MarA, SoxS and Rob function as virulence factors in an Escherichia coli murine model of ascending pyelonephritis

Abstract

MarA, SoxS and Rob are transcription factors belonging to the AraC family. While these proteins have been associated historically with control of multiple antibiotic resistance, and tolerance to oxidative stress agents and organic solvents, only a paucity of experimental data support a role in regulating virulence. Clinical Escherichia coli isolates, and isogenic strains lacking marA, soxS and rob, were studied in a murine model of ascending pyelonephritis, which is a clinically relevant model of urinary tract infection. Organisms lacking all three transcription factors (triple knockouts) were significantly less virulent than parental strains, and complementation studies demonstrated that the addition of marA, soxS and rob individually restored wild-type virulence in the triple-knockout strain. Deletion of soxS or rob alone was more detrimental than the removal of marA. Thus, all three proteins contribute to virulence in vivo. INTRODUCTIONThe AraC family of transcription factors is composed of more than 1000 members (Alekshun & Levy, 2004), many of which have well-known roles as virulence factors (Finlay & Falkow, 1997). ExsA from Pseudomonas aeruginosa regulates a type III secretion system (TTSS) (Hauser et al., 1998), Yersinia spp. LcrF (VirF) and YbtA control a TTSS (Flashner et al., 2004) and yersiniabactin (siderophore) (Fetherston et al., 1996) biosynthesis, respectively, and ToxT from Vibrio cholerae governs the synthesis of cholera toxin and toxin coregulated pili (Champion et al., 1997). Inactivation of genes specifying AraC family members [e.g. BfpT, ToxT, LcrF (VirF), Rv1931c, ExsA, Sp1433 and MarA] attenuates virulence in human subjects (Bieber et al., 1998) and a variety of animal infection models (Champion et al., 1997;Flashner et al., 2004; Frota et al., 2004;Hauser et al., 1998;Hava & Camilli, 2002;Randall & Woodward, 2001).Thus, in addition to a primary role in virulence, it is assumed that many members of the AraC family play larger roles in affecting the overall physiology of the bacterial cell.Notably, genomic array experiments have shown that P. aeruginosa ExsA and V. cholerae ToxT regulate the expression of a large collection of genes termed regulons (Bina et al., 2003;Wolfgang et al., 2003).Escherichia coli MarA and SoxS were originally identified based on their ability to control multiple antibiotic resistance (Mar) (George & Levy, 1983a, b), and susceptibility to superoxide and other oxidative stress agents (Wu & Weiss, 1991), respectively. Experiments with Rob, a MarA and SoxS paralogue, showed that it could function in a similar manner (Ariza et al., 1995). Subsequent data have documented multidrug-resistant clinical strains of E. coli (Linde et al., 2000; Maneewannakul & Levy, 1996) and Salmonella enterica serovar Typhimurium (S. typhimurium) (Koutsolioutsou et al., 2001) that constitutively express AraC family members. It is therefore surmised that these proteins may play a role in the infectious process.Although the soxS and mar loci are expressed by S. typhimurium within macrophag...

The contribution of PmrAB to the virulence of a clinical isolate ofEscherichia coli

“…We hypothesized that expression of pmrAB may play a role in the decreased persistence of strain PC1012, a marA-rob-soxS triple mutant, in the mouse kidney. 5 Therefore we performed single strain infections which compared the bacterial loads of groups of mice infected with strain PC1012 alone, or transformed with plasmid pPmrAB. Upon expression of pmrAB on this multicopy plasmid in strain PC1012 we observed a significant increase in the bacterial loads of this strain on day 4 post-infection when compared with its parent (Fig.…”

“…While these increases in colonization do not bring bacterial loads to those of strain KM-D (~10 4 cfu/g kidney; data not shown), they do show that pmrAB is a contributor to the previously observed persistence attenuation seen in a strain lacking all three transcription regulators. 5 subsequently selected on LB-agar plates containing Cm and Km. Cm R /Km R transformants were screened by PCR, before being used as donors in conjugations with strain KM-D as described previously.…”

“…Cm R /Km R transformants were screened by PCR, before being used as donors in conjugations with strain KM-D as described previously. 5 KM-DpmrAB candidates were screened by PCR for the presence of the disrupted operon, and were confirmed phenotypically by characterization of growth in iron or deoxycholate. Plasmid pPmrAB was transformed into strains KM-DpmrAB and PC1012 via chemical transformation, and successful transformants were cultured on media supplemented with ampicillin.…”

“…[1][2][3][4] The three related transcription factors, MarA, Rob, and SoxS, act in both individual and redundant manners to affect antibiotic resistance and virulence of Escherichia coli, through known and unknown mechanisms. 5,6 These three transcription factors play a key role in the persistence of E. coli in a murine kidney infection model, where deletion of all three genes significantly decreases the bacterial load in the kidney after three days of successful colonization. 5 Previous microarray data show that expression levels of genes associated with the pmrAB regulon 6,7 were decreased in a triple mutant lacking the transcription factors MarA, Rob, and SoxS compared with its parent strain, when extracted from a murine pyelonephritis infection model.…”

“…5,6 These three transcription factors play a key role in the persistence of E. coli in a murine kidney infection model, where deletion of all three genes significantly decreases the bacterial load in the kidney after three days of successful colonization. 5 Previous microarray data show that expression levels of genes associated with the pmrAB regulon 6,7 were decreased in a triple mutant lacking the transcription factors MarA, Rob, and SoxS compared with its parent strain, when extracted from a murine pyelonephritis infection model. 6 The pmrAB operon encodes a two-component regulatory system present in many bacterial species, 8,9 which mediates a wide range of functions, most notably modification of the lipid A moiety of lipopolysaccharide (LPS).…”