Molecular Characterization of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli

Abstract

The vacuolating autotransporter toxin (Vat) contributes to uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Here, we characterized Vat and investigated its regulation in UPEC. We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed that it was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, ST73, and ST95), and these strains secreted the Vat protein. Further analysis of the vat genomic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator; we termed this gene vatX. The vat-vatX genes were present in the UPEC reference strain CFT073, and reverse transcriptase PCR (RT-PCR) revealed that the two genes are cotranscribed. Overexpression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator histone-like nucleoid structuring protein (H-NS); thus, the hns gene was mutated in CFT073 (to generate CFT073 hns). Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073 hns compared to that in wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients infected by vat-containing UPEC strains, demonstrating that Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated immunogenic serine protease autotransporter protein of Enterobacteriaceae (SPATE) secreted by UPEC during infection. IMPORTANCEUropathogenic Escherichia coli (UPEC) is the major cause of hospital-and community-acquired urinary tract infections. The vacuolating autotransporter toxin (Vat) is a cytotoxin known to contribute to UPEC fitness during murine sepsis infection. In this study, Vat was found to be highly conserved and prevalent among a collection of urosepsis clinical isolates and was expressed at human core body temperature. Regulation of vat was demonstrated to be directly repressed by the global transcriptional regulator H-NS and upregulated by the downstream gene vatX (encoding a new MarR-type transcriptional regulator). Additionally, increased Vat-specific IgG titers were detected in plasma from corresponding urosepsis patients infected with vatpositive isolates. Hence, Vat is a highly conserved and tightly regulated urosepsis-associated virulence factor. U rinary tract infections (UTIs) are one of the most common human infections, affecting 40 to 50% of women and approximately 12% of men globally (1). UTIs are ascending infections and can involve infection of the bladder (cystitis), kidneys (pyelonephritis), or dissemination into the bloodstream (urosepsis). Uropathogenic Escherichia coli (UPEC) strains ...

Serine Protease Autotransporters of the Enterobacteriaceae (SPATEs): Out and About and Chopping It Up

“…In a cellulitis infection model, chickens challenged with E. coli Ec222 developed cellulitis, whereas none of the chickens developed cellulitis when infected with the vat mutant [46]. In UPEC, Vat elicits an antibody response in some urosepsis patients, and the titer of Vat-specific IgG was higher in the plasma of patients compared to the titer found in controls and patients infected with vat-negative UPEC strains [115]. Vat contributed to fitness in UPEC during murine systemic infection [116].…”

“…Further, H-NS repressed the expression of vat; in UPEC strain CFT073, as a ∆hns mutant was shown to secrete a significantly higher level of Vat than the wild type strain [115]. Further, sequence analysis of the vat promoter region predicted the presence of three potential H-NS binding sites in the vat promoter region [115]. It seems H-NS could also potentially directly regulate other SPATE genes, as there are predicted putative H-NS binding sites present in SPATE gene promoter regions ( Table 2).…”

“…H-NS has been shown to regulate different trimeric autotransporters [137][138][139]. Further, H-NS repressed the expression of vat; in UPEC strain CFT073, as a ∆hns mutant was shown to secrete a significantly higher level of Vat than the wild type strain [115]. Further, sequence analysis of the vat promoter region predicted the presence of three potential H-NS binding sites in the vat promoter region [115].…”

A Novel Protective Vaccine Antigen from the Core Escherichia coli Genome

“…The peptide fingerprint was evaluated using ProteinPilot software 4.0 in combination with the EC958 protein database. SDS-PAGE and immunoblotting were performed as previously described (54) using a YncE-specific antibody. Rabbit YncE antibodies were raised against purified YncE by the Walter and Eliza Hall Institute Antibody Facility as previously described (55).…”

“…Mouse serum samples were applied at a 1:2 serial dilution, starting from a 1:10 dilution. Subsequent wash, secondary antibody, and development steps were performed as previously described (54), with the exception that peroxidase-conjugated anti-mouse antibodies were applied as the secondary antibody. YncE IgG titers were defined as the logarithmic dilution that produced a significant absorbance (450 nm) in comparison to that of a blank sample.…”