Control of Bacterial Virulence by the RalR Regulator of the Rabbit-Specific Enteropathogenic Escherichia coli Strain E22

Abstract

dAtypical enteropathogenic Escherichia coli (aEPEC) causes endemic diarrhea, diarrheal outbreaks, and persistent diarrhea in humans, but the mechanism by which aEPEC causes disease is incompletely understood. Virulence regulators and their associated regulons, which often include adhesins, play key roles in the expression of virulence factors in enteric pathogenic bacteria. In this study we identified a transcriptional regulator, RalR, in the rabbit-specific aEPEC strain, E22 (O103:H2) and examined its involvement in the regulation of virulence. Microarray analysis and quantitative real-time reverse transcription-PCR demonstrated that RalR enhances the expression of a number of genes encoding virulence-associated factors, including the Ral fimbria, the Aap dispersin, and its associated transport system, and downregulates several housekeeping genes, including fliC. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins and by adherence and motility assays. To investigate the mechanism of RalR-mediated activation, we focused on its most highly upregulated target operons, ralCDEFGHI and aap. By using primer extension, electrophoretic mobility shift assay, and mutational analysis, we identified the promoter and operator sequences for these two operons. By employing promoter-lacZ reporter systems, we demonstrated that RalR activates the expression of its target genes by binding to one or more 8-bp palindromic sequences (with the consensus of TGTGCACA) located immediately upstream of the promoter core regions. Importantly, we also demonstrated that RalR is essential for virulence since infection of rabbits with E22 carrying a knockout mutation in the ralR gene completely abolished its ability to cause disease. Enteropathogenic Escherichia coli (EPEC) is among the most important diarrheal pathogens infecting children and is also a major cause of persistent diarrhea and diarrhea-associated mortality (1, 2). The hallmark of EPEC pathogenicity is colonization of the intestine accompanied by the formation of characteristic "attaching-and-effacing" (A/E) lesions on the surface of intestinal epithelial cells (3,4). A 35-kb chromosomal pathogenicity island, termed the locus of enterocyte effacement (LEE), encodes the genetic determinants required for A/E lesion formation (5-8).EPEC can be further categorized into two subgroups, typical EPEC (tEPEC) and atypical EPEC (aEPEC) (9), which differ from each other in terms of their genetic characteristics, serotypes, and virulence factors (10,11). By definition, all EPEC strains carry the LEE, but tEPEC strains also carry an EPEC adherence factor plasmid (pEAF), which encodes a type IV-like bundle-forming pilus (Bfp) that facilitates the adherence of tEPEC cells to the intestinal mucosa and allows them to form microcolonies on epithelial cells in vitro and in vivo (11). Apart from Bfp, pEAF also encodes two transcriptional activators, PerA and PerC. The former activates transcription of bfpA (the gene for the major structur...

Molecular Characterization and Antimicrobial Resistance of Enteropathogenic Escherichia coli in Children from Ahvaz, Iran

“…The exact mechanisms of aEPEC-induced diarrhea are still not completely understood (43,44). Recently, Afset et al detected a significant association between the prevalence of diarrhea and the presence of the O island 122 (OI-122), including efa1/lifA and several other genes (45).…”

Surface Protein Dispersin of Enteroaggregative Escherichia coli Binds Plasminogen That Is Converted Into Active Plasmin

“…Four of these aap-positive strains produced dispersin in vitro, which could be detected even in the absence of the dispersin transporter system (aatPABCD) present in EAEC 042, which can suggest that there are alternative mechanisms for dispersin regulation and secretion as suggested by Nishi et al (2003). Besides AggR, RalR was described as the dispersin regulator in rabbit-specific enteropathogenic E. coli (Srikhanta et al, 2013), and CfaD/Rns as the regulator of CexE, a dispersin-like protein FIGURE 7 | Detection of dispersin by ELISA in E. coli strains isolated from blood of human patients. Bacterial pellets of overnight cultures in LB broth were used for coating of ELISA plates, followed by incubation with anti-dispersin polyclonal serum (1:200) produced in rabbit.…”

“…The aap gene has already been detected in different E. coli pathotypes (Abe et al, 2008;Monteiro et al, 2009;Crossman et al, 2010;Nazemi et al, 2011;Srikhanta et al, 2013;Riveros et al, 2017). In one study the aap gene was detected in 46.6% of uropathogenic E. coli (UPEC) strains (Nazemi et al, 2011).…”

“…Although dispersin has been firstly described in EAEC, the aap gene has also been detected in extraintestinal E. coli (ExPEC) (Abe et al, 2008;Nazemi et al, 2011;Riveros et al, 2017), and in other diarrheagenic E. coli pathotypes, such as diffusely adherent E. coli and rabbit-specific enteropathogenic E. coli (Monteiro et al, 2009;Srikhanta et al, 2013). Enterotoxigenic E. coli harbors an aap-like gene (cexE), along with the aat operon, encoding its transporter system (Crossman et al, 2010).…”

Control of Acid Resistance Pathways of Enterohemorrhagic Escherichia coli Strain EDL933 by PsrB, a Prophage-Encoded AraC-Like Regulator

“…The PsrB protein belongs to the AraC superfamily of transcriptional regulators which control the expression of genes involved in diverse cellular functions such as carbon metabolism, stress responses, and virulence (35,36). In enteric bacterial pathogens, a number of AraC-like activators including AggR of enteroaggregative E. coli (37), Rns of enterotoxigenic E. coli (38), PerA, RegR, and RalR of enteropathogenic E. coli (39)(40)(41), RegA of Citrobacter rodentium (42,43), and ToxT of Vibrio cholerae (44) play a critical role in bacterial virulence. The genes encoding these virulence activators appear to have coevolved with their principal target virulence genes, which code for secreted factors and surface proteins responsible for intestinal colonization (40,41,45,46).…”