Association of adherence and motility in interleukin 8 induction in human intestinal epithelial cells by Vibrio cholerae

Interleukin 8 (IL-8) mRNA expression in Vibrio cholerae-infected human intestinal epithelial cells Int407 was determined by quantitative real-time RT-PCR and secretion measured by ELISA. Incubation of Int407 with V. cholerae O395 resulted in increased IL-8 mRNA expression as early as within 2 h of infection. Kinetics of IL-8 secretion reached a peak at about 8 h (780 pg/ml) and decreased thereafter. Induction of IL-8 was significantly high among various toxin-producing strains of V. cholerae belonging to serovar O1, O139 and non-O1 compared to non-toxinogenic strains. Induction of IL-8 was maximum in V. cholerae O395, required live cells and was dependent on de novo protein synthesis. The bacterial culture supernatant and crude cell envelope showed IL-8 stimulating activity. Infection of the monolayer with V. cholerae O395 cheY4 null mutant (O395YN), defective in adherence and motility, resulted in highly reduced levels of IL-8 expression, while hyperadherent and hypermotile mutant (O395Y) with the cheY4 gene duplicated also showed very high IL-8 expression. Another hyperadherent icmF insertion mutant (O395F) with reduced motility showed almost half the amount of IL-8 expression compared to O395Y. These results clearly indicate that both motility and adherence to intestinal epithelial cells are possible triggering factors contributing to IL-8 mRNA expression by V. cholerae.     

Human intestinal epithelial cell cytokine mRNA responses mediated by NF-kappaB are modulated by the motility and adhesion process of Vibrio cholerae

Vibro cholerae, the etiological agent of cholera, colonizes the small intestine, produces an enterotoxin and causes acute inflammatory response at intestinal epithelial surface; the signals for such induction are still unknown. We determined the mRNA expression of proinflammatory and anti-inflammatory cytokines in Int407 cells following infection with V. cholerae or its mutants by semi-quantitaive and quantitative real-time RT-PCR. V. cholerae induces the coordinated expression and up-regulation of IL-1alpha, IL-6, GM-CSF and MCP-1 and down-regulation of TGF-beta in Int407 cells. While the pathogenecity of V. cholerae was found to be a possible determinant in modulation of IL-1alpha and TGF-beta, both IL-6 and MCP-1 OmpU might modulate induction. Significant reduction in IL-1alpha, GM-CSF and MCP-1 mRNA expression was observed upon infection with the less motile and less adherent strain O395YN. This association is supported by the absence of nuclear translocation of NF-kappaB (p50 subunit) upon infection with O395YN in contrast to wild-type. Moreover, TPCK treatment prior to V. cholerae infection indicated that proinflammatory cytokine gene expression in Int407 cells is NF-kappaB mediated. Thus, V. cholerae induces proinflammatory cytokine response in Int407 cells, which is mediated by NF-kappaB and is modulated, in part, by adherence or motility of this organism.     

Upregulation of human mitochondrial NADH dehydrogenase subunit 5 in intestinal epithelial cells is modulated by Vibrio cholerae pathogenesis

Cholera still remains an important global predicament especially in India and other developing countries. Vibrio cholerae, the etiologic agent of cholera, colonizes the small intestine and produces an enterotoxin that is largely responsible for the watery diarrheal symptoms of the disease. Using RNA arbitrarily primed PCR, ND5 a mitochondria encoded subunit of complex I of the mitochondrial respiratory chain was found to be upregulated in the human intestinal epithelial cell line Int407 following exposure to V. cholerae. The upregulation of ND5 was not observed when Int407 was infected with Escherichia coli strains. Incubation with heat-killed V. cholerae or cholera toxin or culture supernatant also showed no such upregulation indicating the involvement of live bacteria in the process. Infection of the monolayer with aflagellate non-motile mutant of V. cholerae O395 showed a very significant (59-fold) downregulation of ND5. In contrast, a remarkable upregulation of ND5 expression (200-fold) was observed in a hyperadherent icmF insertion mutant with reduced motility. V. cholerae cheY4 null mutant defective in adherence and motility also resulted in significantly reduced levels of ND5 expression while mutant with the cheY4 gene duplicated showing increased adherence and motility resulted in increased expression of ND5. These results clearly indicate that both motility and adherence to intestinal epithelial cells are possible triggering factors contributing to ND5 mRNA expression by V. cholerae. Interestingly infection with insertion mutant in the gene coding for ToxR, the master regulator of virulence in V. cholerae resulted in significant downregulation of ND5 expression. However, infection with ctxA or toxT insertion mutants did not show any significant changes in ND5 expression compared to wild-type. Almost no expression of ND5 was observed in case of mutation in the gene coding for OmpU, a ToxR activated protein. Thus, infection of Int407 with virulence mutant strains of V. cholerae revealed that the ND5 expression is modulated by the virulence of V. cholerae in a ToxT independent manner. Although no difference in the mitochondrial copy number could be detected between infected and uninfected cells, the modulation of the expression of other mitochondrial genes were also observed. Incidentally, upon V. cholerae infection, complex I activity was found to increase about 3-folds after 6 h. This is the first report of alteration in mitochondrial gene expression upon infection of a non-invasive enteric bacterium like V. cholerae showing its modulation with adherence, motility and virulence of the organism.     

Vibrio vulnificus AphB is involved in interleukin-8 production via an NF-κB-dependent pathway in human intestinal epithelial cells

We previously reported that the aphB gene mutant of Vibrio vulnificus had significantly impaired motility and adherence to host cells. In this study, we investigated the role of V. vulnificus AphB on the production of interleukin-8 (IL-8), a proinflammatory cytokine, as well as its underlying mechanism in human intestinal epithelial INT-407 cells. The aphB gene mutation significantly reduced the ability of V. vulnificus to stimulate IL-8 production and IL-8 gene promoter activation in INT-407 cells. The V. vulnificusaphB mutant also induced lower levels of NF-κB DNA binding activity and NF-κB minimal promoter activation than did the wild-type of V. vulnificus. Importantly, the observed reductions in IL-8 production, IL-8 gene promoter activation and NF-κB DNA binding activity were significantly restored by complementing the aphB gene into the V. vulnificusaphB mutant. These results indicate that V. vulnificus AphB is involved in the IL-8 production via an NF-κB dependent pathway in human intestinal epithelial cells.     

The mannose-sensitive hemagglutinin of Vibrio cholerae promotes adherence to zooplankton.

The bacterium Vibrio cholerae, the etiological agent of cholera, is often found attached to plankton, a property that is thought to contribute to its environmental persistence in aquatic habitats. The V. cholerae O1 El Tor biotype and V. cholerae O139 strains produce a surface pilus termed the mannose-sensitive hemagglutinin (MSHA), whereas V. cholerae O1 classical biotype strains do not. Although V. cholerae O1 classical does not elaborate MSHA, the gene is present and expressed at a level comparable to that of the other strains. Since V. cholerae O1 El Tor and V. cholerae O139 have displaced V. cholerae O1 classical as the major epidemic strains over the last fifteen years, we investigated the potential role of MSHA in mediating adherence to plankton. We found that mutation of mshA in V. cholerae O1 El Tor significantly diminished, but did not eliminate, adherence to exoskeletons of the planktonic crustacean Daphnia pulex. The effect of the mutation was more pronounced for V. cholerae O139, essentially eliminating adherence. Adherence of the V. cholerae O1 classical mshA mutant was unaffected. The results suggest that MSHA is a factor contributing to the ability of V. cholerae to adhere to plankton. The results also showed that both biotypes of V. cholerae O1 utilize factors in addition to MSHA for zooplankton adherence. The expression of MSHA and these additional, yet to be defined, adherence factors differ in a serogroup- and biotype-specific manner.     

Identification of proinflammatory flagellin proteins in supernatants of Vibrio cholerae O1 by proteomics analysis

The genome of Vibrio cholerae contains five flagellin genes that encode proteins (FlaA-E) of 39-41 kDa with 61-82% identity among them. Although the existing live oral attenuated vaccine strains against cholera are protective in humans, there is an intrinsic residual cytotoxic and inflammatory component associated with these candidate vaccine strains. Bacterial flagellins are known to be potent inducers of proinflammatory molecules via activation of Toll-like receptor 5. Here we found that purified flagella from wild type V. cholerae 395 induced significant release of interleukin (IL)-8 from cultured HT-29 human colonic epithelial cells. Furthermore we found that filtered supernatants of KKV90, a DeltaflaA isogenic strain unable to produce flagella, were still able to activate production of IL-8 albeit to significantly lower levels than the wild type, suggesting that other activators of proinflammatory molecules were still present in these supernatants. A comparative proteomics analysis of secreted proteins of V. cholerae 395 and KKV90 identified additional proteins with potential to induce IL-8 release in HT-29 cells. Secreted proteins in the range of 30-45 kDa identified by two-dimensional electrophoresis and mass spectrometry revealed the presence of two additional flagellins, FlaC and FlaD, that appeared to be secreted 3- and 6-fold more, respectively, in the mutant compared with the wild type. Double isogenic mutants flaAC and flaAD were unable to trigger IL-8 release from HT-29 cells. In sum, we have shown that purified flagella and secreted flagellin proteins (FlaC and FlaD) are inducers of IL-8 release from epithelial cells via Toll-like receptor 5. This observation may explain, in part, the observed reactogenicity of cholera vaccine strains in humans.     

Structural and functional importance of outer membrane proteins in Vibrio cholerae flagellum

Vibrio cholerae has a sheath-covered monotrichous flagellum that is known to contribute to virulence. Although the structural organization of the V. cholerae flagellum has been extensively studied, the involvement of outer membrane proteins as integral components in the flagellum still remains elusive. Here we show that flagella produced by V. cholerae O1 El Tor strain C6706 were two times thicker than those from two other Gram-negative bacteria. A C6706 mutant strain (SSY11) devoid of two outer membrane proteins (OMPs), OmpU and OmpT, produced thinner flagella. SSY11 showed significant defects in the flagella-mediated motility as compared to its parental strain. Moreover, increased shedding of the flagella-associated proteins was observed in the culture supernatant of SSY11. This finding was also supported by the observation that culture supernatants of the SSY11 strain induced the production of a significantly higher level of IL-8 in human colon carcinoma HT29 and alveolar epithelial A549 cells than those of the wild-type C6706 strain. These results further suggest a definite role of these two OMPs in providing the structural integrity of the V. cholerae flagellum as part of the surrounding sheath.     

Role of cell-associated N-acetyl-d-glucosamine specific haemagglutinin in the adhesion of Vibrio cholerae O1 to rabbit intestinal epithelial cells in vitro

Abstract Previously a N -acetyl- d -glucosamine specific cell-associated haemagglutinin (HA) had been purified from a Vibrio cholerae O1 strain. This study documents the role of this purified HA as an adhesin of V. cholerae O1. A significant inhibition in the adhesion of V. cholerae O1 bacterial cells to isolated rabbit intestinal brush borders (RIBB) was observed when the latter were pretreated with purified HA in ELISA. Antibody raised against purified HA and Fab (IgG) fragment of this serum inhibited adhesion of the bacteria to isolated rabbit intestinal epithelial cells (RIEC). V. cholerae O1 (both Ogawa and Inaba serovars) showed less adherence to isolated RIEC of animals immunised with the purified HA. Patients convalescing from V. cholerae O1 infection showed high ELISA titres against the purified HA indicating that it is expressed in the host during the disease process.     

Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter

Quorum sensing negatively influences virulence gene expression in certain toxigenic Vibrio cholerae strains. At high cell densities, the response regulator LuxO fails to reduce the expression of HapR, which, in turn, represses the expression of the virulence cascade. A critical regulatory step in the cascade is activation of tcpPH expression by AphA and AphB. We show here that HapR influences the virulence cascade by directly repressing aphA expression. In strain C6706, aphA expression was increased in a delta hapR mutant and decreased in a delta luxO mutant, indicating a negative and positive influence, respectively, of these gene products on the promoter. Overexpression of HapR also reduced aphA expression in both C6706 and Escherichia coli. DNase I footprinting showed that purified HapR binds to the aphA promoter between -85 and -58. Although it appears that quorum sensing does not influence virulence gene expression in strain O395 solely because of a frameshift in hapR, overproduced HapR did not repress expression from the O395 aphA promoter in either Vibrio or E. coli, nor did the protein bind to the promoter. Two basepair differences from C6706 are present in the O395 HapR binding site at -85 and -77. Introducing the -77 change into C6706 prevented HapR binding and repression of aphA expression. This mutation also eliminated the repression of toxin-co-regulated pilus (TCP) and cholera toxin (CT) that occurs in a delta luxO mutant, indicating that HapR function at aphA is critical for density-dependent regulation of virulence genes.     

霍乱弧菌中调控aphB 的基因筛选及其功能

【目的】筛选霍乱弧菌C6706-中调控LysR家族蛋白AphB表达的基因。【方法】将霍乱弧菌埃尔托型菌株C6706-aphB启动子区克隆到2个报告质粒pBBRLux和pKP302上,并将其导入霍乱弧菌C6706-中,以此作为出发菌株。利用出发菌株与转座子pSC123接合构建LZV630-302转座子随机突变文库,通过测定化学发光强度检测aphB启动子的表达水平,筛选aphB表达受影响的突变株。利用随机PCR方法检测转座子插入位点,并测序比对分析基因。【结果】从7个转座子库中(共约4万个突变株)得到能影响aphB表达(均导致下降)的2株突变株T1和T2。测序比对发现T1中转座子插入在vc1585读码框内,T2中转座子插入在距vc1602基因末端7 bp处。【结论】获得aphB表达改变的突变株,基因vc1585和vc1602可能直接或间接影响aphB表达,为进一步研究aphB表达调控影响因素奠定了基础。     

Molecular cloning and characterization of mannitol-1-phosphate dehydrogenase from Vibrio cholerae

Vibrio cholerae utilizes mannitol through an operon of the phosphoenolpyruvate-dependent phosphotransferase (PTS) type. A gene, mtlD, encoding mannitol-1-phosphate dehydrogenase was identified within the 3.9 kb mannitol operon of V. cholerae. The mtlD gene was cloned from V. cholerae O395, and the recombinant enzyme was functionally expressed in E. coli as a 6×His-tagged protein and purified to homogeneity. The recombinant protein is a monomer with a molecular mass of 42.35 kDa. The purified recombinant MtlD reduced fructose 6-phosphate (F6P) using NADH as a cofactor with a K(m) of 1.54 +/- 0.1 mM and V(max) of 320.8 +/- 7.81 micronmol/min/mg protein. The pH and temperature optima for F6P reduction were determined to be 7.5 and 37°C, respectively. Using quantitative real-time PCR analysis, mtlD was found to be constitutively expressed in V. cholerae, but the expression was up-regulated when grown in the presence of mannitol. The MtlD expression levels were not significantly different between V. cholerae O1 and non-O1 strains.