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.     

Lack of umuDC gene functions in Vibrio cholerae cells

Attempts to identify an umuDC analog, using interspecific complementation of Escherichia coli mutants with plasmids containing a gene bank of Vibrio cholerae, were not successful. The DNA from none of the vibrio species examined including marine vibrios hybridized to E. coli umuC and umuD gene sequences. These cells are not mutable by ultraviolet (UV) light and cannot Weigle-reactivate UV-irradiated choleraphages, suggesting that vibrios are deficient in the umuDC operon. This possibility is supported by the fact that when the plasmid pKM101 carrying the mucAB genes is introduced into V. cholerae cells, they acquire the UV-mutable phenotype and UV-irradiated choleraphages can be Weigle-reactivated.     

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.     

Adhesion of Vibrio cholerae 01 to human buccal epithelial cells in vitro

Abstract Adhesive capacity to human buccal cells (HBC) of Vibrio cholerae 01 was expressed optimally in synthetic culture medium. Bacterial adhesion was inhibited efficiently by fetuin and weakly by D -mannose, D -glucose and thyroglobulin. Pretreatment of HBC with neuraminidase, protease or trypsin reduced the adhesion of V. cholerae 01 cells.     

Multipartite regulation of rctB, the replication initiator gene of Vibrio cholerae chromosome II

Replication initiator proteins in bacteria not only allow DNA replication but also often regulate the rate of replication initiation as well. The regulation is mediated by limiting the synthesis or availability of initiator proteins. The applicability of this principle is demonstrated here for RctB, the replication initiator for the smaller of the two chromosomes of Vibrio cholerae. A strong promoter for the rctB gene named rctBp was identified and found to be autoregulated in Escherichia coli. Promoter activity was lower in V. cholerae than in E. coli, and a part of this reduction is likely to be due to autorepression. Sequences upstream of rctBp, implicated earlier in replication control, enhanced the repression. The action of the upstream sequences required that they be present in cis, implying long-range interactions in the control of the promoter activity. A second gene specific for chromosome II replication, rctA, reduced rctB translation, most likely by antisense RNA control. Finally, optimal rctBp activity was found to be dependent on Dam. Increasing RctB in trans increased the copy number of a miniplasmid carrying oriCII(VC), implying that RctB can be rate limiting for chromosome II replication. The multiple modes of control on RctB are expected to reduce fluctuations in the initiator concentration and thereby help maintain chromosome copy number homeostasis.     

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.     

Factors affecting the colonization of isolated rabbit intestinal epithelial cells by Vibrio cholerae 01 in vitro

The adhesive capability of Vibrio cholerae 01 strains to isolated rabbit intestinal epithelial cells was maximally expressed when the bacteria were grown in synthetic broth and was enhanced by the presence of Ca2+ in the growth media. N-Acetyl-D-glucosamine could inhibit the adhesion of the bacteria to rabbit intestinal epithelial cells as could lipopolysaccharide O-antigen from Vibrio cholerae 01 and lectin from Triticum vulgaris. Since the lipopolysaccharide is known to contain N-acetyl-D-glucosamine and because the lectin from Triticum vulgaris shows specificity for this sugar, it is probable that N-acetyl-D-glucosamine is actively involved in the adhesion of Vibrio cholerae 01 to isolated rabbit intestinal epithelial cells.     

The transcriptional activator HIyU of Vibrio cholerae: nucleotide sequence and role in virulence gene expression

HlyU upregulates expression of the haemolysin, HlyA, of Vibrio cholerae. DNA sequence analysis indicates that HlyU is an 11.9 kDa protein containing a putative helix-turn-helix motif and belonging to a family of small regulatory proteins, including NolR (Rhizobium meliloti), SmtB (Synechococcus PCC 7942) and ArsR (piasmids R773, Escherichia coli; p1258, Staphylococcus aureus; and pSX267, Staphylococcus xylosus). An hlyU mutant was constructed by insertional inactivation, and found to be deficient in the production of both the haemolysin and a 28kDa secreted protein. The mutant was assessed for virulence in the infant mouse cholera model, revealing a 100-fold increase in the LD_50.. This suggests that HlyU promotes expression of virulence determinant(s) in vivo.     

A critical role of tropomyosins in TGF-beta regulation of the actin cytoskeleton and cell motility in epithelial cells

We have investigated transforming growth factor beta (TGF-beta)-mediated induction of actin stress fibers in normal and metastatic epithelial cells. We found that stress fiber formation requires de novo protein synthesis, p38Mapk and Smad signaling. We show that TGF-beta via Smad and p38Mapk up-regulates expression of actin-binding proteins including high-molecular-weight tropomyosins, alpha-actinin and calponin h2. We demonstrate that, among these proteins, tropomyosins are both necessary and sufficient for TGF-beta induction of stress fibers. Silencing of tropomyosins with short interfering RNAs (siRNAs) blocks stress fiber assembly, whereas ectopic expression of tropomyosins results in stress fibers. Ectopic-expression and siRNA experiments show that Smads mediate induction of tropomyosins and stress fibers. Interestingly, TGF-beta induction of stress fibers was not accompanied by changes in the levels of cofilin phosphorylation. TGF-beta induction of tropomyosins and stress fibers are significantly inhibited by Ras-ERK signaling in metastatic breast cancer cells. Inhibition of the Ras-ERK pathway restores TGF-beta induction of tropomyosins and stress fibers and thereby reduces cell motility. These results suggest that induction of tropomyosins and stress fibers play an essential role in TGF-beta control of cell motility, and the loss of this TGF-beta response is a critical step in the acquisition of metastatic phenotype by tumor cells.