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Project No:
Responsible investigator: Hin-chung Wong
Title: Characterization of the Stress-induced Factors involved in the Pathogenesis of
Vibrio parahaemolyticus
SPECIFIC AIMS
It is hypothesized that when Vibrio parahaemolyticus enters the gastrointestinal tract of
human, it will induce a number of events to adapt to the new substantially different
environment. Such events may enhance the survival, adherence, colonization and toxin
production of this pathogen in the adverse environment leading to the host’s diarrheal
manifestation. Recently, we identified factors being induced by mild acid and heat shock
treatment and demonstrated significant increase in enteropathogenicity of the
stress-adapted bacteria. To understand the roles of these stress-induced factors on the
pathogenesis of this pathogen, the following specific aims, that are directly derived from
the works carried out during the last project period, are proposed:
1. Biochemical characterization of the mild acid and heat shock induced factors,
specifically those in the outer membrane and extracellular fractions of V.
parahaemolyticus.
2. Determination of specific roles of the mild acid and heat shock induced factors in the
pathogenesis of V. parahaemolyticus.
3. Molecular characterization of the mild acid and heat shock induced factors of V.
parahaemolyticus.
4. Determination of the regulation system in controlling the expression of these inducible
factors.
BACKGROUND
Vibrio parahaemolyticus is a halophilic Gram-negative straight-to-curved rod bacterium,
with a single polar flagellum when grown in liquid medium, or peritrichous flagella on
solid medium. It was first discovered in 1950 and it is now one of the most frequently seen
food-borne pathogens in Taiwan, Japan and other countries {337}. High infection
incidence of this pathogen undoubtedly is originated from frequent consumption of marine
foods in these countries. Clinical manifestations include diarrhea, abdominal cramps,
nausea, vomiting, headache, fever, and chills, with incubation period ranging from 4 to 96
hours {236, 337}. Most clinical isolates are hemolytic on Wagatsuma agar
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(Kanagawa-positive, KP+). In contrast, most environmental isolates are
Kanagawa-negative (KP-) {236}. Takeda {328} reported that KP+ strains of V.
parahaemolyticus produced a thermostable direct hemolysin (TDH). This hemolysin and
other related hemolysins have been purified and characterized {18, 99, 14, 6, 364}.
Although identified in TDH, the enterotoxigenic property in this hemolysin is less active in
general than the heat-labile enterotoxin of Escherichia coli or cholera toxin of V. cholerae.
Also unclear is the virulence factor(s) that is(are) regulated by environmental signals in
this organism.
Enteric pathogens are exposed to substantial environmental changes when they enter a
mammalian host, and they have acquired a number of means to adapt to these changes. The
pathogen can be deprived of certain nutrients, exposed to oxygen radicals and changes in
pH, and bathed in degradative enzymes. To cope with such hostile environment, the
pathogens synthesize stress proteins or heat shock proteins, some of which are associated
with the pathogenesis of these pathogens. Environmental signals stresses controlling the
expression of coordinately regulated virulence determinants have been well characterized
in a number of pathogens, including enteric bacteria {375, 676}.
Among these, acid tolerance response (ATR) has been demonstrated in several
pathogenic bacteria, such as Escherichia coli {783}, Listeria {811}, Streptococcus {808},
Aeromonas {721} and Salmonella {798}. Intensive studies have been carried out on S.
typhimurium {781}. S. typhimurium strains containing two or three different ATR gene
mutants are acid sensitive and also much less virulent {797}. Regulation of virulence by
ATR has also been demonstrated in other pathogenic bacteria. In L. monocytogenes, an
acid-tolerant mutant has been to increase virulence in an intraperitoneally infected mouse
model {811}. Effects of other environmental stresses, such as nutrition starvation, heat and
high level of metallic ions, on survival and physiological aspects, have been studied on
several Vibrio species {52, 775}.
In response to these stresses, the bacteria produce stress proteins and some of these
stress proteins, such as the sigma factor or Hsp60, are conserved in many eukaryotic and
prokaryotic organisms. Some of them are involved in the pathogenesis of bacteria. The
sustained ATR in virulent S. typhimurium requires the presence of alternative sigma factor
s encoded by rpoS that in turn controls the synthesis of seven acid shock proteins {782}.
Similar factors are also present in E. coli and Yersinia enterocolitica and are responsible
for the survival of these bacteria under stress conditions {810, 869}. The sigma factor is
also related to the virulence of some pathogens. In S. typhimurium, one component of a
potential signal transduction system responsible for inducing rpoS expression is the
product of the mouse virulence gene mviA+ {874}. A Hsp60 protein has been recognized
by sera of patients affected by Helicobacter pylori, that is implicated in the induction of
autoimmune disorders in different systems {867}.
Also, there are other species-specific stress-induced factors involved in the pathogenesis
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of some pathogens. In Yersinia pseudotuberculosis mutant strains cured of the virulence
plasmid, culture cell adherence factors (psa locus) are demonstrated to be induced by heat
shock. Three genes in such psa locus, psaE, psaA and psaB, are involved in the synthesis
of a pilus-like surface structure in another closely related species, Y. pestis {880}.
Exposure of the rifampin-resistant mutant of Francisella tularensis to heat stress for 5 h in
vitro resulted in an increase of virulence, and the envelope preparations from such
heat-stressed bacteria show increased levels of several proteins, notably the 16,60 and 75
kDa proteins {870}. In S. dublin, stress induces the expression of virulence proteins (SpvA,
-B, and -C) from the virulence plasmid {634}. In another study, a 66 kDa proteins involved
in binding of S. typhimurium intestinal mucus was induced by heat shock treatment {753}.
These observations suggest that species-specific stress-induced factors present in the outer
membrane and extracellular fractions may have significant roles in the pathogenesis of
these pathogens.
The stress regulatory systems may be closely related to each other. For example, the
ferric uptake regulator ( fur ) is also involved in ATR. In S. typhimurium, fur mutants fail
to mount an effective ATR, and a clear subset of seven proteins are influenced by both acid
and iron and controlled by fur {789}. In A. hydrophila, the level of iron in the culture
medium did not affect the ATR {721}. These studies support the idea that Fur is a major
global regulator and may have profound influences on the pathogenesis of V.
parahaemolyticus.
The effect of high or low temperature and other adverse conditions on the survival of V.
parahaemolyticus have been investigated {472}, but the effect of environmental factors
including temperature on the expression of virulence factors and their role in the
pathogenesis of this pathogen is still unknown.
PRELIMINARY STUDIES
Described below are the results obtained so far including those obtained in the last two
years of the three year project currently being carried out. In V. parahaemolyticus, many
virulence factors have been reported, but their roles in the pathogenesis of this pathogen
have not been clearly elucidated except TDH. We postulated that some unidentified but
important virulence factors may be induced only under stress conditions similar to many
other enteropathogens {275}. Iron, a virulence factor, plays an important role in the
pathogenesis of many enteropathogenic bacteria {343, 319, 339}.We have also
investigated the effect of iron level in the culture medium on the virulence of this pathogen.
Level of iron in the mucus and close proximate region of the epithelium of intestinal gut is
usually limited because of the presence of the host’s iron-binding factors. In our previous
research project that is still being carried out, we examined the ATR and heat shock
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phenomena in this pathogen and identified stress-induced proteins by gel electrophoresis.
Enteropathogenicity of the acid adapted bacteria was significantly enhanced as assayed
with the suckling mouse model {776, 881}.
1. Effect of Iron on the Virulence of V. parahaemolyticus
Roles of iron in the pathogenesis of V. parahaemolyticus are still not well elucidated. In
an in vitro study, the production of siderophore, two outer-membrane proteins (77 and 80
kDa OMP) and TDH of this organism are significantly enhanced in iron-limited culture,
and its hemolytic activities, hydrophobicity, and adherence to HEp-2 cell are also enhanced
in clinical isolates of V. parahaemolyticus {730}. V. parahaemolyticus grown in an
iron-limited medium and intraperitoneally infected showed a better growth at least for four
days in the mose when iron was also supplemented with the infection. The effect of
iron-limitation may be important at least during the early post-infection period and it may
enhance the lethality of some less virulent strains to the mouse {570}.
Therefore, the iron and virulence relationship was further investigated using mutants.
Spontaneous iron-utilization mutants were isolated from a clinical strain ST550.
Comparing with the parental strain, lowered virulence was demonstrated in these mutants
assayed with adult mice as well as suckling mice. The in vivo growth and
enterotoxigenicity of these mutants were lowered in suckling mice. Adherence of these
mutants to the excised mouse intestine was also lowered as demonstrated by scanning
electron microscopy. The iron-regulated outer membrane protein (OMP) profile also
changed in selected mutants. Taking together, these results indicate that the iron-regulated
outer membrane protein and other unknown factors associated with the iron utilization may
have profound influence on the pathogenesis of V. parahaemolyticus {765}.
Since as seen above, presence of sufficient iron or utilization of iron is very important in
the virulence of V. parahaemolyticus, this organism has acquired the ability to use hemin,
hemoglobin, ferritin, lactoferrin and transferrin as iron source in addition to the ferric
ammonium citrate {765}.
2. The Iron Regulation System in V. parahaemolyticus
We next investigated the iron-regulation system and the iron-regulated OMP of V.
parahaemolyticus. Southern blot hybridization with E. coli fur gene probe under stringent
condition yielded positive bands in all strains of V. parahaemolyticus examined. We
confirmed the presence of a Escherichia coli Fur(ferric uptake regulator)-like system in this
pathogen by characterizing its manganese-resistant mutants. Five mutants induced by
N-methyl-N'-nitrosoguanidine mutagenesis were resistant to 3 mM manganese chloride.
Production of iron-regulated 77-kDa OMP and siderophore, and bacterial growth were
changed in these Mn-resistant mutants as compared to wild-type strain, and such changes
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were partially or completely restored by the introduction of E. coli fur gene into these
mutants {678}.
In another molecular study, a complete coding region of the fur-like gene of V.
parahaemolyticus was obtained and sequenced. The Fur protein of this species comprises
of 149 amino acid residues deduced from the nucleotide sequence. Comparing with the E.
coli fur gene, the fur-like gene of this species showed 71.1 and 79.9% homologous in terms
of nucleotide sequence and deduced amino acid sequence, respectively. Comparing with
the V. cholerae fur-like gene, the fur-like gene of this species showed 80.0 and 92.6%
similarity in terms of nucleotide and deduced amino acid sequence, respectively {815}.
On the other hand, the 77-kDa OMP that was negatively regulated by iron was purified
and N-terminal amino acid sequence determined, and the gene encoding this protein was
also cloned {627}. TonB binding sequence (TonB box) which occurs concurrently in many
iron-receptors {627} was identified in the N-terminal sequence of this OMP, it is, therefore,
reasonable to assume that this OMP plays an important role in the uptake of iron in this
pathogen.
3. The Adaptive Acid Tolerance Response and Heat Shock Phenomenon in V.
parahaemolyticus
Recently, we made significant findings on the ATR and heat shock phenomena in V.
parahaemolyticus. In fact, the ATR phenomenon has not been reported in Vibrio species.
In the previous V. parahaemolyticus studies, nutritional starvation was shown to induce
cross-protection against heat, osmotic, or hydrogen peroxide challenge {760}, and enhance
the survival of this pathogen at low temperature {762}. However, enteropathogenic V.
parahaemolyticus may also encounter high acidity in food-handling environment or in
human gastroenteric tract. Logarithmically grown V. parahaemolyticus at pH 7.5 but
shifted to pH 5.0 for 30 min were more resistant to the subsequent acid challenge at pH 4.4.
A two-phase adaptive procedure (pH 5.8 30 min, pH 5.0 30 min) was better than a
single-phase procedure for enhancing the acid tolerance of this pathogen. The acid-adapted
cells were cross-protected against the challenges of low salinity and thermal inactivation.
Cross-protection by the adaptive acid tolerance response was also investigated in S.
typhimurium against the challenge of heat, salt, an activated lactoperoxidase system, and
the surface-active agents crystal violet and polymyxin B {638}. One- and two-dimensional
polyacrylamide gel electrophoresis (PAGE) revealed that 20 species of proteins were
induced or significantly enhanced, while 26 species were inhibited. Using the suckling
mouse, enteropathogenicity of the acid adapted bacteria was shown to significantly
enhance the intestine/body weight ratio and in vivo recovery of infected bacteria {776}.
Since several stress proteins are highly conserved in many organisms, homologous proteins
may be produced during acid tolerance in V. parahaemolyticus . Simply judging from
mobility and position in the 2-D PAGE, DnaK-like protein was observed {635}.
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Identification of these proteins is now being performed.
In the heat shock study, logarithmically grown V. parahaemolyticus, heat shocked at
42C for 30 min, were more resistant to thermal inactivation at 47C than the unshocked
ones. The two-dimensional PAGE analysis showed that after heat shock treatment of the
25C culture, 24 species of proteins were induced while 13 species inhibited. Identical but
fewer number of proteins were regulated during the heat shock of the 37C culture as
compared with that of the 25C culture. DnaJ- and GroEL-like proteins, with molecular size
of 40 and 62 kDa, respectively, were detected by immunoblotting with antibodies against
the Escherichia coli proteins. Comparing the proteins which were enhanced by heat shock
or by adaptive acid tolerance response in this pathogen by 2-D PAGE, 18 species occurred
exclusively in heat shock response and 16 species occurred exclusively in ATR, with only
four protein species occurred in both stress conditions {881}. A 58 kDa Hsp60
(GroEL)-like protein has been demonstrated, by immunoblotting, in V. parahaemolyticus
and in six other Vibrio species after a heat shock from 30 to 42C {764}.
4. Enteropathogenicity of V. parahaemolyticus assayed with Caco2 cells
We have been working with Dr. P.S.Liu, a cytologist, in establishing a new cell model
for the investigation of the cytotoxic and cytotonic virulence factors in this pathogen.
Diarrhea induced by enteropathogen is water efflux from intestinal cells coupled with
secretion of Cl- and Na+ and it is regulated by several factors, such as cAMP, inositol
trisphosphate, and calcium. Caco-2 cells, originating from a human colon adenocarcinoma,
could be an appropriate cell model in studying the cytotoxic and cytotonic activities of
enterotoxins, such as cholera toxin, and thermolabile enterotoxin of E. coli. We measured
the [Na+]i and [H+]i of Caco-2 cells by using florescent dye SBFI and BCECF, respectively,
and enzyme-linked immunosorbent assay (ELISA) kit was used to measure [cAMP]i. We
found that pathogenic strain V. parahaemolyticus ST550 lowed [Na+]i of Caco-2 cells.
After 1 hr, cytosolic sodium concentration was lowered for about 0.2 degree of the ratio of
340 nm and 380 nm excitation and 500 nm emission florescent intensity. TDH also
induced an immediate and sustained suppressing effect on cytosolic sodium concentration.
Dead bacteria had no effects on cytosolic sodium concentration. ST550 also lowered pHi
for about 3.5 degree of the fluorescence ratio within 3 hr. However, TDH had no effects on
pHi. Pathogenic strains of V. parahaemolyticus showed significantly higher shift in pHi
than environmental non-pathogenic strains. Cholera toxin elevated cytosolic cAMP
concentration that was enhanced in the presence of IBMX, phosphodiesterase inhibitor,
and thapsigargin (ER Ca2+ pump blocker). In the presence of IMBX and thapsigargin,
ST550 also slightly elevated cytosolic cAMP concentration. The above results indicates
that pathogenic strains of V. parahaemolyticus have significant effects on the sodium and
proton mobilization and cAMP might be involved (unpublished data).
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RESEARCH DESIGN AND METHODS
A toxRS operon has been shown in V. parahaemolyticus and probably mediates
regulation of the expression of TDH and other virulence factors upon stimulation of
different environmental stresses {600}. Recently we identified factors induced by mild
acid and heat shock treatment in V. parahaemolyticus and upon the induction the
enteropathogenic virulence of the stress-adapted bacteria was significantly enhanced. The
role of the homologous factors (GroEL, DnaJ, RpoS etc.), and species-specific
stress-induced factors especially in the OMP and extracellular fractions, on the
pathogenesis of this pathogen, is next need to be examined. To achieve this, the following
Aims are established. The experiments designed to reach these Aims are described below:
Specific Aim 1. Biochemical characterization of the mild acid and heat shock induced
factors, specifically those in the outer membrane and extracellular fractions of V.
parahaemolyticus.
1. Purification of stress-induced proteins in the OMP and extracellular fractions.
Factors in the OMP are responsible for cellular adherence, protection, uptake of nutrient
or other virulence related functions. We have already demonstrated the regulation of iron
on the expression of OMPs in V. parahaemolyticus {678}. Several species-specific
stress-induced proteins have been purified from OMP and extracellular fractions and are
associated with pathogenesis in some pathogenic bacteria. A 66 kDa protein has also been
purified from the extracellular fraction of S. typhimurium and was responsible for the
aggregation and binding to the intestinal mucus. In V. cholerae, OmpU, a protein positively
regulated by toxR, is associated with adhesion of this bacterium to mammalian cells and
hemagglutination {877}. It is reasonable to suggest that stress-induced factors are also
present in the OMP and extracellular fractions in V. parahaemolyticus. In our present study,
we will attempt to identify the specific stress-induced factors in the OMP and extracellular
fractions in V. parahaemolyticus. These stress-induced factors will be purified by standard
biochemical methods or by affinity column chromatography.
For the purification of OMPs the method of Chakrabarti et al. will be used {876}. The
stressed bacteria are first collected by centrifugation and disrupted in an ultrasonic
disintegrator. Bacterial membrane fraction is then pelleted by centrifugation. The inner
membrane fraction is solubilized by 1% Sarkosyl NL-97 and the outer membrane fraction
separated by centrifugation. Inner membrane contamination of the outer membrane
preparation can be estimated by measuring the cytochrome b content, which is located in
the inner membrane and used as a marker {876}. The OMP will be purified by column
chromatography and analyzed by gel electrophoresis {876}.
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For the extracellular proteins in the culture supernatant, the culture is subjected to stress
treatment and collected by ammonium sulfate precipitation and centrifugation. The
stress-induced proteins in this fraction will be purified by a different column
chromatography and analyzed by gel electrophoresis.
Among the stress proteins, the GroEL- and DnaJ-like protein have been demonstrated in
stress adapted V. parahaemolyticus {881}. These stress-proteins which are homologous to
proteins of other species could be purified by affinity column chromatography that is
prepared by incorporating the commercial or laboratory-made antibody into the column
mattrix. The whole bacterial fraction or fractionated preparations will be subjected to the
affinity column chromatography and the purified proteins analyzed by gel electrophoresis.
2. Biochemical characterization of these stress-induced proteins.
The purified proteins are next characterized biochemically. Thus, molecular weight,
isoelectric point, amino acid composition and amino acid sequencing of these purified
proteins will be determined by conventional methods {876}. Amino acid sequencing will
be performed with an automated amino acid sequencer.
Specific Aim 2. Determination of specific roles of the mild acid and heat shock
induced factors in the pathogenesis of V. parahaemolyticus.
From the biochemical characteristics of the stress induced proteins, some roles of the
proteins may be inferred but here we will attempt to determine their specific roles. The
extracellular factors may have cytotoxic/cytotonic activities or may induce the immune
response of the infected host {867}. The factors in the OMP fractions may be a toxic factor,
involved in the cellular adherence and/or play other functions during the pathogenesis of
the pathogen {877}. Roles of these stress-induced factors will thus be examined by in vitro
and in vivo methods described below.
1. Preparation of polyclonal antibody against the stress-induced factors
Anti-factors antibodies are useful for the detection as well as functional test for these
factors. Polyclonal antibody against these purified stress-induced factors will therefore be
prepared by immunizing rabbit and purified by ammonium sulfate precipitation,
DEAE-column or protein A column chromatography {624}. ELISA or immunoblot
procedures with these polyclonal antibody will be employed for the quantification of these
stress-induced factors.
2. Cytotoxic or cytotonic activity of the stress-induced factors
Next, the cytotoxic or cytotonic activity of the purified stress-induced factors will be
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determined. We have established a Caco-2 cell model which is described earlier for the
examination of toxicity of virulence factors in V. parahaemolyticus at cellular level. We
will measure the [Na+]i and [H+]i of Caco-2 cells by using florescent dye SBFI and BCECF,
respectively. We will use ELISA kit for the determination of [cAMP]i which will
accumulate in the presence of a typical enterotoxin, such as cholera toxin, and the factors
may also show a similar event.
We will also use the Chinese hamster ovary (CHO) cell and Vero cells for the
determination of cytotoxic or cytotonic activity of these stress-induced factors and the
methods for such tests have already been established and published by our laboratory
{414}.
3. Roles of the stress-induced factors in bacterial adhesion during pathogenesis
The stress-induced factors in the OMP fraction may be involved in the bacterial
adhesion during pathogenesis {877}. Roles of the OMP factors in bacterial adhesion will
therefore be determined in in vitro assay and cell culture model.
These factors are first adhered to immobilized matrix proteins (fibronectin, laminin, and
collagen) and the arginine-glycine-aspartic acid tripeptide is then examined employing the
method of Sperandio et al. {877}. Microplate wells are coated with these mattrix proteins,
incubated with these stress-induced factors, washed, and the quantity of the adhered factors
are determined by ELISA method using the polyclonal antibody prepared in this study.
Also, the role of these factors on adhesion will be examined in vitro using HEp-2 or
Caco-2 cell as previously described {730}. These factors will be added to the cell culture,
incubated for about 30 min, and washed with fresh culture medium. The amount of the
stress-induced factor attached on the cell culture will be determined by ELISA method
using the polyclonal antibody prepared in this project. A dose response curve will be
determined using various concentration of these stress-induced factors.
The anti-stress proteins antibodies can be used to inhibit bacterial adhesion and the
inhibition can be assayed using the HEp-2 or Caco-2 cells {877}. V. parahaemolyticus will
be added to the cell monolayer. Inhibition of bacterial adhesion to the cell culture will be
examined by preincubating the cell culture or the bacteria for 20 min at room temperature
with specific antibodies at various dilutions. Number of bacteria adhered after washing is
determined by lysing the cells and count the colony formed on tryptic soy agar plate {877}.
4. Animal model for passive protection
When the function of a virulence factor on a live pathogen is blocked by a drug or
attachment of antibody, the pathogen is rendered avirulent, and the infected animal would
be protected. For example, if the adhesin is blocked by the antibody, the bacteria will be
unable to adhere to the tissue or cells, and thus render the bacteria avirulent and the animal
protected. Such passive protection effect can be examined by the infant mouse model
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{877}. The infant mouse model has been successfully used in the study of pathogenesis in
this pathogen in our laboratory {765}. The test is therefore performed as follows. Bacterial
suspension is mixed with the antibody, and infant mice (3 to 5 days old) will be inoculated
orally with 50 µl of this vibrio-antibody mixture. Protection is determined by mouse
survival after incubation for 24-48 h {877}.
Specific Aim 3. Molecular characterization of the mild acid and heat shock induced
factors of V. parahaemolyticus.
We will next characterize molecularly the stress-induced factors but only on specific
stress-induced factors that have been proved in our study to be important in the
pathogenesis of this pathogen. Specific genes will be cloned and characterized. Presence of
these genes in clinical and environmental strains of V. parahaemolyticus will be examined,
because these genes may be absent in the latter strains. Isogenic mutants for these genes
will be made and the changes in the virulence of these mutants will be investigated.
1. Cloning and genetic analysis of the stress-induced factors
In order to examine genetically the stress-induced factors, their genes, wild-type or
mutants, must be isolated. Thus, genes of these stress-induced factors of V.
parahaemolyticus will be cloned into E. coli by standard molecular techniques, and
nucleotide sequences of the selected clones will be determined by standard dideoxy
method {620}. Degenerate probes derived from the N-terminal end sequences (determined
in Specific Aim 1) of these factors will be prepared and used in screening of the clones.
The nucleotide sequences of the clones will be analyzed by PCgene software and also
compare with published gene sequences of E. coli, V. cholerae and other enteric bacteria.
2. Presence of the stress-induced factors in clinical and environmental strains of
Vibrio parahaemolyticus.
Environmental strains are generally avirulent and therefore clinical strains may contain
virulence genes that may be absent in the environmental strains. Therefore, we will be
design primers for polymerase chain reaction (PCR) from the nucleotide sequences of these
stress-induced factors. Special gene fragments will then be amplified by PCR using these
primers. Such amplified fragments will also be used as confirmation probes. Strains of V.
parahaemolyticus collected from the clinical and environmental sources will be assayed for
the presence of these factors by PCR using the same primers and confirmed by Southern
hybridization using the confirmation probes {620}. Certain major virulence factors that are
present in the clinical strains but absent in the environmental non-pathogenic strains may
be found.
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3. Determination of the roles of the stress-induced factors by using isogenic mutants.
To examine the roles of the stress-induced factors in the virulence, isogenic mutants
with a mutation in their genes of these factors will be isolated. To do this, an antibiotic or
beta-galactosidase gene will be inserted by in vitro manuipulation into the cloned genes.
Such cloned genes with the insertion will be transformed by electroporation established in
our laboratory {660} into wild-type V. parahaemolyticus and the antibiotic or
beta-galactosidase genes will be allowed to integrate into the corresponding genes of the
wild-type strains via homologous recombination. The antibiotic or beta-galactosidase is
served as a marker and their integration is served as mutation. The pathogenicity of these
isogenic mutants will then be determined by methods described in Specific Aim 2.
Specific Aim 4. Determination of the regulation system in controlling the expression
of these inducible factors.
Regulation of these stress-induced factors that are obviously important in pathogenesis
of this pathogen will be studied in several ways. One is to examine expression of these
factors under different environmental stresses. Role of the regulatory gene (e.g. the toxRS)
in the regulation of these factors will also be investigated. These are described below.
1. Effect of various stresses on the expression of the stress-induced factors
To examine the effect, V. parahaemolyticus at late-exponential phase will be exposed to
various stress conditions, such as heat shock, mild acid treatment, osmotic shock, nutrient
starvation, and heavy metal treatment {760, 761}, and expression of the selected factors
determined. First, total cellular proteins will be collected and the relative quantity of these
factors determined by ELISA or immunoblot procedure using specific antibody prepared in
Specific Aim 2. Increased protein amount would indicate induction by the stress. We will
isolate and measure the amount of total mRNA of the stress-treated bacterial culture using
conventional method {620}. The transcription level, another measure of expression for
these genes, will also be determined by northern blotting procedure with hybridization
probes derived from the nucleotide sequences {620}.
2. Role of the toxRS in the regulation of the stress-induced factors
The cloned toxRS gene will be mutated by site directed mutagenesis and introduced
into V. parahaemolyticus by electroporation {679}. Isogenic
mutants with defective toxRS genes, that are formed presumately by homologous
recombination, will be selected and the defect confirmed by nucleotide sequencing.
Expression of these stress-induced factors in such toxRS mutant strain under different
stress conditions will be examined by ELISA, immunoblot and northern blot procedures.
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Survival of such mutant strain under various stress conditions will also be determined {776,
881}. Because of the defect in toxRS, these mutants should fail to regulate the expression
of these stress-induced factors.
ANTICIPATED RESULTS
The experimental design and method of this application is written in a sequential order
together with anticipated results. Results obtained in an Aim will facilitate the planing and
execution of the succeeding experiments. Specific Aim 1 proposes to purify and
characterize the factors in the OMP and/or extracellular fractions induced by heat shock or
mild acid treatment. The pathogenic roles of these factors can in turn be clarified by in
vitro and in vivo experiments, the second Specific Aim. Knowing these factors, the genes
encoding these factors will be cloned, sequenced and analyzed, the third Specific Aim.
Nucleotide sequence data obtained will facilitate the study of regulations of these factors.
In the last part of this project, regulation of toxRS on the expression of the specific
stress-induced factors will be elucidated. These results on the stress-induced factors should
add to further biological, and can be utilized in the devising of methods, including various
forms of vaccines, for the control of this pathogen.
HUMAN SUBJECTS
In this study, no human subjects will be involved.
POTENTIAL HAZARDOUSNESS
V. parahaemolyticus causes diarrhea when certain amount of viable bacteria are
ingested. Personnel in the laboratory is well trained with aseptic techniques and all the
laboratory materials and labwares containing or having contacted with this bacterium will
be autoclaved before disposal or sterilized for reuse.
ANIMAL INVESTIGATION
Adult New Zealand white rabbits will be used in the production of polyclonal
antibodies. Suckling ICR mice will be used in assaying the virulence properties of the
stress-induced factors. These animals are bred and raised in the National Laboratory of
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Animal Breeding and Research (National Science Council, ROC). The investigators will
follow the "Guide for Laboratory Animal Facilities and Care" promulgated by the
Committee of the Guide for Laboratory Animal Facilities and Care of the Institute of
Laboratory Animal Resources, National Academy of Sciences, the National Research
Council, USA, throughout this research.
REFERENCES
1. Albertson, N. H., T. Nystroem, and S. Kjelleberg. 1990. Macromolecular synthesis
during recovery of the marine Vibrio sp. S14 from starvation. J. Gen. Microbiol.
136:2201-2207.
2. Badger, J. L., and V. L. Miller. 1995. Role of RpoS in survival of Yersinia
enterocolitica to a variety of environmental stresses. J. Bacteriol. 177:5370-5373.
3. Bearson, S. M. D., W. H. Benjamin,Jr., W. E. Swords, and J. W. Foster. 1996. Acid
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