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.

Importance: Uropathogenic 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 vat-positive isolates. Hence, Vat is a highly conserved and tightly regulated urosepsis-associated virulence factor.

FIG 1

(A) BLAST alignment demonstrating the level of nucleotide sequence conservation (gray shading) for vat and vatX (red) and the other surrounding genes (blue). The VAT-PAI (defined by the proA and yagU genes [yellow]) was identified in 14 of 77 complete E. coli genomes examined. These sequences were compared to the VAT-PAI originally identified in the avian pathogenic E. coli strain Ec222 (top). (B) Immunodetection of the Vat passenger domain (Vatα) from supernatant fractions prepared from overnight cultures of the well-characterized UPEC strains CFT073, IHE3034, and 536. Vat expression by MG1655(pVat) is shown as a positive control, while MG1655(pSU2718) and CFT073 vat were included as a negative controls.

FIG 2

Phylogram demonstrating the relationship between representative E. coli MarR-type regulator proteins. The scale represents the number of amino acid substitutions per site over 194 positions.

FIG 3

(A) qRT-PCR analysis of vat transcription in CFT073 vatX and CFT073 vatX(pVatX) compared to that in wild-type CFT073. The transcription of vat was significantly increased in CFT073 vatX(pVatX) compared to that in CFT073 (**, P < 0.01; error bars indicate standard deviations). (B) Western blot analyzing the effect of VatX on Vat expression. Supernatant fractions were prepared from overnight cultures of MG1655(pVat), MG1655(pSU2718), CFT073(pSU2718), CFT073(pVatX), CFT073 vatX(pSU2718), and CFT073 vatX(pVatX). The overexpression of VatX led to an increase in the amount Vat detected in the culture supernatant. Molecular mass markers are given on the left.

FIG 4

(A) Schematic of the vat-vatX gene operon in CFT073. The positions of the promoter and primers used to identify vat-vatX and vatX transcripts are indicated. The inset shows the vat gene transcriptional start site (+1), which was mapped to 80 bp upstream of the ATG start codon (arrow). Also indicated are the consensus −10 and −35 promoter elements (underlined) and the three putative H-NS nucleation sites (shown in bold). (B) Immunodetection of the Vat passenger domain from the supernatant fractions of CFT073, CFT073 vat, CFT073 vatX, CFT073 hns, and CFT073 vatX hns. The level of Vat was increased in CFT073 hns and CFT073 vatX hns compared to that in CFT073. (C) EMSA demonstrating the direct interaction of H-NS with the vat promoter region. The assay was performed using a 252-bp fragment encompassing the vat promoter region (indicated by an asterisk), a 218-bp fragment containing the bla promoter region amplified from pBR322 (positive control; indicated by an arrow head), and three additional DNA fragments amplified from pBR322 (negative controls; 152 bp, 312 bp, and 479 bp). Native H-NS protein was incubated with the DNA in increasing concentrations (0 μM, 0.1 μM, 0.5 μM, and 1.0 μM H-NS). (D) Transcriptional analysis of the vat and vatX genes. Total RNA was extracted during exponential growth of CFT073 hns and converted to cDNA. Shown are the PCR products (vat-vatX [1,112 bp] or vatX [404 bp]) amplified from CFT073 hns gDNA (positive control), total RNA (negative control), and cDNA.

FIG 5

(A) Diagram depicting the full-length Vat primary protein sequence, including three protein domains typical for SPATES: (i) the extended signal peptide (SP); (ii) the passenger domain comprising the immunoglobulin A1 protease-like domain, which contains the serine protease motif, as well as the upstream aspartate (D158) and histidine (H130) residues of the catalytic triad; and (iii) the translocation domain, which is cleaved at the α-helical linker region. Class II SPATEs are characterized by the presence of a small additional domain termed domain 2. Two variable regions (VR1 and VR2) located within the passenger domain were identified. (B) Alignment of the Vat amino acid sequence in VR1 and VR2 from CFT073 and the 10 strains representing the diverse STs examined. Residues identical to those in Vat from CFT073 are indicated by dots; residues that differed from the CFT073 sequence are indicated and highlighted in gray. Vat secretion was determined by Western blotting of the supernatant fractions from each strain following overnight growth in LB broth at 37°C. All strains secreted an ∼110-kDa protein that cross-reacted with the Vat-specific polyclonal antibody (indicated by +).

FIG 6

Immunoreactivity of Vat. Blood plasma was collected from 45 urosepsis patients at the time of admittance to the hospital. Paired UPEC strains were also isolated from the blood of each patient, and the presence of the vat gene was determined by PCR. Plasma samples were subsequently grouped by their association with vat-positive (Vat⁺) or vat-negative (Vat⁻) strains. The presence of IgG-specific antibodies was determined by ELISA, and the results were compared to those obtained from 42 healthy volunteers with no recent history of UTI. Significantly higher IgG titers were detected in plasma samples from patients infected with Vat⁺ strains than in those from patients infected with Vat⁻ strains and healthy controls. Values for individual plasma samples are represented, together with the means (horizontal lines) and standard errors of the means (error bars).