6-Deoxyheptose is found within the surface polysaccharides of several bacterial pathogens. In Yersinia pseudotuberculosis, it is important for the barrier function of the O-antigen in vitro and for bacterial dissemination in vivo. The putative C6 dehydratase DmhA and C4 reductase DmhB, that were identified as responsible for 6-deoxyheptose synthesis based on genetics data, represent potential therapeutical targets. Their detailed biochemical characterization is presented herein. The substrate, GDP-D-glycero-D-manno-heptose, was synthesized enzymatically from sedoheptulose 7-phosphate using overexpressed and purified GmhA/B/C/D enzymes from Aneurinibacillus thermoaerophilus. Overexpressed and purified DmhA used this substrate with high efficiency, as indicated by its K(m) of 0.23 mM and k(cat) of 1.1 s(-1). The mass spectrometry (MS) analysis of the reaction product was consistent with a C6 dehydration reaction. DmhB could readily reduce this compound in the presence of NAD(P)H to produce GDP-6-deoxy-D-manno-heptose, as indicated by MS and NMR analyses. DmhA also used GDP-mannose as a substrate with a K(m) of 0.32 mM and a k(cat) of 0.25 min(-1). This kinetic analysis indicates that although the K(m) values for GDP-mannose and GDP-manno-heptose were similar, the genuine substrate for DmhA is GDP-manno-heptose. DmhB was also able to reduce the GDP-4-keto-6-deoxymannose produced by DmhA, although with poor efficiency and exclusively in the presence of NADPH. This study is the first complete biochemical characterization of the 6-deoxyheptose biosynthesis pathway. Also, it allows the screening for inhibitors, the elucidation of substrate specificity determinants, and the synthesis of carbohydrate antigens of therapeutic relevance.
BackgroundThe pathogenesis of enterohemorrhagic Escherichia coli (EHEC) O157∶H7 infection is attributed to virulence factors encoded on multiple pathogenicity islands. Previous studies have shown that EHEC O157∶H7 modulates host cell signal transduction cascades, independent of toxins and rearrangement of the cytoskeleton. However, the virulence factors and mechanisms responsible for EHEC-mediated subversion of signal transduction remain to be determined. Therefore, the purpose of this study was to first identify differentially regulated genes in response to EHEC O157∶H7 grown in the presence of epithelial cells, compared to growth in the absence of epithelial cells (that is, growth in minimal essential tissue culture medium alone, minimal essential tissue culture medium in the presence of 5% CO2, and Penassay broth alone) and, second, to identify EHEC virulence factors responsible for pathogen modulation of host cell signal transduction.Methodology/Principal FindingsOvernight cultures of EHEC O157∶H7 were incubated for 6 hr at 37°C in the presence or absence of confluent epithelial (HEp-2) cells. Total RNA was then extracted and used for microarray analyses (Affymetrix E. coli Genome 2.0 gene chips). Relative to bacteria grown in each of the other conditions, EHEC O157∶H7 cultured in the presence of cultured epithelial cells displayed a distinct gene-expression profile. A 2.0-fold increase in the expression of 71 genes and a 2.0-fold decrease in expression of 60 other genes were identified in EHEC O157∶H7 grown in the presence of epithelial cells, compared to bacteria grown in media alone.Conclusion/SignificanceMicroarray analyses and gene deletion identified a protease on O-island 50, gene Z1787, as a potential virulence factor responsible for mediating EHEC inhibition of the interferon (IFN)-γ-Jak1,2-STAT-1 signal transduction cascade. Up-regulated genes provide novel targets for use in developing strategies to interrupt the infectious process.
Enterohemorrhagic Escherichia coli serotype O157:H7 is a food- and waterborne pathogen that causes significant morbidity and mortality in both developing and industrialized nations. The present review focuses on the history, epidemiology and evolution of the pathogen; provides a mechanistic overview of major virulence factors (including Shiga toxins, locus of enterocyte effacement pathogenicity island and pO157 plasmid); discusses host immune responses to infection; considers available animal models; and provides an overview of current and potential future management considerations.
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