An activator of glutamate decarboxylase genes regulates the expression of enteropathogenic Escherichia coli virulence genes through control of the plasmid‐encoded regulator, Per

Abstract

SummaryEnteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in a number of developing countries and is the prototype of pathogenic bacteria that cause attaching and effacing (A/E) intestinal lesions. A chromosomal pathogenicity island, termed the locus of enterocyte effacement (LEE), contains all the genes necessary for the A/E phenotype as well as genes for a type III secretion system and intimate adhesion. Genes in the LEE and genes involved in the synthesis of bundle-forming pili (BFP) are positively regulated by the plasmid-encoded regulator (Per) and comprise the per regulon. In order to identify factors that control the per regulon, we screened an EPEC genomic library for clones that modulate the expression of per. A plasmid clone that decreased the expression of per was isolated using a lacZ reporter gene fused to the per promoter. Subcloning revealed that YhiX, a putative AraC/XylR family transcriptional regulator, was the effector of per repression. Through downregulation of per, a plasmid overproducing YhiX reduced the synthesis of intimin, BfpA, Tir, and CesT, factors important for EPEC virulence. yhiX is located downstream of gadA, which encodes glutamate decarboxylase, an enzyme involved in acid resistance of E. coli. YhiX was found to be an activator of gadA, and the cloned yhiX gene increased production of glutamate decarboxylases (GAD) and activated the transcription of the gadA and gadB promoters. Therefore, yhiX was renamed gadX. Analysis of a gadX mutant grown in the different culture media with acidic and alkaline pH showed that regulation of perA, gadA and gadB by GadX was altered by the external pH and the culture media condition. Under conditions in which EPEC infects cultured epithelial cells, GadX negatively regulated perA expression, and the derepression in the gadX mutant increased translocation of Tir into epithelial cells relative to wild-type EPEC. DNA mobility shift experiments showed that purified GadX protein bound to the perA, gadA and gadB promoter regions in vitro, indicating that GadX is a transcriptional regulator of these genes. On the basis of these results, we propose that GadX may be involved in the appropriate expression of genes required for acid resistance and virulence of EPEC. Our data are consistent with a model in which environmental changes resulting from passage from the stomach to the proximal small intestine induce the functional effect of GadX on per and GAD expression in order to prevent inappropriate expression of the products of these two systems.

Genetic Features Differentiating Bovine, Food, and Human Isolates of Shiga Toxin-Producing Escherichia coli O157 in The Netherlands

“…The capacity to withstand extreme acidity is critical for STEC O157 isolates in order to successfully colonize cattle. In addition, increased expression of the central activator of the glutamate decarboxylase acid resistance system negatively regulates expression of LEE (39).…”

Acid-stress-induced changes in enterohaemorrhagic Escherichia coli O157 : H7 virulence

“…Acid-stress-induced changes in EHEC O157 : H7 virulence showed recovery and/or increase after prolonged acute acid stress and acid-adapted acid stress. Expression of LEE genes has been shown to be regulated by environmental factors including culture conditions, temperature, biocarbonate ion and nutrient levels (Abe et al, 2002;Kenny et al, 1997;Nakanishi et al, 2006;Puente et al, 1996;Rosenshine et al, 1996;Sperandio et al, 2002) and in EPEC it is also regulated by acid-tolerance response mechanisms (Shin et al, 2001).…”

“…In the related pathogen, enteropathogenic E. coli (EPEC), the plasmid-encoded regulator, Per, which regulates expression of many EPEC virulence factors, is negatively regulated at pH 5.5 and positively regulated at pH 8.0, suggesting that virulence gene expression is repressed during mild acid stress and enhanced at neutral to alkaline pH, typical of the small intestine, which it colonizes (Shin et al, 2001). In another study, a gadE (encoding an acidresistance regulator) mutation resulted in increased adhesion of E. coli O157 : H7 to colonic epithelial cells, again suggesting negative regulation of one or more adhesins during acid stress (Tatsuno et al, 2003).…”

The glutamate-dependent acid resistance system in Escherichia coli: essential and dual role of the His–Asp phosphorelay RcsCDB/AF

“…Highlighting the importance of the AR2 system is the plethora of regulators involved in its regulation, the relative importance of each regulator depending on environmental pH and growth phase. These regulators include: two phosphorelays, EvgAS (Masuda & Church, 2002) and TorRS (Bordi et al, 2003), the stationary phase s s -factor (De Biase et al, 1999), the global regulators Crp ) and H-NS (Hommais et al, 2001), three AraC-like regulators, GadW (Ma et al, 2002), GadX (Shin et al, 2001) and YdeO (Masuda & Church, 2003), and the Era-like GTPase TrmE (Gong et al, 2004). Central to the AR2 system is the activity of the LuxRlike transcription regulator GadE, as most of the regulators listed target gadE expression.…”