Comparative Proteomic Analysis of Extracellular Proteins of Enterohemorrhagic and Enteropathogenic Escherichia coli Strains and Their ihf and ler Mutants

Abstract

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains are closely related human pathogens that are responsible for food-borne epidemics in many countries. Integration host factor (IHF) and the locus of enterocyte effacement-encoded regulator (Ler) are needed for the expression of virulence genes in EHEC and EPEC, including the elicitation of actin rearrangements for attaching and effacing lesions. We applied a proteomic approach, using two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry and a protein database search, to analyze the extracellular protein profiles of EHEC EDL933, EPEC E2348/69, and their ihf and ler mutants. Fifty-nine major protein spots from the extracellular proteomes were identified, including six proteins of unknown function. Twenty-six of them were conserved between EHEC EDL933 and EPEC E2348/69, while some of them were strain-specific proteins. Four common extracellular proteins (EspA, EspB, EspD, and Tir) were regulated by both IHF and Ler in EHEC EDL933 and EPEC E2348/69. TagA in EHEC EDL933 and EspC and EspF in EPEC E2348/69 were present in the wild-type strains but absent from their respective ler and ihf mutants, while FliC was overexpressed in the ihf mutant of EPEC E2348/69. Two dominant forms of EspB were found in EHEC EDL933 and EPEC E2348/69, but the significance of this is unknown. These results show that proteomics is a powerful platform technology for accelerating the understanding of EPEC and EHEC pathogenesis and identifying markers for laboratory diagnoses of these pathogens.Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains are closely related human pathogens (7). EHEC strains, especially those of serotype O157:H7, which produce Shiga-like toxin (Stx), are a common cause of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome, while EPEC is the most common bacterial cause of infant diarrhea (29). Both have been implicated in food-borne outbreaks in many countries and cause diarrhea by colonizing the intestinal mucosa (29, 30). EHEC and EPEC strains are distinguished from other pathogenic E. coli strains by their ability to produce a characteristic histopathological feature known as attaching and effacing (AE) lesions on the mucosa (12, 29). AE lesions are characterized by the destruction of the microvilli and the induction of actin-based pedestal formation underneath the eukaryotic membrane at the site of attachment (6). EHEC and EPEC secrete many extracellular proteins (ECPs), and the type III secretion system (TTSS) is a major secretion apparatus for secreting virulence factors which interact directly with the host (20, 25). The TTSS is located within a chromosomal pathogenicity island designated the locus of enterocyte effacement (LEE) which is necessary for the formation of AE lesions (18,19). The LEE-encoded regulator (Ler) activates most of the genes within the LEE region and is centr...

Type 2 Secretion Promotes Enterohemorrhagic Escherichia coli Adherence and Intestinal Colonization

“…A number of omptin family members have been implicated in virulence, including Pla from Yersinia pestis and NspA from Neisseria meningitidis (21). The Z3065 protein was previously identified in the culture supernatant of EHEC grown in the tissue culture medium DMEM (29). The Z3065 protein is 99% identical to YodA from E. coli K-12, and the Z3065 gene and yodA are flanked by the same genes in their respective genomes.…”

Proteins differentially expressed by Shiga toxin-producing Escherichia coli strain M03 due to the biliar salt sodium deoxycholate

“…Proteomic analysis has become increasingly important, allowing global analysis of protein expression patterns and providing a better understanding of physiological changes related to various genetic and/or environmental conditions (Li et al, 2004;Sanchez et al, 2005;Weber et al, 2006;Xia et al, 2008).…”

Molecular Characterization of GrlA, a Specific Positive Regulator of ler Expression in Enteropathogenic Escherichia coli

“…It has been well documented that the master positive regulator of LEE genes, as well as of genes located outside the LEE, is the LEE-encoded regulator (Ler), whose main function is to counteract the repression exerted by H-NS on these genes (1,3,9,17,23,39,48,57,62,65,69). It has also been reported that Ler represses its own expression in a concentration-dependent fashion (4) and that it can also act as a negative regulator for other genes outside the LEE (1,61).…”