Project description:Enterohaemorrhagic E. coli (EHEC) is a significant human pathogens that cause outbreaks of haemorrhagic colitis and haemolytic uremic syndrome. During infection, pathogens compete for iron with the host, and one mechanism by which EHEC obtains iron is through haem uptake and utilitisation which is encoded by the chu operon. We have demonstrated that the haem receptor chuA is regulated by the Crp-cAMP-dependent sRNA CyaR. We further demonstrate that activation of chuA by CyaR is independent of the chuA RNA-thermometer and termination by Rho. These results highlight the ability of regulatory sRNAs to integrate multiple environmental signals into a layered hierarchy of signal input.
Project description:Integrating laterally acquired virulence genes into the backbone regulatory network is important for the pathogenesis of Escherichia coli O157:H7, which has captured many virulence genes through horizontal transfer during evolution. GadE is an essential transcriptional activator of the glutamate decarboxylase (GAD) system, the most efficient acid resistance (AR) mechanism in E. coli. The full contribution of GadE to the AR and virulence of E. coli O157:H7 remains largely unknown. We inactivated gadE in E. coli O157:H7 Sakai and compared global transcription profiles of the mutant with that of the wild type in the exponential and stationary phases of growth. Inactivation of gadE significantly altered the expression of 60 genes independently of the growth phase and of 122 genes in a growth phase-dependent manner. Inactivation of gadE markedly downregulated the expression of gadA, gadB, and gadC and of many acid fitness island genes. Nineteen genes encoded on the locus of enterocyte effacement (LEE), including ler, showed a significant increase in expression upon gadE inactivation. Inactivation of ler in the DeltagadE strain reversed the effect of gadE deletion on LEE expression, indicating that Ler is necessary for LEE repression by GadE. GadE is also involved in downregulation of LEE expression under conditions of moderately acidic pH. Characterization of AR of the DeltagadE strain revealed that GadE is indispensable for a functional GAD system and for survival of E. coli O157:H7 in a simulated gastric environment. Altogether, these data indicate that GadE is critical for the AR of E. coli O157:H7 and that it plays an important role in virulence by downregulating expression of LEE.
Project description:Transcript abundance in Escherichia coli O157:H7 was determined in the presence or absence of pulsed expression of the small RNA, AsxR.
Project description:Comparative analysis of EHEC O157:H7 Sakai vs. TW14359 secreted and cytosolic protein using iTRAQ and mass spectrometry on an LTQ Orbitrap. Samples were fractionated through strong cation exchange and HPLC before analsyis. For TW14359 sepD secretome analysis, strains were similarly prepared and analyzed. All samples were searched against a TW14359 specific protein database (Uniprot) coupled with randomized protein sequences.