Author
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Sharma, Vijay |
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Submitted to: Zoonoses and Public Health
Publication Type: Proceedings Publication Acceptance Date: 1/20/2012 Publication Date: 3/20/2012 Citation: Sharma, V.K. 2012. Quorum sensing transcriptional regulator QseA is essential for the expression of multiple virulence regulons of enterohemorrhagic Escherichia coli O157:H7. Zoonoses and Public Health. 59:(Suppl. 1), 2-18. Interpretive Summary: Technical Abstract: Introduction and Objectives: QseA is one of several transcriptional regulators that regulates the virulence gene expression in enterohemorrhagic Escherichia coli (EHEC) O157:H7 through quorum sensing. QseA has been shown to regulate the expression of the locus of enterocyte effacement (LEE), non-LEE encoded effectors, and some putative fimbrial gene clusters. The LEE encodes for a type three secretion system to secrete proteins necessary for the adherence of EHEC O157:H7 to intestinal epithelial cells, tissue colonization, and persistence of these foodborne pathogenic bacteria in bovine intestines. In this study, we demonstrate that QseA is essential not only for the LEE expression but also for the expression of flagellar and curli fimbrial regulons that play important role in the adherence of EHEC O157:H7 to epithelial cells and formation of biofilms on abiotic surfaces and food matrices. Material and Methods: A one-step QRT-PCR was used to assess the expression of select genes encoded by LEE, flagellar, and curli fimbrial regulons. DNA-free RNA prepared from EHEC O157:H7 strain 86-24 and its isogenic qseA deletion mutant, constructed by an allelic replacement method, was amplified and detected by incorporating gene-specific sense and anti-sense primers and a TaqMan probe. Bacterial motility was determined by inoculating overnight cultures or cultures grown to an A600 of 1.0 on 0.3% motility agar plates with or without supplementation with 50 microM epinephrine. Motility zones were measured after 12 to 48 hours of incubation of these plates at 37degree C. Weaned calves (n = 4 per group) were orally inoculated with 10**10 cfu of the wild-type or qseA mutant strains and these calves were monitored for fecal shedding of the inoculated strains for 27-days. Results: The QRT-PCR analysis revealed that the transcriptional levels of ler, flhDC, and csgD, the master regulators of LEE, flagellar, and curli fimbrial regulons, respectively, were several fold lower in the qseA mutant than the wild-type EHEC O157:H7 strain. The reduced expression of the three master regulators correlated with the reduced expression of espA, fliC, and csgA of LEE, flagellar, and curli fimbrial regulons, respectively. The reduced expression of flagellar genes resulted in reduced motility of the qseA mutant compared to the wild-type strain regardless of the presence or absence of epinephrine in the motility medium. In calves, orally inoculated with the wild-type or the qseA mutant strains, the qseA mutant strain showed reduced persistence and magnitude of fecal shedding. Conclusions: The results described in this study indicate that QseA is an important transcriptional factor that directly and in conjunction with other quorum sensing signaling pathways regulates the effects of quorum sensing signaling on virulence gene expression and persistence of EHEC O157:H7 in cattle. |
