS contains polysaccharides,
proteins, nucleic acids, lipids and other biopolymers. This matrix may aid in desiccation,
oxidizing agents, form a protective barrier against some antibiotics, and ultraviolet
radiation. The question to ask is whether Campylobacter produces extracellular
polymeric substances and what constitutes C. jejuni biofilms. Polysaccharides are a major
fraction of the EPS matrix (Frolund, B., et al., 1996, Wingender. J., et al., 2001). Several
polysaccharides have been visualized by electron microscopy as fine strands that are
attached to the cell surface and form complex networks. However, the genome sequence
of C. jejuni does not show any obvious candidates for a biofilm polysaccharide. It is
possible, that the constitution of the biofilm may be composed of capsular
polysaccharides or poly-amino acid (Ornek et al., 2002).
We can speculate that upon mutation of Cj1343c gene, C. jejuni overexpresses
constituents of the biofilm thus reflecting an increase in biofilm formation. Thus further
analysis of the dynamics and composition of C. jejuni biofilm must be performed in order
to elucidate the exact role of Cj1343c in biofilm formation. A way to approach this is by
confocal laser scanning microscopy (Lawrence, J. R., et al., 2007), which is a common
tool for detection of components of the EPS in biofilms. Using fluorescently labeled
lectins, exopolysaccharides are visualized according to their interaction with specific
75 targets. For example, extracellular DNA can be detected with dyes specific for nucleic
acids, thus facilitating the identification of the major constituents of the EPS.
Here we have identified two genes required for cecal colonization leading to
commensalism in the chick gastrointestinal tract. However, it is unclear whether these
genes are required for pathogenesis in other disease models of C. jejuni gastroenteritis,
such as the ferret, and piglets in which C. jejuni inflammatory responses and pathology
are evident. Little is known about the common features that C. jejuni uses during
commensal colonization and pathogenic models of infection.
76 V. Chapter IV Tables and Figures
Table 6C. Strains and plasmids used in this study
Strain or plasmid
Bacteria
C. jejuni NCTC 11168
TOP10
C. jejuni ΔCj0248
C. jejuni ΔCj1475c
Plasmids
pCR2.1-TOPO
pCR0248
pCR1475c
pUOA23
Characteristic
Source
Clinical isolate
F- mcrA Δ(mrr-hsdRMSmcrBC) Φ80lacZΔM15
ΔlacΧ74 recA1 araD139
Δ(araleu) 7697 galU galK
rpsL (StrR) endA1 nupG
Mutant strain carrying a
deletion on gene Cj0248
This study
Invitrogen
Mutant strain carrying a
deletion on gene Cj1475c
This study
Cloning vector
Contains both flanking
regions and CmR marker
Contains both flanking
regions and CmR marker
Contains C. coli plasmid C589 chloramphenicol
acetyltransferase (cat) gene
Invitrogen
This study
This study
This study
Dr. Van der Walls
Table 7C. Oligonucleotides used in this study
Primer
Sequence
Cj0248-F1
Cj0248-R1
Cj0248-F2
Cj0248-R2
Cj1475c-F1
Cj1475c-R1
Cj1475c-F2
Cj1475c-R2
Cat-F
Cat-R
Sequence (5’ > 3’)
tgacaaGGATCCgttcttcaaaagtgcagtta
aaggataaattaaatatgatGCTAGCgatgct
tatgacGCTAGCaggctctaaattgtagtgat
ttaatttgaagtatttttgcGTCGACttcgtc
caaataaagacaatggtgct
aaagaaaatcgtataaatttGCTAGCgt
ttGCTAGCtactaaagagattaaattcc
tctattattaactttctttc
atGCTAGCtaacccattgctcggcggtgttcctttcca
tcgcactgataaaaaccctttaggaaGCTAGCaa
Restriction
site
BamHI
NheI
NheI
SalI
N/A
NheI
NheI
N/A
NheI
NheI
77 Cj1470c
Cj1471c
Cj1472c
Cj1473c
Cj1474c
-­‐29 -­‐29
Cj1475c
Cj1476c
24 24
Figure. 7C. Analysis of the location of gene Cj1475c in the NCTC 11168 genome. The
query gene (Cj1475c) is shown in light blue. Nearby genes upstream and downstream of
the Cj1475c are shown in white. The arrowhead indicates the transcription direction. The
numbers appear below the genes indicate the intergenic distance between two adjacent
genes. Negative numbers indicate overlapping genes.
78 Figure 8C. Scanning electron micrograph of mutants. A.) Biofilms of C. jejuni ΔCj0248.
B). Biofilms of C. jejuni ΔCj1475c.
1.00E+09 1.00E+08 1.00E+07 Log10CFU/g 1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 Negative NCTC::Cj0248 NCTC WT Figure 9C. Effects of ΔCj0248 mutation on in vivo poultry colonization, first trial. Error
bar indicates standard deviation, n = 15 chickens per group, p = 0.01.
79 1.00E+09 1.00E+08 Log10CFU/g 1.00E+07 1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 Negative NCTC::Cj0248 NCTC WT Figure 10C. Effects of ΔCj0248 mutation on in vivo poultry colonization, second trial.
Error bar indicates standard deviation, n = 15 chickens per group, p = 0.02.
1.00E+08 1.00E+07 Log10CFU/g 1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 Negative NCTC::Cj0248 NCTC WT Figure 11C. Effects of ΔCj0248 mutation on in vivo poultry colonization, third trial.
Error bar indicates standard deviation, n = 15 chickens per group, p = 0.01.
80 1.00E+09 1.00E+08 1.00E+07 Log10CFU/g 1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 Negative NCTC::Cj1475c NCTC WT Figure 12C. Effects of ΔCj1475c mutation on in vivo poultry colonization, first trial.
Error bar indicates standard deviation, n = 15 chickens per group, p = 0.05.
1.00E+09 1.00E+08 Log10CFU/g 1.00E+07 1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 Negative NCTC::Cj1475c NCTC WT Figure 13C. Effects of ΔCj1475c mutation on in vivo poultry colonization, second trial.
Error bar indicates standard deviation, n = 15 chickens per group, p = 0.05.
81 1.00E+09 1.00E+08 Log10CFU/g 1.00E+07 1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 Negative NCTC::Cj1475c NCTC WT Figure 14C. Effects of ΔCj1475c mutation on in vivo poultry colonization, third trial.
Error bar indicates standard deviation, n = 15 chickens per group, p > 0.05.
0.200 0.180 0.160 0.140 A570 0.120 0.100 0.080 24hrs 0.060 0.040 0.020 0.000 Figure 15C. Effects of Cj0248, Cj1475c and Cj1343c mutation in biofilm formation, at
24 h. Error bar indicates standard deviation.
82 1.200 1.000 A570 0.800 0.600 0.400 48hr
0.200 0.000 Figure 16C. Effects of Cj0248, Cj1475c and Cj1343c mutation in biofilm formation, at
48 h. Error bar indicates standard deviation.
0.600 0.500 A570 0.400 0.300 0.200 72hrs 0.100 0.000 Figure 17C. Effects of Cj0248, Cj1475c and Cj1343c mutation in biofilm formation, at
72 h. Error bar indicates standard deviation.
83 1.00 % of agglutination cells 0.80 0.60 0.40 0.20 0.00 NCTC::Cj0248 NCTC::Cj1474c NCTC::Cj1343c NCTC WT Figure 18C. Effects of Cj0248, Cj1475c and Cj1343c mutation in agglutination assays.
Error bar indicates standard deviation.
3.50 3.00 % adherent cells 2.50 2.00 1.50 1.00 0.50 0.00 Figure 19C. Adherence assays. Effects of Cj0248, Cj1475c and Cj1343c mutations on
the ability to adhere to INT 407 cells. Error bar indicates standard deviation.
84 30.00 % invasive cells 25.00 20.00 15.00 10.00 5.00 0.00 Figure 20C. Invasion assays. Effects of Cj0248, Cj1475c and Cj1343c mutations on the
ability to invade to INT 407 cells. Error bar indicates standard deviation.
3.50 % surviving cells 3.00 2.50 2.00 1.50 1.00 0.50 0.00 Figure 21C. Macrophage survival assays. Effects of Cj0248, Cj1475c and Cj1343c
mutations in their ability to survive intracellularly within macrophages (24 h). Graph
85 represent the percent of surviving cells. Error bar indicates standard deviation.
3.50 % surviving cells 3.00 2.50 2.00 1.50 1.00 0.50 0.00 Figure 22C. Macrophage survival assays. Effects of Cj0248, Cj1475c and Cj1343c
mutations in their ability to survive intracellularly within macrophages (48 h). Graph
represent the percent of surviving cells.
3.50 % surviving cells 3.00 2.50 2.00 1.50 1.00 0.50 0.00 Figure 23C. Macrophage survival assays. Effects of Cj0248, Cj1475c and Cj1343c
mutations in their ability to survive intracellularly within macrophages (72 h). Graph
represents the percent of surviving cells.
86 CHAPTER V: ATTENUATED SALMONELLA AS VECTOR TO DELIVER
VACCINE (Se:0248) AGAINST C. JEJUNI INFECTION
I. Abstract
The Gram-negative bacterium Campylobacter is the leading cause of bacterial
gastroenteritis in humans and is associated with the occurrence of life-threatening
autoimmune-based neurological disorders such as GBS and Reiter’s arthritis. Chickens,
which are often heavily colonized with Campylobacter without signs of pathology, are
considered the most important source for human infection. Chickens usually encounter
Campylobacter at a young age and fecal shedding results in a rapid spread throughout the
entire flock. Depending on the geographical region and season, the percentage of
Campylobacter-positive flocks in Europe reaches up to 90% (Newell et al., 2003). The
bacterium colonizes the caeca, and large intestine and cloaca of chicken in numbers up to
109 CFU per gram of feces (Beery et al., 1988, Dhillon et al., 2006) without apparent
signs of pathology.
Several strategies have been applied to reduce Campylobacter counts on chicken
meat, including attempts to eliminate Campylobacter from the farms by increasing
biosecurity and the separation of contaminated flocks, and by improving hygiene during
the process of slaughtering. In addition, several experimental approaches like the
reduction of colonization by competitive exclusion, antibacterial agents, or phage therapy
are being investigated for their efficacy (Loc Carrillo et al., 2005; Wagenaar et al., 2005).
Although these measures may or may not help in controlling shedding of Campylobacter
by the poultry, vaccination of poultry against Campylobacter would be the most effective
87 means of reducing or eliminating Campylobacter infections and remains a major goal for
researchers. The molecular basis for the apparent different pathology of Campylobacter
in poultry and human host remains unclear. However, it has been demonstrated that
effective colonization of the chicken gut require several bacterial proteins including
flagellin, CadF and CiaB, as well as a functional protein N-glycosylation machinery.
Based on our mutational analysis of Cj0248, there was a remarkable reduction in
colonization as well as reduction of initial biofilm formation in C. jejuni. Therefore, we
have hypothesized that Cj0248 represents a potential vaccine target for C. jejuni
infection.
For the construction of the vaccine, we used an attenuated Salmonella strain that
has been previously shown to be an effective tool for vaccination. Attenuated Salmonella
vaccines have been constructed by introduction of mutations in the genes required for
virulence, including the cyclic AMP receptor protein gene (crp) (Curtiss, R., III et al.,
1987). Also, attenuated Salmonella harbors a deletion of the asd gene, which is essential
for synthesis of the bacterial cell wall component diaminopimelic acid (DAP). This
Salmonella strain was used to develop a recombinant vaccine. Attenuated Salmonella
vaccine strains have been genetically modified to express the antigens from pathogens by
multicopy plasmids this allows the inducing of immunity to the pathogen whose gene is
being expressed as well as to Salmonella. Previous studies performed in our lab showed
that attenuated Salmonella is not detectable in the birds seven days after vaccination, thus
reducing the concern of having Salmonella being shed by birds among commercial
flocks. This chapter will address the following aims:
88 1. Construction of a vaccine using attenuated Salmonella strain as an expressing
vector
2. Confirm expression of Cj0248 gene by Salmonella vector
3. Vaccinate chicks with generated vaccine and challenge with C. jejuni
This study supports the potential used of live vaccines for control of Campylobacter in
poultry, however, the protective effect differed remarkably between trials. Thus
something occurred within the studies affected the outcome of these results.
II. Material and Methods
A. Culture of Bacterial Strains
Escherichia coli χ6212 pYA3493 was maintained on Luria Bertani (LB) agar
(BD) supplemented with 0.1% glucose (LB-G). E. coli χ6212 used in plasmid
electroporation was grown on LB-G agar containing 50 μg/ml diaminopimelic acid
(DAP) at 37°C. S. enterica strain χ9992 was routinely cultivated on LBG agar with DAP
supplemented with 0.2% mannose and 0.05% arabinose and was grown at 37°C. Strains
containing pYA3493 were grown on LB-G agar containing mannose and arabinose and
were grown at 37°C. All strains were subcultured at 48 h intervals. All strain and
plasmids used in this study are listed in Table 8D.
B. Construction of an S. enterica serovar Typhimurium vaccine strain
Plasmid construct was developed in the E. coli χ6212 strain and later introduce into
the S. enterica strain. Salmonella enterica expression plasmid pYA3493 was used as the
backbone for the gene expression. To construct the vaccine, DNA for Cj0248 was PCR
amplified. The gene was inserted into the pYA3493 expression plasmid in the proper
89 reading frame; generating pYA3493+Cj0248. This construct was later introduced into S.
enterica serovar Typhimurium strain χ9088. In-frame cloning was confirmed by
nucleotide sequencing. Primers used in this study are listed in Table 9D.
C. Expression of Cj0248
In brief protein samples were boiled for 10 min and then separated by
discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The gel was mounted in a vertical electrophoresis apparatus (Mini-Protean 3, Bio-Rad),
and 1X running buffer was added. The running conditions were set as follows: 150V until
the sample was at the end of the gel. Protein bands were visualized by GelCode Blue
(Sigma, St. Louis, Mo.) staining. Band corresponding to the Cj0248 protein size was
excised from the gel and sent for mass spectrometry analysis (University of Arizona
Genetics Core).
D. Challenge of Birds
Chicken studies were performed in accordance with the policies of the
Institutional Animal Care and Use Committee of the University of Arizona (IACUC).
One-day old chicks were obtained from a commercial hatchery (Murray McMurray,
Webster City, Iowa). To determine if the birds were free of C. jejuni at arrival the chicks
were swabbed and the fecal sample direct plated onto Cefex plates. Vaccinations were
performed at 3, 10 and 16 days post hatching.
E. Vaccination using Salmonella enterica
The vaccine was prepared as follows: Salmonella strain χ9088 was grown in LBG broth overnight at 37°C with aeration (200 rpm). Following incubation, the overnight
90 culture was diluted 1:10 with fresh LB-G broth and grown at 37°C with aeration for 24 h.
Cultures were centrifuge at 4000 rpm for 15 min, the supernatant removed and the
pelleted cells re-suspended in 20 ml PBS. Serial dilutions were performed to determine
the exact titer of the vaccine. On the day of vaccination, eight hours prior to the
vaccination of the chickens, feed was removed, and the feed was returned to the birds one
hour after vaccination. Chickens were orally vaccinated with 1 ml of vaccine. At day 26,
birds were challenged by gavage with 1 ml of a C. jejuni suspension (105 CFU/ml) in
phosphate buffered saline (PBS). A negative control group was challenged with PBS
only. Ten days after challenge, chickens were euthanized by carbon dioxide asphyxiation.
Ceca were collected, the contents were vortex and sampled (0.25 g per ceca into 4.5ml of
PBS) and serially diluted and direct plated to quantify the C. jejuni in each bird.
III. Results:
A. Construction of an S. enterica vaccine strain
To construct the Salmonella vaccine (Se:0248) the open reading frame of Cj0248
was amplified by PCR using primers listed in Table 9D. Each primer was developed with
a built in cut site, and each cut site corresponded to the cut sites on the cloning vector so
that cloning into the expressing vector could be achieved. The plasmid map of the
cloning vector used in this study can be found in Figure 28D.
B. Expression of C. jejuni 0248 gene in the Salmonella vaccine strain
To ensure Salmonella expression of the C. jejuni 0248 gene, both Salmonella and
the YA3493 plasmid (SalEV) were grown overnight and then passed into LB broth
containing arabinose, mannose and glucose to allow expression of inserted gene
91 (Cj0248). Samples were later separated on a SDS-PAGE to see if Salmonella was
expressing the protein of interest. Our results demonstrated a 37 kDa protein present in
the Se:0248 vector and absent in the SalEV, Figure 24D. This band was excised and sent
for mass spectrometry analysis to confirm the identity of the sample.
C. Oral Vaccination with Salmonella
To examine the ability of Salmonella-Cj0248 vaccine (Se:0248) to protect against
C. jejuni infection, chicks at 3, 10 and 16 days of age were immunized orally with S.
enterica pYA3493-0248 at a dose of 1.0 x109 CFU. Birds did not show any weakness or
signs of disease after immunization, thus ensuring that use of attenuated Salmonella as a
vector was a safe procedure. During the first oral vaccination trial for the Se:0248 birds
our results indicated that 10 out of 15 birds were colonized with C. jejuni to wild type
levels, Table10D. However, our control groups, both the SalEV (empty vector) and those
that received no immunization, were colonized at levels lower than the vaccinates (Table
10D). During the second oral vaccination trial for the Se:0248 birds our results indicated
that 15 out of 15 birds were colonized with C. jejuni to wild type levels, as well the other
two control groups (Table 11D). During the third oral vaccination trial for the Se:0248
birds our results indicated that 9 out of 15 birds were colonized with lower levels of C.
jejuni. However, our control groups, both the SalEV and those that received no
immunization, were colonized at levels lower than the non-vaccinated controls (Table
12D). A summary of each vaccine trial in a graph format can be found in Appendix D
(Figures 25D, 26D and 27D).
92 IV. Discussion
In our previous work, it was shown that the C. jejuni 0248 gene, encoding a
hypothetical protein with a putative signal transduction function was shown to have
significant reduction in colonization when the gene was deleted from the genome. Also, a
significant decrease in initial biofilm formation was observed at 24 h.
In addition
mutation of this gene causes a significant reduction in the mutant ability to invade and
survive intracellularly. Thus, Cj0248 represents a new potential virulence factor
important in the poultry colonization. Based on this information, this protein was selected
for expression in a recombinant attenuated Salmonella Typhimurium vector for the
vaccination of broiler chickens against C. jejuni challenge.
The first and second oral vaccination trial results indicate that birds that received
the vaccine were colonized with C. jejuni to wild type levels; however, it is important to
note that in both SalEV group and those that received no immunization, birds were
colonized at lower levels when infected with C. jejuni. In the third oral vaccination trial,
9 out of 15 birds were colonized with lower levels of C. jejuni. However, we cannot
compare broilers chicks vaccinated with Se:0248 to unvaccinated groups because
unvaccinated groups show colonization levels lower than the level seen in our wild type
group. A chicken colonized with C. jejuni usually excretes large amounts of bacteria, up
to 10^8, thus something occurred within the studies affected the outcome of the results.
We can speculate whether the age of the birds at the time of challenge will affect the
minimum infectious dose required. Generally, birds 1 to 14 days of age are inoculated
with a defined amount of Campylobacter, and then given several days to weeks for the
93 bacteria to establish the colonization (Biswas et al., 2007; Nachamkin and Blaser, 2000).
Potentially at the time of challenge day 26, chicks are indeed in need of higher doses thus
affecting the final counts. Previously, it has been shown that the time of infection affects
the shedding patterns observed. The older the chicken is at the time of infection, the
shorter the duration of shedding and a higher dose is required for successful colonization
(Kaino et al., 1988), however this holds true if all chickens were not to colonized at all.
Further analysis needs to be performed in order to eliminate the possibility of getting
lower colonization levels in the control groups. The results from broiler chickens
vaccinated with Sal0248 are inconclusive.
We suggest to perform a more comprehensive study in which loads of C. jejuni in
the cecum of birds are monitored overtime, for example, fives day after each
immunization. This would allow us to monitor the protective response in a real-time
manner, including whether protective responses are present at the time of challenge. In
addition, we suggest studying the Cj0248 expression under different promoters; this
would allow us to determine whether the vaccine is been expressed efficiently and which
conditions are more favorable for the expression of this particular gene. We also suggest
studies prior first immunization, to detect whether birds at the time of hatch do have a
protective immune response against Salmonella and Campylobacter. It is plausible that
pre-existing antibody to the vector strain or to the C. jejuni antigen are affecting the
outcome of the experiment.
94 V. Chapter V Tables and Figures
Table 8D. Strains and plasmids used in this study
Strain or plasmid
Bacteria
C. jejuni NCTC 11168
E. coli Χ6212
S. enterica Χ9992
Plasmids
pYA3493
pCj0248
Characteristic
Source
Challenge strain
φ80d lacZ ΔM15 deoR
Δ(lacZYA-argF)U169 supE44
λ− gyrA96 recA1 relA1
endA1 Δasd Δzhf-2::Tn10
hsdR17
Δpmi-2426 Δ(gmd-fcl)-26
ΔPfur77::TT
araC PBADfur ΔPcrp527::TT
araC PBAD crp
ΔasdA27::TT araC PBADc2
ΔaraE25
ΔaraBAD23 ΔrelA198::araC
PBADlacI TT
L.A. Joens
R. Curtiss 3rd
Asd+, β-lactamase signal
sequence
Asd+, β-lactamase signal
sequence
R. Curtiss 3rd
R. Curtiss 3rd
This study
Table 9D. Oligonucleotides used in this study
Primer Sequence
SalCj0248-F1
Sequence (5’ > 3’)
gactGAATTCtcataaaaaatgcaataag
Restriction site
EcoRI
SalCj0248-R1
ttgtagtgatgaaaagtttaGTCGACcgg
SalI
Table 10D. First Oral Vaccination with Se:0248
Negatives*
Se:0248*
SalEV*
C. jejuni NCTC*
1.00E+00
2.80E+08
2.20E+04
5.50E+03
1.00E+00
8.00E+04
5.30E+04
1.00E+00
1.00E+00
1.90E+06
5.00E+03
3.00E+04
1.00E+00
1.00E+03
2.00E+03
9.00E+02
1.00E+00
2.30E+05
6.00E+03
9.00E+02
1.00E+00
3.80E+08
7.00E+02
2.00E+03
8.20E+06
1.40E+04
1.00E+02
Average
* = represent
95 8.80E+07
1.00E+06
3.00E+02
1.30E+09
6.00E+05
9.00E+03
4.00E+02
1.00E+04
4.00E+04
3.40E+04
9.00E+04
6.50E+08
1.90E+05
1.40E+05
1.90E+05
3.60E+08
1.10E+04
4.00E+08
3.00E+03
2.31E+08
1.46E+05
8.87E+03
the number of bacteria recovered 10 days after challenge of birds.
Table 11D. Second Oral Vaccination with Se:0248
Negatives*
Se:0248*
SalEV*
C. jejuni NCTC*
1.00E+00
3.70E+08
4.40E+07
3.60E+04
1.00E+00
8.00E+07
3.80E+05
6.00E+07
1.00E+00
8.00E+07
1.40E+05
2.00E+03
1.00E+00
5.00E+05
2.40E+03
8.10E+05
1.00E+00
7.10E+08
5.90E+06
5.50E+05
1.00E+00
1.20E+08
2.60E+08
7.00E+08
5.00E+06
2.80E+08
3.90E+07
2.00E+05
4.80E+06
6.90E+04
2.80E+08
1.40E+04
4.70E+04
4.20E+08
1.80E+07
5.30E+07
3.10E+06
1.40E+06
3.60E+08
2.20E+07
3.60E+08
9.30E+05
8.00E+07
1.00E+07
1.50E+06
1.10E+04
5.40E+07
2.40E+08
1.50E+06
8.16E+07
8.48E+07
Average
* = Represent
1.67E+08
the number of bacteria recovered 10 days after challenge of birds.
Table 12D. Third Oral Vaccination with Se:0248
Negatives*
Se:0248*
SalEV*
C. jejuni NCTC*
1.00E+00
4.40E+04
3.00E+02
1.00E+00
1.00E+00
6.00E+02
2.10E+05
5.90E+03
1.00E+00
4.70E+06
2.90E+07
7.00E+02
1.00E+00
7.00E+07
1.40E+08
1.00E+00
96 1.00E+00
2.70E+04
1.60E+08
1.00E+00
1.00E+00
6.60E+06
3.80E+04
1.80E+04
3.50E+04
1.00E+00
2.00E+03
3.20E+04
1.00E+02
3.80E+05
2.70E+04
1.30E+05
6.50E+06
1.50E+08
2.70E+08
6.50E+04
2.00E+07
3.40E+08
4.00E+03
1.40E+03
2.30E+08
8.00E+02
7.00E+04
8.70E+07
7.00E+03
5.20E+05
5.00E+02
2.10E+03
4.30E+04
8.00E+07
9.00E+02
1.68E+07
8.91E+07
4.66E+05
Average
* = Represent
the number of bacteria recovered 10 days after challenge of birds.
1
2
3
kDa
250
37
25
Figure 24D. SDS-PAGE. 1.) Attenuated Salmonella without insert. 2.) Attenuated
Salmonella vector expressing C. jejuni 0248 gene. 3.) Molecular weight. Arrow indicates
97 band of appropriate molecular weight that might correspond to C. jejuni hypothetical
protein encoded by Cj0248.
1.00E+09 1.00E+08 Log10CFU/g 1.00E+07 1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 Negative SalCj0248 SalEV NCTC Figure 25D. First oral vaccination with S. enterica (pYA3494) expressing Cj0248
(Se:0248). Error bar indicates standard deviation, p > 0.05.
1.00E+09 1.00E+08 1.00E+07 Log10CFU/g 1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 Negative SalCj0248 SalEV NCTC Figure 26D. Second oral vaccination with S. enterica (pYA3494) expressing Cj0248
98 (Se:0248). Error bar indicates standard deviation. Error bar indicates standard deviation,
p > 0.05.
1.00E+09 1.00E+08 1.00E+07 1.00E+06 Log10CFU/g 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 Negative SalCj0248 SalEV NCTC Figure 27D. Third oral vaccination with S. enterica (pYA3494) expressing Cj0248
(Se:0248). Error bar indicates standard deviation, p > 0.05.
Figure 28D. Plasmid map of cloning vector used in this study.
99 REFERENCES
Allos B. M., 2001. Campylobacter jejuni Infections: update on emerging issues and
trends. Clin Infect Dis 32:1201–1206.
Chang N. and Taylor D. E., 1990. Use of pulsed-field agarose gel electrophoresis to
size Campylobacter spp. genomes and to construct a SalI map of Campylobacter jejuni
UA580, J. Bacteriol. 172, pp. 5211–5217.
Nuijten, P. J. M., Bartels, C., Bleumink-Pluym, N. M. C., Gaastra, W. & van der
Zeijst, B. A. M., 1990. Size and physical map of the Campylobacter jejuni chromosome.
Nucleic Acids Res 18, 6211–6214.
Rollins D. M., and Colwell R. R., 1986. Viable but non-culturable stage of
Campylobacter jejuni and its role in survival in the natural aquatic environment. Applied
and Environmental Microbiology 52, 531-538.
de Jong AE, Verhoeff-Bakkenes L, Nauta MJ, de Jonge R., 2008. Crosscontamination in the kitchen: effect of hygiene measures. J Appl Microbiol. 105(2):61524.
CDC: Campylobacter (2010). Retrieved June, 2011, from
http://www.cdc.gov/nczved/divisions/dfbmd/diseases/campylobacter/
Tauxe R. V., 1992. Epidemiology of Campylobacter jejuni infections in the United
States and other industrial nations. In: I Nachamkin, MJ Blaser, and LS Tompkins, (Ed.).
Campylobacter jejuni: current and future trends (pp 9-12). Washington: American
Society for Microbiology.
CDC. FoodNet 2007 Surveillance Report. Atlanta: U.S. Department of Health and
Human Services, 2009.
CDC. Foodbome Diseases Active Surveillance Network (FoodNet): FoodNet
Surveillance Report for 2008 (Final Report). Atlanta, Georgia: U.S. Department of
Health and Human Services, CDC. 2010.
CDC. Foodborne Diseases Active Surveillance Network (FoodNet): FoodNet
Surveillance Report for 2009 (Final Report). Atlanta, Georgia: U.S. Department of
Health and Human Services, CDC. 2011.
Evans S., and A. R. Sayers., 2000. A longitudinal study of Campylobacter infection of
broiler flocks in Great Britain. Prevent. Vet. Med. 46:209–223.
100 Lindblom, G.B., Sjorgren, E., Kaijser, B., 1986. Natural Campylobacter colonization
in chickens raised under different environmental conditions. The Journal of Hygiene; 96,
385-391.
Harris NV, Thompson D, Martin DC, Nolan CM., 1986. A survey of Campylobacter
and other bacterial contaminants of pre-market chicken and retail poultry and meats,
King County, Washington. Am J Public Health;76:401-6.
Adak G. K, Cowden J. M., Nicholas S., Evans H. S., 1995. The Public Health
Laboratory Service national case-control study of primary indigenous sporadic cases of
Campylobacter infection. Epidemiol Infect; 115:15–22.
van Gerwe, T., Miflin, J. K., Templeton, J. M., Bouma, A., Wagenaar, J. A., JacobsReitsma, W. F., Stegeman, A., Klinkenberg, D., 2009. Quantifying Transmission of
Campylobacter jejuni in Commercial Broiler Flocks. Appl. Environ. Microbiol. 75: 625628.
Pattison, M., 2001. Practical intervention strategies for Campylobacter. Symp. Ser. Soc.
Appl. Microbiol. 30:121S–125S.
Fernie, D., and R. Park., 1977. The isolation and nature of Campylobacters
(microaerophilic vibrios) from laboratory and wild rodents. J. Med. Micro- biol. 10:325–
329.
Pearson, A. D., M. H. Greenwood, R. K. Feltham, T. D. Healing, J. Donald- son, D.
M. Jones, and R. R. Colwell., 1996. Microbial ecology of Campylobacter jejuni in a
United Kingdom chicken supply chain: intermittent common source, vertical
transmission, and amplification by flock propagation. Appl. Environ. Microbiol.
62:4614–4620.
Ruiz-Palacios, Torres G. M. J., Torres N. I., Escanilla E., Ruiz-Palacios B. R., and
Tamayo J., 1983. Cholera-like enterotoxin produced by Campylobacter jejuni
characterization and clinical significance. Lancet ii. 250-253.
Scherer K., Bartelt E., Sommerfeld C., and Hildebrandt G., 2006. Comparison of
different sampling techniques and enumeration methods for the isolation and
quantification of Campylobacter spp. in raw retail chicken legs. Int. J. Food Microbiol.
108, 115–119.
Stern, N. J., P. Fedorka-Cray, J. S. Bailey, N. A. Cox, S. E. Craven, K. L. Hiett, M.
T. Musgrove, S. Ladely, D. E. Cosby, and G. C. Mead., 2001. Distribution of
Campylobacter spp. in selected US poultry production and processing operations. J. Food
Prot. 64:1705–1710.
101 Nachamkin, I., Blaser, M.J. 2000. Campylobacter (Washington D.C., ASM Press),
p.545.
Biswas, D., Fernando, U.M., Reiman, C.D., Willson, P.J., Townsend, H.G., Potter,
A.A., Allan, B.J., 2007. Correlation Between In Vitro Secretion of Virulence-Associated
Proteins of Campylobacter jejuni and Colonization of Chickens. Curr. Microbiol.
Ringoir, D.D., Szylo, D., Korolik, V., 2007. Comparison of 2-day-old and 14-day-old
chicken colonization models for Campylobacter jejuni. FEMS Immunology and Medical
Microbiology 49, 155-158.
Biswas, D., Fernando, U., Reiman, C., Willson, P., Potter, A., Allan, B., 2006. Effect
of cytolethal distending toxin of Campylobacter jejuni on adhesion and internalization in
cultured cells and in colonization of the chicken gut. Avian diseases 50, 586-593.
Udayamputhoor, R.S., Hariharan, H., Van Lunen, T.A., Lewis, P.J., Heaney, S.,
Price, L., Woodward, D., 2003. Effects of diet formulations containing proteins from
different sources on intestinal colonization by Campylobacter jejuni in broiler chickens.
Canadian Journal of Veterinary Research = Revue Canadienne de Recherche Veterinaire
67, 204-212.
Blaser M. J., Wells J. G., Feldman R. A., Pollard R. A., Allen J. R., the
Collaborative Diarrheal Disease Study Group. 1983. Campylobacter enteritis in the
United States: a multicenter study. Ann Intern Med; 98:360–5.
Black M. J., Perlman D., Clements M. L., Levine M. M., and Blaser M. J., 1992.
Human volunteer studies with Campylobacter jejuni. In Campylobacter jejuni: Current
Status Future Trends ed.
Black, R. E., M. M. Levine, M. L. Clements T. P., Hughes and Blaser M. J., 1988.
Experimental Campylobacter jejuni infection in humans. J. Infect. Dis. 157:472-479.
Skirrow, M., J. Blaser., 1992. Clinical and Epidemiologic Considerations. In:
Nachamkin, I, Blaser, J, Tompkins, L, Campylobacter jejuni: Current status and future
trends. American Society for Microbiology. Washington: pp 3-8.
Wassenaar, T. M., and M. J. Blaser., 1999. Pathophysiology of Campylobacter jejuni
infections of humans. Microbes Infect. 1:1023–1033.
Wassenaar, T. M., B. A. van der Zeijst, R. Ayling, and D. G. Newell., 1993.
Colonization of chicks by motility mutants of Campylobacter jejuni demonstrates the
importance of flagellin A expression. J. Gen. Microbiol. 139:1171–1175.
102 Parkhill J, Wren BW, Mungall K, et al. 2000. The genome sequence of the foodborne
pathogen Campylobacter jejuni reveals hypervariable sequences. Nature; 403; 66–8.
Pickett, C. L., D. L. Cottle, E. C. Pesci, and G. Bikah., 1994. Cloning, sequencing, and
expression of the Escherichia coli cytolethal distending toxin
Scott, D. A., and J. B. Kaper., 1994. Cloning and sequencing of the genes encoding
Escherichia coli cytolethal distending toxin. Infect. Immun. 62:244–251.
Slutsker LA, Ries AA, Greene KD, Wells JG, Hutwagner L, Griffin PM., 1997.
Escherichia coli O157:H7 diarrhea in the United States: clinical and epidemiological
features. Ann Intern Med; 126:505–13.
Cope, L. D., S. Lumbley, J. L. Latimer, J. Klesney-Tait, M. K. Stevens, L. S.
Johnson, M. Purven, R. S. Munson, Jr., T. Lagergard, J. D. Radolf, and E. J.
Hansen., 1997. A diffusible cytotoxin of Haemophilus ducreyi. Proc. Natl. Acad. Sci.
USA 94:4056–4061.
Okuda, J., H. Kurazono, and Y. Takeda., 1995. Distribution of the cytolethal
distending toxin A gene (cdtA) among species of Shigella and Vibrio, and cloning and
sequencing of the cdt gene from Shigella dysenteriae. Microb. Pathog. 18:167–172.
Okuda, J., M. Fukumoto, Y. Takeda, and M. Nishibuchi., 1997. Examination of
diarrheagenicity of cytolethal distending toxin: suckling mouse response to the products
of the cdtABC genes of Shigella dysenteriae. Infect. Immun. 65:428–433.
Young, V. B., K. A. Knox, and D. B. Schauer., 2000. Cytolethal distending toxin
sequence and activity in the enterohepatic pathogen Helicobacter hepaticus. Infect.
Immun. 68:184–191.
Eyigor A., K. A. Dawson, B. E. Langlois, and C. L. Pickett., 1999. Cytolethal
distending toxin genes in Campylobacter jejuni and Campylobacter coli isolates:
detection and analysis by PCR. J. Clin. Microbiol. 37:1646–1650.
Pickett, C. L., E. C. Pesci, D. L. Cottle, G. Russell, A. N. Erdem, and H. Ringoir, D.
D., and V. Korolik., 2002. Colonization phenotype and colonization differences in
Campylobacter jejuni strains in chickens before and after passage in vivo. Vet.
Microbiol. 92:225–235.
Zeytin., 1996. Prevalence of cytolethal distending toxin production in Campylobacter
jejuni and relatedness of Campylobacter spp. cdtB gene. Infect. Immun. 64:20 2070–
2078.
103 Purdy, D., Buswell, C. M., Hodgson, A. E., McAlpine, K., Henderson, I. & Leach, S.
A., 2000. Characterisation of cytolethal distending toxin (CDT) mutants of
Campylobacter jejuni. J Med Microbiol 49, 473–479
Whitehouse, C.A., Balbo, P.B., Pesci, E.C., Cottle, D.L., Mirabito, P.M. and Pickett,
C.L., 1998. Campylobacter jejuni cytolethal distending toxin causes a G2-phase cell cycle
block. Infect. Immun. 66: 1934-1940.
Hendrixson, D.R., DiRita, V.J., 2004. Identification of Campylobacter jejuni genes
involved in commensal colonization of the chick gastrointestinal tract. Molecular
Microbiology 52, 471-484.
Nuijten, P. J. M., Bartels, C., Bleumink-Pluym, N. M. C., Gaastra, W. & van der
Zeijst, B. A. M., 1990. Size and physical map of the Campylobacter jejuni chromosome.
Nucleic Acids Res 18, 6211–6214.
Guerry P, Ewing CP, Hickey TE, Prendergast MM, Moran AP., 2000. Sialylation of
lipooligosaccharide core affects immunogenicity and serum resistance of Campylobacter
jejuni. Infect Immun, 68:6656-6662.
Wosten MM, Wagenaar JA, van Putten JP., 2004. The FlgS/FlgR two-component
signal transduction system regulates the fla regulon in Campylobacter jejuni. J Biol
Chem, 279:16214-16222.
Hiett KL, Stintzi A, Andacht TM, Kuntz RL, Seal BS., 2008. Genomic differences
between Campylobacter jejuni isolates identify surface membrane and flagellar function
gene products potentially important for colonizing the chicken intestine. Funct Integr
Genomics, 8:407-420.
Konkel ME, Klena JD, Rivera-Amill V, Monteville MR, Biswas D, Raphael B,
Mickelson J., 2004. Secretion of virulence proteins from Campylobacter jejuni is
dependent on a functional flagellar export apparatus. J Bacteriol, 186:3296-3303.
Beery JT, Hugdahl MB, Doyle MP., 1988. Colonization of gastrointestinal tracts of
chicks by Campylobacter jejuni. Appl Environ Microbiol, 54:2365-2370.
Hartley-Tassell LE, Shewell LK, Day CJ, Wilson JC, Sandhu R, Ketley JM, Korolik
V., 2010. Identification and characterization of the aspartate chemosensory receptor of
Campylobacter jejuni. Mol Microbiol, 75:710-730.
Pizarro-Cerda J., and Cossart P., 2006: Bacterial Adhesion and Entry into Host Cells.
Cell 124, 715–727.
104 Konkel ME, Garvis SG, Tipton SL, Anderson DE Jr, Cieplak W Jr., 1997.
Identification and molecular cloning of a gene encoding a fibronectin binding protein,
(CadF), from Campylobacter jejuni. Mol Microbiol, 24:953-963.
Quaroni A, Isselbacher K J, and Ruoslahti E., 1978. Fibronectin synthesis by
epithelial crypt cells of rat small intestine PNAS 75 (11) 5548-5552.
Konkel, M. E. et al. 2005. Identification of a fibronectin binding domain within the
Campylobacter jejuni CadF protein. Mol. Microbiol. 57, 1022–1035.
Monteville, M. R., Yoon, J. E. & Konkel, M. E., 2003. Maximal adherence and
invasion of INT 407 cells by Campylobacter jejuni requires the CadF outermembrane
protein and microfilament reorganization. Microbiology 149, 153–165.
Mamelli, L., Pages, J. M., Konkel, M. E. & Bolla, J. M., 2006. Expression and
purification of native and truncated forms of CadF, an outer membrane protein of
Campylobacter. Int. J. Biol. Macromol. 39, 135–140.
Jin, S. et al., 2001. JlpA, a novel surface-exposed lipoprotein specific to Campylobacter
jejuni, mediates adherence to host epithelial cells. Mol. Microbiol. 39, 1225–1236
Jin, S., Song, Y. C., Emili, A., Sherman, P. M. & Chan, V. L., 2003. JlpA of
Campylobacter jejuni interacts with surface-exposed heat shock protein 90α and triggers
signalling pathways leading to the activation of NF‑κB and p38 MAP kinase in epithelial
cells. Cell. Microbiol. 5, 165–174 .
Ashgar, S. S. et al., 2007. CapA, an autotransporter protein of Campylobacter jejuni,
mediates association with human epithelial cells and colonization of the chicken gut. J.
Bacteriol. 189, 1856–1865.
Leon-Kempis Mdel, Guccione R., Mulholland E., Williamson F., M. P. Kelly, D. J.,
2006. The Campylobacter jejuni PEB1a adhesin is an aspartate/glutamatebinding protein
of an ABC transporter essential for microaerobic growth on dicarboxylic amino acids.
Mol. Microbiol. 60, 1262–1275.
Pei, Z. et al., 1998. Mutation in the peb1A locus of Campylobacter jejuni reduces
interactions with epithelial cells and intestinal colonization of mice. Infect. Immun. 66,
938–943.
Kakuda T, DiRita VJ., 2006. Cj1496c encodes a Campylobacter jejuni glycoprotein that
influences invasion of human epithelial cells and colonization of the chick
gastrointestinal tract. Infect Immun 74:4715-4723.
105 Shreeve J. E., Toszeghy M., Ridley A., and Newell D. G., 2002. The carry-over of
Campylobacter isolates between sequential poultry flocks. Avian Dis. 46:378–385.
Fronzes, R., 2008. Architectures and biogenesis of non-flagellar protein appendages in
Gram-negative bacteria. EMBO J. 27, 2271– 2280
Ottow, J. C. 1975. Ecology, physiology, and genetics of fimbriae and pili. Annu. Rev.
Microbiol. 29, 79 – 108.
Craig, L. and Li, J., 2008. Type IV pili: paradoxes in form and function. Curr. Opin.
Struct. Biol. 18, 267 – 277.
Craig, L., Pique, M. E. and Tainer, J. A., 2004. Type IV pilus structure and bacterial
pathogenicity. Nat. Rev. Microbiol. 2, 363 – 378.
Mattick J. S., 2002. Type IV pili and twitching motility. Annu Rev Microbiol 56:289–
314.
Proft T., Baker E. N., 2009. Pili in Gram-negative and Gram-positive bacteria structure,
assembly and their role in disease. Cell Mol Life Sci 66:613–635.
Strom, M. S. and Lory, S., 1993. Structure-function and biogenesis of the type IV pili.
Annu. Rev. Microbiol. 47, 565 –596.
Wolfgang, M., VanPutten, J.P. ,Hayes, S.F., Dorward, D. and Koomey, M., 2000.
Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for
bacterial pili. EMBO J 19, 6408 – 6418.
Ellemant C. & Stewartd J., 1988. Efficacy against footrot of a Bacteroides nodosus 265
(serogroup H) pilus vaccine expressed in Pseudomonas aeruginosa. Infection and
Immunity 56, 595-600.
Doig, P., Yao, R., Burr, D.H., Guerry, P., and Trust, T.J., 1996. An environmentally
regulated pilus-like appendage involved in Campylobacter pathogenesis. Mol Microbiol
20: 885±894.
Gaynor E. C., Ghori N., and Falkow S., 2001. Bile-induced `pili' in Campylobacter
jejuni are bacteria-independent artifacts of the culture medium Molecular Microbiology
39(6), 1546±1549
Wiesner R. S., Hendrixson D. R., and DiRita V. J., 2003. Natural Transformation of
Campylobacter jejuni Requires Components of a Type II Secretion System. Journal of
Bacteriology, p. 5408-5418.
106 Syrovy, L. and Hodny, Z., 1991. Staining and quantification of proteins separated by
polyacrylamide gel electrophoresis.
Schägger H., 2006. Tricine–SDS-PAGE. Nature Protocols 1, 16 – 22.
Proft T., and Baker E. N., 2009. Pili in Gram-negative and Gram-positive bacteria
structure, assembly and their role in disease. Cell Mol Life Sci;66(4):613-35.
Olsen, A., Jonsson, A. & Normark, S., 1989. Fibronectin binding mediated by a novel
class of surface organelles on Escherichia coli. Nature 338, 652–655.
Kikuchi, T., Mizunoe, Y., Takade, A., Naito, S. & Yoshida, S. 2005. Curli fibers are
required for development of biofilm architecture in Escherichia coli K-12 and enhance
bacterial adherence to human uroepithelial cells. Microbiol. Immunol. 49, 875–884.
Hahn, E. et al., 2002. Exploring the 3D molecular architecture of Escherichia coli type 1
pili. J. Mol. Biol. 323, 845–85.
Craig, L., Pique, M. E. & Tainer, J. A., 2004. Type IV pilus structure and bacterial
pathogenicity. Nature Rev. Microbiol. 2, 363–378.
de Jong AE, Verhoeff-Bakkenes L, Nauta MJ, de Jonge R., 2008. Crosscontamination in the kitchen: effect of hygiene measures. J Appl Microbiol. 105(2):61524.
DiRita et al., 2008. Genetic Manipulation of Campylobacter jejuni. Current Protocols.
Reeser, R. J., Medler R. T., Billington S. J., Jost B. H., and Joens L. A., 2007.
Characterization of Campylobacter jejuni biofilms under defined growth conditions.
Appl. Environ. Microbiol. 73:1908-1913
Misawa, N., and M. J. Blaser., 2000. Detection and characterization of
autoagglutination activity by Campylobacter jejuni. Infect. Immun. 68:6168–6175.
Malik-Kale, P., Raphael, B. H., Parker, C. T., Joens, L. A., Klena, J. D., Quinones,
B., Keech, A. M. & Konkel, M. E., 2007. Characterization of genetically matched
isolates of Campylobacter jejuni reveals that mutations in genes involved in flagellar
biosynthesis alter the organism's virulence potential. Appl Environ Microbiol 73, 3123–
3136.
Chiang, S. L., Taylor R. K., Koomey M., and Mekalanos J. J., 1995. Single amino
acid substitutions in the N-terminus of Vibrio cholerae TcpA affect colonization,
autoagglutination, and serum resistance. Mol. Microbiol. 17: 1131–1142.
Knutton, S., Shaw R. K., Anantha R. P., Donnenberg M. S., and Zorgani A. A.,
1999. The type IV bundle-forming pilus of enteropathogenic Escherichia coli undergoes
107 dramatic alterations in structure associated with bacterial adherence, aggregation and
dispersal. Mol. Microbiol. 31:499–509.
Roggenkamp, A., Neuberger H. R., Flugel A., Schmoll T., and Heesemann J., 1995.
Substitution of two histidine residues in YadA protein of Yersinia enterocolitica
abrogates collagen binding, cell adherence and mouse virulence. Mol. Microbiol.
16:1207–1219.
Lacroix, F. J., Cloeckaert, A., Grepinet, O., Pinault, C., Popoff, M. Y., Waxin, H. &
Pardon, P., 1996. Salmonella typhimurium acrB-like gene: identification and role in
resistance to biliary salts and detergents and in murine infection. FEMS Microbiol Lett
135, 161–167.
Bina JE, Mekalanos JJ., 2001. Vibrio cholerae tolC is required for bile resistance and
colonization. Infect Immun 69:4681–4685
Hendrixson D. R., Akerley B. J., and DiRita V. J., 2001. Transposon mutagenesis of
Campylobacter jejuni identifies a bipartite energy taxis system required for motility.
Molecular Microbiology, 40(1), 214-224.
Hendrixson D. R., and DiRita V. J., 2004. Identification of Campylobacter jejuni genes
involved in commensal colonization of the chick gastrointestinal tract. Molecular
Microbiology, 52 (2), 471-484.
Falkow S., Isberg R.R., and Portnoy D. A., 1992. The interaction of bacteria with
mammalian cells. Annu. Rev. Cell. Biol. 8:333-363
Takeuchi A., 1967. Penetration into the intestinal epithelium by Salmonella
Typhimurium. Am. J. Pathol. 50:109-136
Sansonetti P.J., 1991. Genetic and molecular basis of epithelial cell invasion by Shigella
species. Rev. Infect. Dis. 13:S285-292.
Guerry, P., Alm R. A., Power M. E., Logan S. M., and Trust T. J., 1991. Role of two
flagellin genes in Campylobacter motility. J. Bacteriol. 173:4757–4764.
Wassenaar, T. M., Bleumink-Pluym N. M., and van der Zeijst., 1991. Inactivation of
Campylobacter jejuni flagellin genes by homologous recombination demonstrates that
flaA but not flaB is required for invasion. EMBO J. 10:2055–2061.
Yao, R., Burr D. H., Doig P., Trust T. J., Niu H., and Guerry P., 1994. Isolation of
motile and nonmotile insertional mutants of Campylobacter jejuni: the role of motility in
adherence and invasion of eukaryotic cells. Mol. Microbiol. 14:883–893.
108 Hu, L., and Kopecko D. J., 1999. Campylobacter jejuni 81–176 associates with
microtubules and dynein during invasion of human intestinal cells. Infect. Immun.
67:4171–4182.
Frølund, B., Palmgren, R., Keiding, K. & Nielsen, P.-H., 1996. Extraction of
extracellular polymers from activated sludge using a cation exchange resin. Water Res.
30, 1749–1758.
Wingender. J., Strathmann, M., Rode, A., Leis, A. & Flemming, H.-C., 2001.
Isolation and biochemical characterization of extracellular polymeric substances from
Pseudomonas aeruginosa. Methods Enzymol. 336, 302–314
Lawrence, J. R., Swerhone, G. D. W., Kuhlicke, U. & Neu, T. R., 2007. In situ
evidence for microdomains in the polymer matrix of bacterial microcolonies. Can. J.
Microbiol. 53, 450–458.
Newell DG, Fearnley C. 2003. Sources of Campylobacter colonization in broiler
chickens. Appl Environ Microbiol; 69(8):4343–51.
Beery JT, Hugdahl MB, Doyle MP. 1988. Colonization of gastrointestinal tracts of
chicks by Campylobacter jejuni. Appl Environ Microbiol; 54(10):2365–70.
Dhillon AS, Shivaprasad HL, Schaberg D, Wier F, Weber S, Bandli D., 2006.
Campylobacter jejuni infection in broiler chickens. Avian Dis, 50(1):55–8.
Loc Carrillo C, Atterbury RJ, el-Shibiny A, Connerton PL, Dillon E, Scott A, et al.,
2005. Bacteriophage therapy to reduce Campylobacter jejuni colonization of broiler
chickens. Appl Environ Microbiol; 71(11):6554–63.
Wagenaar JA, Van Bergen MA, Mueller MA, Wassenaar TM, Carlton RM., 2005.
Phage therapy reduces Campylobacter jejuni colonization in broilers. Vet Microbiol,
109(3–4):275–83.
Curtiss, R., III, and S. M. Kelly. 1987. Salmonella typhimurium deletion mutants
lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and
immunogenic. Infect. Immun. 55:3035–3043.