T6-04 Down-Regulation of Flagellin in CytR Mutant Leads to an Attenuation in Virulence of Escherichia coli O157:H7

Tuesday, August 2, 2016: 9:15 AM
242 (America's Center - St. Louis)
Haiqing Yu, University of Missouri-Columbia, Columbia, MO
Yuanxi Xu, University of Missouri-Columbia, Columbia, MO
Fanding Gao, University of Missouri-Columbia, Columbia, MO
Azlin Mustapha, University of Missouri-Columbia, Columbia, MO
Hongmin Sun, University of Missouri-Columbia, Columbia, MO
Introduction: Escherichia coli O157:H7 is a foodborne pathogen causing an estimated annual incidence of 73,000 cases of diarrheal illness in the United States. Cytidine repressor (CytR) plays a crucial role in transcription initiation from several operons in pathogens. However, little is known about its contribution to the virulence of E. coli O157:H7.

Purpose: To evaluate the possible molecular mechanism for CytR in pathogenesis of E. coli O157:H7.

Methods: The CytR insertion mutant strain (ΔCytR) was constructed by using TargeTron® Gene Knockout System. E. coli O157:H7 wild-type strain (WT, ATCC 43894) and ΔCytR strain were cultured for 16 h in THY medium at 37°C. The prominent differential expression protein between the two strains was analyzed by NanoLC-Nanospray QTOF MS-MS/MS. The transcription level of differential genes was analyzed by using quantitative RT-PCR (qRT-PCR), which was repeated three times. The virulence of the two strains was investigated via an oral infection mouse model with 3-5×1010 CFU.

Results: Compared with the WT strain, SDS-PAGE and NanoLC-Nanospray QTOF MS-MS/MS analyses indicated that flagellin was significantly decreased in ΔCytR strain (P<0.01). qRT-PCR analysis indicated that the transcription level of flagellin was dramatically inhibited in ΔCytR strain compared to WT strain (P<0.01). The survival rate in WT group and ΔCytR group was 60% and 100% (P<0.05), respectively.

Significance: The expression of flagellin, a principal component of bacterial flagella being responsible for adhesion and virulence of E. coli, was down-regulated in CytR-deficient strain, leading to the attenuation of virulence. Our result suggests that CytR or flagellin could serve as a novel drug target in controlling the pathogenesis of E. coli O157:H7. To identify small compounds as candidates for inhibition of E. coli O157:H7 attachment, a high-throughput screening assay is developing based on a chloramphenicol resistance gene under control of the CytR or flagellin promoter.