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.