Interleukin-8 gene regulation in epithelial cells by Vibrio cholerae: role of multiple promoter elements, adherence and motility of bacteria and host MAPKs.

Interleukin (IL)-8 is an important mediator in neutrophil-mediated acute inflammation. We previously demonstrated that incubation of intestinal epithelial cells with Vibrio cholerae O395 resulted in increased IL-8 mRNA expression and IL-8 secretion, which was associated with the adherence and motility of bacteria. However, the mechanisms responsible for transcriptional regulation of the IL-8 gene in epithelial cells during V. cholerae infections were not explored. Transient transfection analysis of 5' deletions and mutations of the IL-8 promoter driving expression of the luciferase reporter gene indicates that multiple binding sites contribute to IL-8 promoter induction in response to V. cholerae and that cooperation among these different sites is required for full activation of the promoter. Stimulation with V. cholerae O395 insertional mutants, defective in adherence and motility, significantly reduced IL-8 promoter activity compared with the wild-type strain. We further demonstrate maximal involvement of extracellular signal-regulated kinase 1/2/mitogen-activated protein kinase pathways to regulate IL-8 gene transcription. This study will help to design agents which could reduce the V. cholerae-induced inflammatory response and in the generation of safe vaccines.

The cytoskeleton-associated Ena/VASP proteins are unanticipated partners of the PMR1 mRNA endonuclease.

The PMR1 mRNA endonuclease catalyzes the selective decay of a limited number of mRNAs. It participates in multiple complexes, including one containing c-Src, its activating kinase, and one containing its substrate mRNA. This study used tandem affinity purification (TAP) chromatography to identify proteins in HeLa cell S100 associated with the mature 60-kDa form of Xenopus PMR1 (xPMR60). Unexpectedly, this identified a number of cytoskeleton-associated proteins, most notably the Ena family proteins mammalian Enabled (Mena) and vasodilator-stimulated phosphoprotein (VASP). These are regulators of actin dynamics that distribute throughout the cytoplasm and concentrate along the leading edge of the cell. xPMR60 interacts with Mena and VASP in vivo, overexpression of Mena has no impact on mRNA decay, and Mena and VASP are recovered together with xPMR60 in each of the major complexes of PMR1-mRNA decay. In a wound-healing experiment induced expression of active xPMR60 in stably transfected cells resulted in a twofold increase in cell motility compared with uninduced cells or cells expressing inactive xPMR60 degrees . Under these conditions xPMR60 colocalizes with VASP along one edge of the cell.

Differential expression and glycosylation of anaplasma phagocytophilum major surface protein 2 paralogs during cultivation in sialyl Lewis x-deficient host cells.

Many microbial pathogens alter expression and/or posttranslational modifications of their surface proteins in response to dynamics within their host microenvironments to retain optimal interactions with their host cells and/or to evade the humoral immune response. Anaplasma phagocytophilum is an intragranulocytic bacterium that utilizes sialyl Lewis x (sLe(x))-modified P-selectin glycoprotein ligand 1 as a receptor for infecting myeloid cells. Bacterial populations that do not rely on this receptor can be obtained through cultivation in sLe(x)-defective cell lines. A. phagocytophilum major surface protein 2 [Msp2(P44)] is encoded by members of a paralogous gene family and is speculated to play roles in host adaptation. We assessed the complement of Msp2(P44) paralogs expressed by A. phagocytophilum during infection of sLe(x)-competent HL-60 cells and two HL-60 cell lines defective for sLe(x) expression. Multiple Msp2(P44) and N-terminally truncated 25- to 27-kDa isoforms having various isoelectric points and electrophoretic mobilities were expressed in each cell line. The complement of expressed msp2(p44) paralogs and the glycosyl residues modifying Msp2(P44) varied considerably among bacterial populations recovered from sLe(x)-competent and -deficient host cells. Thus, loss of host cell sLe(x) expression coincided with both differential expression and glycosylation of A. phagocytophilum Msp2(P44). This reinforces the hypothesis that this bacterium is able to generate a large variety of surface-exposed molecules that could provide great antigenic diversity and result in multiple binding properties.

IL-1beta expression in Int407 is induced by flagellin of Vibrio cholerae through TLR5 mediated pathway.

Vibrio cholerae, a noninvasive enteric bacterium, causing inflammatory diarrheal disease cholera, is associated with the secretion of proinflamammatory cytokines including IL-1beta in cultured epithelial cells. Incubation of Int407 with live V. cholerae resulted in increased IL-1beta mRNA expression as early as 2h of infection, reached a peak at approximately 3.5h and decreased thereafter. The identity of the effector molecule(s) is largely unknown. The bacterial culture supernatant showed IL-1beta stimulating activity. An engineered aflagellate V. cholerae flaA mutant (O395FLAN) resulted in highly reduced level of IL-1beta expression in Int407. The crude flagellar protein of V. cholerae as well as recombinant FlaA induced IL-1beta expression in Int407. Infection of Toll-like receptor 5 (TLR5) transfected HeLa cells with O395FLAN showed reduced expression of IL-1beta compared to wild-type. Unlike wild-type V. cholerae, O395FLAN did not activate the NF-kappaB while the recombinant flagellin could activate NF-kappaB. Finally, the mitogen activated protein kinases (ERK1 and 2, p38) were phosphorylated in wild-type and recombinant flagellin treated Int407 cells and inhibition of the p38 and ERK pathways significantly decreased the IL-1beta response induced by wild-type V. cholerae as well as recombinant flagellin. Our data clearly indicate that flagellin of V. cholerae could induce IL-1beta expression by recognizing TLR5 that activate NF-kappaB and MAP kinase in Int407.

Anaplasma phagocytophilum MSP2(P44)-18 predominates and is modified into multiple isoforms in human myeloid cells.

Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis. MSP2(P44), the bacterium's major surface protein, is encoded by a paralogous gene family and has been implicated in a variety of pathobiological processes, including antigenic variation, host adaptation, adhesion, porin activity, and structural integrity. The consensus among several studies performed at the DNA and RNA levels is that a heterogeneous mix of a limited number of msp2(p44) transcripts is expressed by A. phagocytophilum during in vitro cultivation. Such analyses have yet to be extended to the protein level. In this study, we used proteomic and molecular approaches to determine that MSP2(P44)-18 is the predominant if not the only paralog expressed and is modified into multiple 42- to 44-kDa isoforms by A. phagocytophilum strain HGE1 during infection of HL-60 cells. The msp2(p44) expression profile was homogeneous for msp2(p44)-18. Thus, MSP2(P44)-18 may have a fitness advantage in HL-60 cell culture in the absence of selective immune pressure. Several novel 22- to 27-kDa MSP2 isoforms lacking most of the N-terminal conserved region were also identified. A. phagocytophilum MSP2(P44) orthologs expressed by other pathogens in the family Anaplasmataceae are glycosylated. Gas chromatography revealed that recombinant MSP2(P44)-18 is modified by glucose, galactose, xylose, mannose, and trace amounts of other glycosyl residues. These data are the first to confirm differential modification of any A. phagocytophilum MSP2(P44) paralog and the first to provide evidence for expression of truncated versions of such proteins.

Sialyl-Lewis x-independent infection of human myeloid cells by Anaplasma phagocytophilum strains HZ and HGE1.

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that infects neutrophils and neutrophil precursors. Bacterial recognition of P-selectin glycoprotein ligand-1 (PSGL-1) and the alpha2,3-sialylated- and alpha1,3-fucosylated-moiety sialyl-Lewis x (sLe(x)), which modifies the PSGL-1 N terminus, is important for adhesion to and invasion of myeloid cells. We have previously demonstrated that A. phagocytophilum organisms of the NCH-1 strain that utilize an sLe(x)-modified PSGL-1-independent means of entry can be enriched for by cultivation in undersialylated HL-60 cells that are unable to construct sLe(x). Because it was unknown whether other A. phagocytophilum isolates share this ability, we extended our studies to the geographically diverse strains HZ and HGE1. HL-60 A2 is a clonal cell line that is defective for sialylation and alpha1,3-fucosyltransferase. HL-60 A2 cell surfaces, therefore, not only lack sLe(x) but also are virtually devoid of any other sialic acid- and/or alpha1,3-fucose-modified glycan. By cultivating HZ and HGE1 in HL-60 A2 cells, we enriched for bacterial subpopulations (termed HZA2 and HGE1A2) that bind and/or infect myeloid cells in the absence of sialic acid and alpha1,3-fucose and in the presence of antibody that blocks the N terminus of PSGL-1. Thus, multiple A. phagocytophilum isolates share the ability to use sLe(x)-modified PSGL-1-dependent and -independent routes of entry into myeloid cells. HZA2 and HGE1A2 represent enriched bacterial populations that will aid dissection of the complexities of the interactions between A. phagocytophilum and host myeloid cells.

Transcriptional upregulation of inflammatory cytokines in human intestinal epithelial cells following Vibrio cholerae infection.

Coordinated expression and upregulation of interleukin-1alpha, interleukin-1beta, tumor necrosis factor-alpha, interleukin-6, granulocyte-macrophage colony-stimulating factor, interleukin-8, monocyte chemotactic protein-1 (MCP-1) and epithelial cell derived neutrophil activator-78, with chemoattractant and proinflammatory properties of various cytokine families, were obtained in the intestinal epithelial cell line Int407 upon Vibrio cholerae infection. These proinflammatory cytokines also showed increased expression in T84 cells, except for interleukin-6, whereas a striking dissimilarity in cytokine expression was observed in Caco-2 cells. Gene expression studies of MCP-1, granulocyte-macrophage colony-stimulating factor, interleukin-1alpha, interleukin-6 and the anti-inflammatory cytokine transforming growth factor-beta in Int407 cells with V. cholerae culture supernatant, cholera toxin, lipopolysaccharide and ctxA mutant demonstrated that, apart from cholera toxin and lipopolysaccharide, V. cholerae culture supernatant harbors strong inducer(s) of interleukin-6 and MCP-1 and moderate inducer(s) of interleukin-1alpha and granulocyte-macrophage colony-stimulating factor. Cholera toxin- or lipopolysaccharide-induced cytokine expression is facilitated by activation of nuclear factor-kappaB (p65 and p50) and cAMP response element-binding protein in Int407 cells. Studies with ctxA mutants of V. cholerae revealed that the mutant activates the p65 subunit of nuclear factor-kappaB and cAMP response element-binding protein, and as such the activation is mediated by cholera toxin-independent factors as well. We conclude that V. cholerae elicits a proinflammatory response in Int407 cells that is mediated by activation of nuclear factor-kappaB and cAMP response element-binding protein by cholera toxin, lipopolysaccharide and/or other secreted products of 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.

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.