![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Infection and Immunity, November 2002, p. 5990-5996, Vol. 70, No. 11
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.11.5990-5996.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Zentrum für Infektionsforschung, Universität Würzburg, 97070 Würzburg, Germany,1 University of Texas Health Science Center, San Antonio, Texas 78229-39002
Received 8 May 2002/ Returned for modification 8 July 2002/ Accepted 5 August 2002
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
30-fold reduction in colonization of the infant mouse small intestine, indicating that the presence of both LPS O side chain and CPS is important during the colonization process. The strain lacking both CPS and LPS O side chain and a CPS-positive, LPS O side chain-negative core OS mutant were both essentially unable to colonize. To characterize the role of surface polysaccharides in survival in the host intestine, resistance to several antimicrobial substances was investigated in vitro. These investigations revealed that the presence of CPS protects the cell against attack of the complement system and that an intact core OS is necessary for survival in the presence of bile. |
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The encapsulated O139 pathogen emerged in late 1992, causing cholera epidemics in Bangladesh and India (23). Molecular and epidemiological analysis as well as whole-genome comparison revealed that O139 strains are very similar to O1 El Tor strains, supporting the hypothesis that pathogenic O139 isolates evolved from the recent seventh pandemic El Tor strains (3, 7, 9, 10).
One genetic difference between O1 El Tor and O139 strains corresponds to the surface polysaccharide biosynthesis (wb*) gene cluster. In O139 strains, the genes encoding the O1 antigen are deleted and replaced by the O139 antigen (wbf) locus, encoding the lipopolysaccharide (LPS) O side chain and the capsular polysaccharide (CPS) (34). The structures of the LPS O side chain and the CPS are identical, and it was found that one O139 side chain unit is linked to lipid A-core oligosaccharide (OS), whereas the LPS-unlinked CPS is formed by polymerized O139 side chain repeat units (15, 16, 41).
Consistent with the structural data, mutations in putative O139 side chain biosynthetic genes led to LPS O side chain- and CPS-negative mutants (5, 41). On the other hand strains with mutations in putative capsule translocation genes are still able to express the LPS O side chain but they are CPS-negative (4). No defined LPS O side chain-negative but CPS-positive mutants linked to the wbf cluster have been described; only spontaneous mutants with unknown mutation(s) have been isolated so far (2).
Other gram-negative bacteria are known that, like O139 strains, express side chain repeat units as a high-molecular-weight capsular and a low-molecular-weight lipid A-core-linked form. These strains are classified based on their biosynthetic gene clusters as expressing either group 1 or group 4 capsule (44). The current biosynthetic model for these groups proposes that individual repeat units are assembled on a lipid carrier in the cytoplasm and then transferred across the inner membrane by a polysaccharide exporter protein. The repeat units can serve as a substrate for the polymerization and capsule translocation machinery or for the WaaL enzyme, which ligates one repeating unit onto the lipid A-core OS (44).
Given the similarity, it is likely that V. cholerae O139 strains use a similar pathway. The structures of the LPS core OS of O1 and O139 strains were reported to be fairly similar (13), and we showed recently that they have identical core OS biosynthetic (wav) gene clusters (25). The putative lipid A core-surface polymer ligase WaaL is also encoded by the wav gene cluster, and mutations within this gene led to an O side chain-negative O1 El Tor mutant (25). Since the O1 and O139 waaL genes are identical (25), it is likely that ligation of the O139 side chain to the lipid A core is also catalyzed by this enzyme.
The role of the O139 surface polysaccharides in V. cholerae pathogenesis is still not completely understood. The CPS layer is hydrophobic and very thin, and it provides only partial protection against phagocytosis (1, 21). However, synthesis of the capsular material is required for efficient colonization of the small intestine in suckling mice (41). CPS is also required for serum resistance (41), and this phenotype might be relevant for intestinal colonization of humans by O139 strains, since complement proteins are detectable within the small intestine (29).
Expression of the LPS O side chain also seems to be important for colonization and complement resistance, as investigated with undefined spontaneous LPS O side chain-negative, CPS-positive mutants (2). The influence of the LPS core OS has not been investigated for O139 strains. Evidence for contribution of the core OS to V. cholerae virulence comes from our recent observation that strains of different serogroups associated with clinical cholera (O1, O139, and O37) have the same wav gene cluster, which differs markedly from those of environmental isolates (25). Indeed, preliminary data with V. cholerae O1 LPS core OS mutants suggested that an intact core OS contributes to resistance against bile salts, short-chain organic acids, and antimicrobial peptides, which should improve survival in the human small intestine (26). O139 strains can also produce another exopolysaccharide (EPS) which is important for biofilm formation but interferes with intestinal colonization (43).
In this study, we constructed a set of defined CPS, LPS O side chain, and LPS core OS V. cholerae O139 mutants and investigated their behavior in intestinal colonization in vivo and their resistance to several antimicrobial compounds in vitro.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
pir (22) and XL-1 Blue (New England Biolabs, Schwalbach, Germany) were used for all genetic constructions. V. cholerae strains and plasmids used in this study are listed in Table 1. All strains were grown in Luria broth (LB; Difco) at 37°C, except as noted otherwise. For optimal cholera toxin and toxin-coregulated pilin (TCP) expression, the strains were incubated under inducing conditions. Cultures grown overnight in AKI medium (14) were diluted 1:100 in AKI without NaHCO3, grown for 4 h at 37°C under static growth conditions, and then transferred to vigorous shaking for 4 h at 37°C (20). Expression of waaL from the araBAD promoter was induced by supplementing the medium with 0.01% arabinose for in vitro assays; for the in vivo colonization assay, no induction was necessary. Antibiotics (Sigma, Deisenhofen, Germany) were used at the following concentrations: kanamycin, 50 µg/ml; ampicillin, 50 and 100 µg/ml for V. cholerae and E. coli, respectively; and chloramphenicol, 2 and 20 µg/ml for V. cholerae and E. coli, respectively.
|
The downstream DNA regions of the corresponding genes were PCR amplified with primer pairs waaLdownSalI (5'-TACGCGTCGACAAACAGGCTCTCCAATCAGC-3') and waaLdownApaI (5'-GTAAGGGCCCGCCAAAGCAACATACCTTTCC-3'), and galKdownSalI (5'-TACGCGTCGACCACCCGAAGCAAGTAGAAGC-3') and galKdownApaI (5'-GTAAGGGCCCATTTGCCAGCGACGACGATC-3'). The upstream and downstream PCR products of waaL and galK were digested with SalI and ligated overnight. The ligation mix was digested with SmaI and ApaI, purified, and ligated into pKEK229 (6) that had been digested with SmaI and ApaI to give plasmids pKEK
galK and pKEKwaaL.
For the construction of complementing plasmid pSSwavB, we used plasmid pAKwavB, in which wavB was subcloned under the transcriptional control of the tet promoter (25). To make the plasmid usable for this study, we ligated a BamHI fragment containing a kanamycin resistance gene (kanR) (39) (Pharmacia, Biotech Europe, Freiburg, Germany) into the unique BamHI site of pAKwavB to obtain pSSkanwavB1. We deleted the tetracycline resistance gene (tetR) by first digesting pSSkanwavB1 with restriction enzymes SnaBI and NdeI, then treating with Klenow fragment (Sigma), and religating, yielding plasmid pSSwavB2, which had lost most of the tetR gene (341-bp internal fragment) and constitutively expressed wavB.
Mutant strain constructions. The chromosomal mutations in V. cholerae O139 strain MO10 were constructed with different suicide plasmids (Table 1) as described previously (25, 26). The correct chromosomal insertion for all mutants was confirmed by Southern blot analysis (data not shown).
Analysis of cell surface polysaccharides. Cell surface polysaccharides from proteinase K-digested whole-cell lysates were isolated as described by Hitchcock and Brown (11) and analyzed by electrophoresis on sodium dodecyl sulfate-15% polyacrylamide gels. LPS was visualized by silver staining (38). CPS and LPS O side chain expression was assessed by Western blot analysis. Samples were electrophoretically transferred to nitrocellulose membranes (37) and probed with a polyclonal rabbit anti-O139 serum (25).
In vitro resistance assays. Serum resistance was determined under laboratory conditions. Briefly, cells were grown to mid-logarithmic phase in LB broth, washed, and mixed to a final concentration of 20% with normal human serum, obtained and pooled from four donors, or 20% heat-inactivated normal human serum in phosphate-buffered saline (PBS) with 0.1% peptone. Approximately 108 CFU were used in each assay. After incubation at 37°C for 1 h, the cells were harvested, washed, and resuspended in PBS-0.1% peptone. The viable cells were determined by plating serial dilutions onto LB agar. The minimal bactericidal concentration (MBC) of protamine (Sigma), ranging from 600 µg/ml to 9.3 µg/ml, was determined as described by Steinberg et al. (33). The MBC was defined as the lowest concentration of drug that eliminated viable cells after 18 h of incubation at 37°C without shaking. The sensitivity to bile salts was tested on thiosulfate-citrate-bile-sucrose (TCBS) agar plates, prepared as instructed for the commercially available TCBS (Difco), containing no or 0.8% bile.
In vivo colonization assay. The infant mouse colonization assay for O139 strains has been described previously (43). Briefly, mutant strains (lacZ+) were mixed with strain PW96 (MO10 wild type lacZ) and given as an oral inoculum at a ratio of approximately 105 CFU of mutant to 105 CFU of wild type to 5- to 6-day-old CD-1 suckling mice. After a 22-h period of colonization, intestinal homogenates were collected, and the ratio of mutant to wild-type cells was determined by plating appropriate dilutions on LB agar containing streptomycin and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal).
We also determined the in vitro competition index. The same inoculum which was used for the mice was added to 5 ml of LB and allowed to grow at 37°C overnight, and then cells were plated on X-Gal-LB agar medium to determine the cell counts.
CTX-Km
transduction and GM1-ELISA.
CTX-Km utilizes TCP as its receptor, and therefore TCP expression was investigated by determining CTX-Km phage transduction frequency (40). The level of cholera toxin in culture supernatants was measured by the GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) (35) with purified cholera toxin to generate a standard curve.
TCP Western blot analysis. Whole-cell lysates were matched by optical density at 600 nm, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane (37), and probed with TcpA polyclonal antiserum (kindly provided by J. Mekalanos).
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
LPS extracted from the waaF mutant displayed in SDS-PAGE analysis, as expected, a faster-migrating core OS and no LPS O side chain (Fig. 1B, lane 8). The presence of the CPS was confirmed in Western blot analysis (Fig. 1A, lane 8). Inactivation of wavB resulted in a truncated LPS core OS with attached O side chain, but the amount of O side chain was significantly less compared to the wild type (Fig. 1B, lane 6). This was in contrast to the O1 wavB mutant (25), in which no significant reduction of O side chain was observed. The synthesis of the CPS was not affected in the O139 wavB strain (Fig. 1A, lane 6). Deletion of waaL led to an LPS O side chain-negative strain (Fig. 1A and B, lane 1), which was consistent with the proposed function of WaaL as the lipid A core-surface polymer ligase. Furthermore, the waaL deletion did not affect capsule biogenesis (Fig. 1A, lane 1).
|
Recently, we showed that mutations in galE (VCA0774) did not interfere with LPS biosynthesis and intestinal colonization in O1 El Tor strains (26). In contrast, O139 strains do contain Gal residues in the O139 side chain (15, 16), and hence we concluded that the inability to produce UDP-Gal should prevent the biosynthesis of both LPS O side chain and CPS. A strain mutated in galE could still synthesize LPS O side chain and CPS (data not shown), presumably because it can utilize exogenous galactose present in the LB medium. To prevent the utilization of exogenous galactose, we additionally inactivated galK. The galEK double mutant was unable to synthesize CPS and LPS O side chain (Fig. 1A and B, lane 3) and was also unable to form a biofilm (data not shown).
Finally, we constructed a wbfF waaL double mutant, which, like the galEK mutant, was deficient in CPS and LPS O side chain synthesis (Fig. 1A and B, lane 10). Both mutant combinations allowed us to determine whether there was any other UDP-Gal-dependent complex carbohydrate structure not associated with O139 side chain synthesis that might affect intestinal colonization of the galEK mutant. In the presence of plasmids encoding galE, waaL, wavB, or waaF, the phenotypes of the corresponding mutants could be restored (Fig. 1A and B).
In vivo colonization phenotypes of LPS and CPS mutants.
The ability to colonize the small intestine was investigated in the infant mouse model. In a direct comparison of wild-type versus mutant strains, the colonization phenotype for all mutants was determined. In these competition assays, all mutants investigated were attenuated in their ability to successfully colonize the suckling mouse small intestine (Fig. 2). When CPS (wbfF, Fig. 2, data set no. 1) or LPS O side chain synthesis (waaL, Fig. 2, data set no. 4) was prevented, similar decreases in colonization were observed for each of these mutants (30-fold). Mutants unable to express O side chain and capsule (galEK, no. 2; waaL wbfF, no. 10) were severely defective for colonization, as indicated by a competitive index of less than 0.0001. No mutant bacteria were recovered for the waaL wbfF strain (competitive index <0.0001), and very few were recovered for the galEK strain (competitive index 0.0001), which was inoculated at a higher mutant/wild-type ratio. We believe that these two mutant strains are equally defective in colonization, because when they were coinoculated, they were recovered at extremely low but equal frequencies (data not shown).
|
Next, we examined whether the alteration of the surface polysaccharides caused other deleterious effects which could explain the attenuation in intestinal colonization. None of the mutants demonstrated any general growth defects in in vitro competition assays, with the exception of the waaF mutant, which had an approximately 100-fold attenuation (competitive index of 0.014 for this strain). We also tested the abilities of these strains to induce virulence factors known to be important for intestinal colonization. The formation of TCP is critical for colonization (36), and all strains were able to induce TCP expression under in vitro inducing conditions.
TcpA-directed Western blot analysis revealed that only the waaF mutant expressed slightly less TcpA (Fig. 3A, lane 7), which could be restored in the presence of a waaF expressing plasmid (Fig. 3A, lane 8). However, the waaF mutant assembled functional TCP on the surface, since it could be infected with phage CTX-Km, which is known to use TCP as its receptor (40) (data not shown). All mutants also expressed and secreted relatively normal amounts of cholera toxin, indicating that the mutations affected neither cholera toxin induction nor the type II protein secretion pathway (Fig. 3B). In summary, these investigations indicated that the alterations of the surface polysaccharides caused no detectable cell damage or alteration in virulence factor induction that might explain the colonization defects, with the possible exception of the waaF mutation.
|
|
Survival in the presence of bile was tested on TCBS plates containing 0.8% bile. Growth defects were only observed for mutants with defects in core OS assembly (Fig. 4A) and not for the waaL, wbfF, galEK (Fig. 4C, sets 6 to 8), and waaL wbfF (data not shown) mutants. The waaF mutant could not grow (Fig. 4A, set 3), while the wavB mutant grew poorly on these plates (Fig. 4A, 5). The growth defects could be complemented in the presence of waaF- and wavB-expressing plasmids (Fig. 4A, sets 2 and 4).
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Pathogenic V. cholerae O139 strains have a complex life cycle, including survival and growth in the estuarine environment and the human intestine. We were interested in the contribution of surface polysaccharides to these processes. The exopolysaccharide VPS seems to be mainly important for survival in aquatic environments by stabilization of biofilms (43), while the role of the other surface polysaccharides, LPS core OS, LPS O side chain, and CPS, during the life cycle is less clear. The behavior of LPS O side chain and CPS mutants in intestinal colonization of O139 isolates was addressed previously, but these investigations were performed with spontaneous mutants (2, 41). With the intention of establishing a comprehensive analysis, we investigated the role of surface polysaccharides in intestinal colonization with a set of genetically defined LPS O side chain-CPS mutants and two LPS core OS mutants.
What is the role of LPS O side chain and CPS during O139 intestinal colonization? To investigate this further, we constructed mutants defective in ligation of the O139 side chain to lipid A core (waaL; LPS O side chain-negative, CPS-positive), capsule translocation (wbfF; LPS O side chain-positive, CPS-negative), biosynthesis of UDP-galactose (galEK; LPS O side chain-negative, CPS-negative), and lipid A core ligation and capsule translocation (waaLwbfF; LPS O side chain-negative, CPS-negative) mutants; the last two mutants behaved similarly under all conditions tested, indicating that in both mutants the loss of CPS and O side chain is responsible for the observed effects.
The galEK and waaL wbfF mutants were essentially unable to colonize the small intestine of infant mice (i.e., no mutant bacteria were recovered in competition experiments with a wild-type strain), which is in accordance with earlier reports (2, 41). The observed 30-fold decrease in colonization of strains lacking either the LPS O side chain or the CPS alone (waaL and wbfF mutants, respectively) is significant and, in the case of the CPS-negative mutant, similar to results reported previously (2, 41). Our defined waaL mutant colonized better than a spontaneous LPS O side chain-negative but CPS-positive mutant described previously (2), for which a three- to fourfold decrease in intestinal colonization was reported. We believe the discrepancy may be due to the nature of the mutation in the two studies. In the present study, a strain with a nonpolar deletion in waaL was used, which should have no effect on the expression of other genes and, as shown here, have no effect on capsule translocation, representing a true LPS O side chain-negative but CPS-positive mutant.
What is the mechanism of the attenuation? We excluded the possibility that the observed colonization deficiency was caused simply by a loss of functional pili, since Western blot and CTX phage transduction experiments (data not shown) demonstrated the presence of TCP. It has been proposed that the colonization defect of CPS- and/or LPS O side chain-negative V. cholerae mutants is mainly due to increased complement sensitivity (2, 41). However, we found that the presence of CPS alone protects against the complement system, independent of the LPS defect. When the CPS was absent, the LPS O side chain could partially compensate for this loss. Increased complement sensitivity would therefore explain only the attenuated colonization of the CPS (wbfF) and the CPS-LPS O side chain double mutants (galEK and waaL wbfF) but not of the CPS-positive LPS O side chain-negative mutant (waaL).
It is not clear how much the complement system contributes to defense within the intestine, e.g., by bacteriolysis of gram-negative bacteria, since there is only evidence for activation of initial factors, such as C3, but not the terminal components of the complement cascade (29). Intestinal colonization likely constitutes a multifactorial process, and the mucus lining of the small intestine probably serves as the initial matrix to which V. cholerae cells adhere. During this stage of colonization, the ToxR regulatory cascade (including TCP expression) is probably not fully operative, as suggested by several lines of research (18, 31). In this scenario, O139 CPS or O139 side chain molecules might be involved in initial adhesion (45). However, there is still a lack of experiments addressing the function of surface polysaccharides as adhesins, e.g., measurement of mucus binding by defined surface polysaccharide mutants or conjugated LPS or CPS material.
The contribution of the V. cholerae LPS core OS to V. cholerae virulence was neglected for a long time. In a genetic study, we recently found evidence that clinical V. cholerae strains of different serogroups have a common core OS structure that was distinguishable from those of environmental isolates (25). This finding prompted us to investigate O139 LPS core OS mutants in intestinal colonization. Two defined knockout mutants were addressed in this study. The waaF mutant, which lacked core material and LPS O side chain but was still encapsulated, could not be recovered from the mouse intestine, indicating a severe defect in colonization. Notably, alteration of the core OS of the waaF mutant resulted in a 1.000-fold reduction in colonization compared to the O side chain-negative, CPS-positive waaL mutant.
The waaF mutant was sensitive to both bile and polycationic peptides, a phenotype which was observed exclusively in this core OS mutant. In addition, the waaF mutation caused pleiotropic effects, such as a noticeable growth defect and decreased TcpA expression, which could explain the deleterious colonization defect. These findings indicate that the LPS core OS structure synthesized by the waaF mutant was not able to maintain the integrity of the outer membrane. The other core OS mutant (wavB) was constructed with the intention of investigating the role of the LPS core OS in a strain that expresses the LPS O side chain and CPS. This mutation had no pleiotropic effects on growth rate or expression of TCP and OmpU but, unfortunately, affected O side chain attachment, leading to a decreased amount of LPS O side chain in this mutant compared to the wild-type strain. For this reason, we cannot exclude the decrease in LPS O side chain as the cause of the attenuated colonization behavior of this mutant. However, we also observed that the wavB strain grew poorly in the presence of bile, a phenotype which was not associated with the LPS O side chain-negative (waaL) mutant, and thus identifying at least one exclusive phenotype associated with the LPS core OS that may contribute to colonization.
In summary, our results presented here and our previous results with O1 El Tor LPS core OS mutants (26) provide evidence that the LPS core OS structure might be critical for survival of pathogenic V. cholerae strains in the mammalian intestine, but the nature of its role and whether a specific core OS structure is important for colonization will be addressed in more detail in future investigations. A detailed understanding of core OS distribution among pathogenic strains and its contribution to virulence could be beneficial for the development of a vaccine providing protection against pathogenic V. cholerae of different serogroups.
|
ACKNOWLEDGMENTS |
|---|
This work was funded by BMBF grant 01KI8906 and Nachwuchsgruppenförderprogramm Bayern to J.R. and NIH AI43486 to K.E.K.
Jutta Nesper, Stefan Schild, and Crystal M. Lauriano contributed equally to this work.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
| 1. | Albert, M. J., F. Qadri, N. A. Bhuiyan, S. M. Ahmad, M. Ansaruzzaman, and A. Weintraub. 1999. Phagocytosis of Vibrio cholerae O139 Bengal by human polymorphonuclear leukocytes. Clin. Diagn. Lab. Immunol. 6:276-278. |
| 2. | Attridge, S. R., A. Fazeli, P. A. Manning, and U. H. Stroeher. 2001. Isolation and characterization of bacteriophage-resistant mutants of Vibrio cholerae O139. Microb. Pathog. 30:237-246.[CrossRef][Medline] |
| 3. | Berche, P., C. Poyart, E. Abachin, H. Lelievre, J. Vandepitte, A. Dodin, and J. M. Fournier. 1994. The novel epidemic strain O139 is closely related to the pandemic strain O1 of Vibrio cholerae. J. Infect. Dis. 170:701-704.[Medline] |
| 4. | Bik, E. M., A. E. Bunschoten, R. J. Willems, A. C. Chang, and F. R. Mooi. 1996. Genetic organization and functional analysis of the otn DNA essential for cell-wall polysaccharide synthesis in Vibrio cholerae O139. Mol. Microbiol. 20:799-811.[CrossRef][Medline] |
| 5. | Comstock, L. E., J. A. Johnson, J. M. Michalski, J. G. Morris, and J. B. Kaper. 1996. Cloning and sequence of a region encoding a surface polysaccharide of Vibrio cholerae O139 and characterization of the insertion site in the chromosome of Vibrio cholerae O1. Mol. Microbiol. 19:815-826.[CrossRef][Medline] |
| 6. | Correa, N. E., C. M. Lauriano, R. McGee, and K. E. Klose. 2000. Phosphorylation of the flagellar regulatory protein FlrC is necessary for Vibrio cholerae motility and enhanced colonization. Mol. Microbiol. 35:743-755.[CrossRef][Medline] |
| 7. | Dziejman, M., E. Balon, D. Boyd, C. M. Fraser, J. F. Heidelberg, and J. J. Mekalanos. 2002. Comparative genomic analysis of Vibrio cholerae: genes that correlate with cholera endemic and pandemic disease. Proc. Natl. Acad. Sci. USA 99:1556-1561. |
| 8. | Guzman, L. M., D. Belin, M. J. Carson, and J. Beckwith. 1995. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177:4121-4130. |
| 9. | Hall, R. H., F. M. Khambaty, M. Kothary, and S. P. Keasler. 1993. Non-O1 Vibrio cholerae. Lancet i:342-430. |
| 10. | Higa, N., Y. Honma, J. M. Albert, and M. Iwanaga. 1993. Characterization of Vibrio cholerae O139 synonym Bengal isolated from patiens with cholera-like disease in Bangladesh. Microbiol. Immunol. 37:971-974.[Medline] |
| 11. | Hitchcock, P. J., and T. M. Brown. 1983. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154:269-277. |
| 12. | Holmgren, J., and A. M. Svennerholm. 1977. Mechanisms of disease and immunity in cholera: a review. J. Infect. Dis. 136:105-112. |
| 13. | Holst, O. 1999. Chemical structure of the core region of lipopolysaccharides, p. 115-154. In H. Brade, S. M. Opal, S. N. Vogel, and D. C. Morrison (ed.), Endotoxin in health and disease. Marcel Dekker Inc., New York, N.Y. |
| 14. | Iwanaga, M., K. Yamamoto, N. Higa, Y. Ichinose, N. Nakasone, and M. Tanabe. 1986. Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor. Microbiol. Immunol. 30:1075-1083.[Medline] |
| 15. | Knirel, Y. A., L. Paredes, P.-E. Jansson, A. Weintraub, G. Widmalm, and M. J. Albert. 1995. Structure of the capsular polysaccharide of Vibrio cholerae O139 synonym Bengal containing D-galactose-4,6-cyclophosphate. Eur. J. Biochem. 232:391-396.[Medline] |
| 16. | Knirel, Y. A., G. Widmalm, S. N. Senchenkova, P.-E. Jansson, and A. Weintraub. 1997. Structural studies on the short-chain lipopolysaccharide of Vibrio cholerae O139 Bengal. Eur. J. Biochem. 247:402-410.[Medline] |
| 17. | Koch, R. 1884. An address on cholera and its bacillus. Br. Med. J. 2:403-407. |
| 18. | Lee, S. H., D. L. Hava, M. K. Waldor, and A. Camilli. 1999. Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection. Cell 99:625-634.[CrossRef][Medline] |
| 19. | Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 20. | Medrano, A. I., V. J. DiRita, G. Castillo, and J. Sanchez. 1999. Transient transcriptional activation of the Vibrio cholerae El Tor virulence regulator ToxT in response to culture conditions. Infect. Immun. 67:2178-2183. |
| 21. | Meno, Y., M. K. Waldor, J. J. Mekalanos, and K. Amako. 1998. Morphological and physical characterization of the capsular layer of Vibrio cholerae O139. Arch Microbiol. 170:339-344.[CrossRef][Medline] |
| 22. | Miller, V. L., and J. J. Mekalanos. 1988. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170:2575-2583. |
| 23. | Morris, J. G. 1994. Vibrio cholerae O139 Bengal, p. 95-102. In I. K. Wachsmuth, P. A. Blake, and O. Olsvik (ed.), Vibrio cholerae and cholera: molecular to global perspectives. ASM Press, Washington, D.C. |
| 24. | Nesper, J., J. Blaß, M. Fountouloakis, and J. Reidl. 1999. Characterization of the major control region of Vibrio cholerae bacteriophage K139: immunity, exclusion and integration. J. Bacteriol. 181:2902-2913. |
| 25. | Nesper, J., A. Kraiss, S. Schild, J. Blass, K. E. Klose, J. Bockemühl, and J. Reidl. 2002. Comparative and genetic analysis of the putative Vibrio cholerae lipopolysaccharide core oligosaccharide biosynthesis (wav) gene cluster. Infect. Immun. 70:2419-2433. |
| 26. | Nesper, J., C. M. Lauriano, K. E. Klose, D. Kapfhammer, A. Kraiss, and J. Reidl. 2001. Characterization of Vibrio cholerae O1 El Tor galU and galE mutants: influence on lipopolysaccharide structure, colonization, and biofilm formation. Infect. Immun. 69:435-445. |
| 27. | Provenzano, D., and K. E. Klose. 2000. Altered expression of the ToxR-regulated porins OmpU and OmpT diminishes Vibrio cholerae bile resistance, virulence factor expression, and intestinal colonization. Proc. Natl. Acad. Sci. USA 97:10220-10224. |
| 28. | Ramamurthy, T., S. Grag, and R. Sharma. 1993. Emergence of novel strain of Vibrio cholerae with epidemic potential in southern and eastern India. Lancet 341:703-704.[Medline] |
| 29. | Riordan, S. M., C. J. McIver, D. Wakefield, P. C. Andreopoulos, V. M. Duncombe, T. D. Bolin, and M. C. Thomas. 1997. Local and systemic complement activity in small intestinal bacterial overgrowth. Dig. Dis. Sci. 42:1128-1136.[CrossRef][Medline] |
| 30. | Rivas, M., C. Toma, E. Miliwebsky, M. I. Caffer, M. Galas, P. Varela, M. Tous, A. M. Bru, and N. Binsztein. 1993. Cholera isolates in relation to the eight pandemic. Lancet 342:926-927.[Medline] |
| 31. | Schumacher, D. A., and K. E. Klose. 1999. Environmental signals modulate ToxT-dependent virulence factor expression in V. cholerae. J. Bacteriol. 181:1508-1514. |
| 32. | Shimada, T., G. B. Nair, B. C. Deb, M. J. Albert, R. B. Sack, and Y. Takeda. 1993. Outbreak of Vibrio cholerae non-O1 in India and Bangladesh. Lancet 341:1347.[CrossRef][Medline] |
| 33. | Steinberg, D. A., M. A. Hurst, C. A. Fujii, A. H. Kung, J. F. Ho, F.-C. Cheng, D. J. Loury, and J. C. Fiddes. 1997. Protegrin-1: a broad-spectrum, rapidly microbicidal peptide with in vivo activity. Antimicrob. Agents Chemother. 41:1738-1742.[Abstract] |
| 34. | Stroeher, U. H., K. E. Jedani, and P. A. Manning. 1998. Genetic organization of the regions associated with surface polysaccharide synthesis in Vibrio cholerae O1, O139 and Vibrio anguillarum O1 and O2: a review. Gene 223:269-282.[CrossRef][Medline] |
| 35. | Svennerholm, A. M., and J. Holmgren. 1978. Identification of Escherichia coli heat-labile enterotoxin by means of a ganglioside immunosorbant assay (GM1-ELISA) procedure. Curr. Microbiol. 1:19-23. |
| 36. | Tacket, C. O., R. K. Taylor, G. Losonsky, Y. Lim, J. P. Nataro, J. B. Kaper, and M. M. Levine. 1998. Investigation of the roles of toxin-coregulated pili and mannose-sensitive hemagglutinin pili in the pathogenesis of Vibrio cholerae O139 infection. Infect. Immun. 66:692-695. |
| 37. | Towbin, H., T. Staehlin, and J. Gordon. 1979. Electrophoretic transfer of proteins. Proc. Natl. Acad. Sci. USA 76:4350-4354. |
| 38. | Tsai, C. M., and C. E. Frasch. 1982. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119:115-119.[CrossRef][Medline] |
| 39. | Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259-268.[CrossRef][Medline] |
| 40. | Waldor, K. W., and J. J. Mekalanos. 1996. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272:1910-1914.[Abstract] |
| 41. | Waldor, M. K., R. Colwell, and J. J. Mekalanos. 1994. The Vibrio cholerae O139 serogroup antigen includes an O antigen capsule and lipopolysaccharide virulence determinants. Proc. Natl. Acad. Sci. USA 91:11388-11392. |
| 42. | Waldor, M. K., and J. J. Mekalanos. 1994. ToxR regulates virulence gene expression in non-O1 strains of Vibrio cholerae that cause epidemic cholera. Infect. Immun. 62:72-78. |
| 43. | Watnick, P. I., C. M. Lauriano, K. E. Klose, L. Croal, and R. Kolter. 2001. The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139. Mol. Microbiol. 39:223-235.[CrossRef][Medline] |
| 44. | Whitfield, C., and I. S. Roberts. 1999. Structure, assembly and regulation of expression of capsules in Escherichia coli. Mol. Microbiol. 31:1307-1319.[CrossRef][Medline] |
| 45. | Yamamoto, T., M. J. Albert, and R. B. Sack. 1994. Adherence to human small intestines of capsulated Vibrio cholerae O139. FEMS Microbiol. Lett. 119:229-235.[CrossRef][Medline] |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
