ISOLATION OF POTENTIAL ANTIMICROBIAL METABOLITE FROM ENDOPHYTIC BACILLUS AMYLOLIQUEFACIENS DL06 OF CARNIVOROUS PLANT DROSERA BURMANNII VAHL.

Objectives: Exploitation of bacterial endophytes for production of antimicrobial substances has led to the discovery of novel natural metabolites of diverse chemical nature. The present study focuses attention toward optimization of cultural conditions for production of antimicrobial compound(s) by an endophytic bacterium DL06 followed by its extraction and partial purification. Methods: The leaf endophytic bacterium Bacillus amyloliquefaciens DL06 (GenBank Accession no. MK696415, Microbial Culture Collection Accession no. 4186) isolated from carnivorous plant Drosera burmannii has been identified as a potent producer of antimicrobial metabolite following agar cup assay against several test bacterial and fungal strains. Cultural conditions for production of antimicrobials were optimized by “one variable at a time” method. The active fraction was isolated and purified partially using solvent extraction, thin-layer chromatography, and high performance liquid chromatography (HPLC) analysis. Results: B. amyloliquefaciens DL06 produced maximum antimicrobial compound in tryptic soy broth and Davis–Mingioli’s medium when grown under shake culture. Production of the antimicrobial metabolite has been optimized for the inoculum density, aeration, temperature, pH as well as carbon, and nitrogen sources. The antimicrobial metabolite was extracted from the cell-free culture filtrate in butanol and partially purified by silica gel column chromatography and HPLC. Conclusions: The antimicrobial metabolite, tentatively identified as quercetin showed broad spectrum bioactivity affecting several fungi and a number of Gram-positive and Gram-negative bacteria

Transcriptional Upregulation of Inflammatory Cytokines in Human Intestinal Epithelial Cells Following Vibrio cholerae Infection

Coordinated expression and upregulation of interleukin-1a, interleukin-1b, tumor necrosis factor-a, interleukin-6, granulocyte–macrophage colonystimulating 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-1a, interleukin- 6 and the anti-inflammatory cytokine transforming growth factor-b 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- 1a and granulocyte–macrophage colony-stimulating factor. Cholera toxin- or lipopolysaccharide-induced cytokine expression is facilitated by activation of nuclear factor-jB (p65 and p50) and cAMP response elementbinding protein in Int407 cells. Studies with ctxA mutants of V. cholerae revealed that the mutant activates the p65 subunit of nuclear factor-jB 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-jB and cAMP response element-binding protein by cholera toxin, lipopolysaccharide and ⁄ or other secreted products of V. cholerae

IL-1b 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-1b in cultured epithelial cells. Incubation of Int407 with live V. cholerae resulted in increased IL-1b mRNA expression as early as 2 h of infection, reached a peak at �3.5 h and decreased thereafter. The identity of the effector molecule(s) is largely unknown. The bacterial culture supernatant showed IL-1b stimulating activity. An engineered aflagellate V. cholerae flaA mutant (O395FLAN) resulted in highly reduced level of IL-1b expression in Int407. The crude flagellar protein of V. cholerae as well as recombinant FlaA induced IL-1b expression in Int407. Infection of Toll-like receptor 5 (TLR5) transfected HeLa cells with O395FLAN showed reduced expression of IL-1b compared to wild-type. Unlike wild-type V. cholerae, O395FLAN did not activate the NF-kB while the recombinant flagellin could activate NF-kB. 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-1b response induced by wild-type V. cholerae as well as recombinant flagellin. Our data clearly indicate that flagellin of V. cholerae could induce IL-1b expression by recognizing TLR5 that activate NF-kB and MAP kinase in Int407