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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.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.

SUBMITTER: Nichols KB

PROVIDER: S-EPMC4859599 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Molecular Characterization of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli.

Nichols Katie B KB Totsika Makrina M Moriel Danilo G DG Lo Alvin W AW Yang Ji J Wurpel Daniël J DJ Rossiter Amanda E AE Strugnell Richard A RA Henderson Ian R IR Ulett Glen C GC Beatson Scott A SA Schembri Mark A MA

Journal of bacteriology 20160428 10

<h4>Unlabelled</h4>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 ...[more]

PMID: 26858103

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Project description:Urinary tract infections (UTIs) affect more than 150 million people, with a cost of over 3.5 billion dollars, each year. Escherichia coli is associated with 70-80% of UTIs. Uropathogenic E. coli (UPEC) has virulence factors including adhesins, siderophores, and toxins that damage host cells. Vacuolating autotransporter toxin (Vat) is a member of serine protease autotransporter proteins of Enterobacteriaceae (SPATEs) present in some uropathogenic E. coli (UPEC) strains. Vat has been identified in 20-36% of UPEC and is present in almost 68% of urosepsis isolates. However, the mechanism of action of Vat on host cells is not well-known. Thus, in this study the effect of Vat in a urothelium model of bladder cells was investigated. Several toxin concentrations were tested for different time periods, resulting in 15-47% of cellular damage as measured by the LDH assay. Vat induced vacuole formation on the urothelium model in a time-dependent manner. Vat treatment showed loss of the intercellular contacts on the bladder cell monolayer, observed by Scanning Electron Microscopy. This was also shown using antibodies against ZO-1 and occludin by immunofluorescence. Additionally, changes in permeability of the epithelial monolayer was demonstrated with a fluorescence-based permeability assay. Cellular damage was also evaluated by the identification of cytoskeletal changes produced by Vat. Thus, after Vat treatment, cells presented F-actin distribution changes and loss of stress fibers in comparison with control cells. Vat also modified tubulin, but it was not found to affect Arp3 distribution. In order to find the nature of the vacuoles generated by Vat, the Lysotracker deep red fluorescent dye for the detection of acidic organelles was used. Cells treated with Vat showed generation of some vacuoles without acidic content. An ex vivo experiment with mouse bladder exposed to Vat demonstrated loss of integrity of the urothelium. In conclusion, Vat induced cellular damage, vacuole formation, and urothelial barrier dysregulation of bladder epithelial cells. Further studies are needed to elucidate the role of these vacuoles induced by Vat and their relationship with the pathogenesis of urinary tract infection.

Project description:Some strains of Helicobacter pylori are known to produce an extracellular cytotoxin that causes vacuolization in various mammalian cells. In this study, we found that concentrated culture supernatants from four Helicobacter strains isolated from patients infected with the bacterium, but having normal gastric mucosa, lacked cytotoxic activity. We also show that a higher percentage of strains isolated from patients with polymorphonuclear leukocyte infiltration of gastric mucosa are toxin positive (78%) versus those isolated from patients lacking such infiltration (33%). In addition to examining the relationship between pathology and cytotoxic activity, we used the previously published N-terminal sequence of the protein to clone and characterize vacA, the structural gene encoding the cytotoxin. Briefly, three oligonucleotides capable of encoding the first nine amino acids corresponding to the sense strand and four oligonucleotides corresponding to the noncoding strand of the last seven known amino acids of the cytotoxin protein were made. They were used in all 12 possible combinations in 12 different PCR reactions, with DNA from a cytotoxin-positive strain as template. In four combinations, the expected 69-bp fragment was seen. The sequence of this 69-bp fragment confirmed that it encoded the known N-terminal sequence of the cytotoxin. This gene is capable of encoding a 136-kDa protein with a 33-amino-acid signal peptide, whereas the purified cytotoxin is only 87 kDa, suggesting processing in the C-terminal region of the protein. A single copy of the vacA gene encodes the cytotoxin in H. pylori. Consequently, the insertion of a kanamycin resistance marker in the vacA gene produced an isogenic mutant lacking the cytotoxic activity. This mutant provides genetic evidence that vacA encodes the cytotoxin. Sequence analysis of the DNA adjacent to the vacA gene demonstrated that this gene is next to a putative cysteinyl tRNA synthetase gene. From the sequence arrangement, we predict that there are no other genes transcribed together with vacA. We also show that five of seven cytotoxin-negative strains examined still carry the sequences encoding it whereas the other two have suffered a deletion of the vacA gene. We further show that in at least one cytotoxin-negative but vacA-positive strain (MO19), there are variations in the length of the vacA gene that could explain the cytotoxin-negative phenotype in this strain.

Dataset Information

Molecular Characterization of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli.

Publications

Molecular Characterization of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli.

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