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.
INTRODUCTION
Urinary 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 are the primary etiological agent of UTIs and cause 70 to 90% of all such infections (2). UPEC can survive in the urinary tract and cause disease due to a diverse range of virulence factors, including fimbriae (3–6), autotransporter (AT) proteins (7–10), surface polysaccharides, such as the O antigen and capsule (11–13), iron acquisition systems (14–16), and toxins (17–21).
AT proteins constitute a large family of proteins from Gram-negative bacteria that are translocated by a dedicated type V secretion system (reviewed in references 22 and 23–26). AT translocation also requires accessory proteins, including the β-barrel assembly module (BAM) and the translocation and assembly module (TAM) (27–30). AT proteins consist of three major domains: (i) a signal peptide that targets the protein to the secretory apparatus for inner membrane translocation, (ii) a passenger domain that comprises the functional domain of the protein, and (iii) a translocator domain that inserts into the outer membrane (reviewed in references 22, 23, 25, and 31–33). One major subgroup of AT proteins is the serine protease AT proteins of Enterobacteriaceae (SPATEs). SPATEs are characterized by the presence of an immunoglobulin A1-like protease domain (PF02395) within the passenger domain that contains the conserved serine protease motif GDSGS (34, 35). The first serine within this motif comprises the catalytic triad in conjunction with upstream conserved histidine and aspartate residues. SPATEs can be phylogenetically grouped into two classes (reviewed in references 34, 36, and 37). Class I SPATEs represent the major group of these proteins and exhibit cytotoxic activity (37–43). Class II SPATEs recognize a more diverse range of substrates, including mucins (reviewed in references 34, 36, and 37) and immunomodulatory host proteins (44).
The vacuolating AT toxin (Vat) of E. coli is a class II SPATE (34, 36, 45) that exhibits cytotoxicity to chicken embryonic fibroblast cells and contributes to avian cellulitis infection (46). The vat gene was originally identified within a pathogenicity island (PAI) designated the VAT-PAI from the avian pathogenic E. coli (APEC) strain Ec222 (46). The VAT-PAI is integrated into the Ec222 chromosome at the thrW tRNA site between the proA and yagU genes (45, 46). The VAT-PAI from Ec222 consists of 33 open reading frames (ORFs), with the vat gene residing at ORF27. Only five additional ORFs in this PAI were reported to share homology with other previously known protein sequences. This includes the ORF located downstream of vat (ORF26), which shares 44% amino acid identity to the P pilus-associated transcriptional regulatory protein PapX from UPEC strain CFT073 (46). PapX belongs to the family of multiple antibiotic resistance (MarR) regulators of Enterobacteriaceae and contributes to flagellar regulation by binding to the promoter region of the flhDC master regulator genes (47–49). In UPEC, the vat gene is associated with virulence and contributes to survival during murine systemic infection (50).
The full-length Vat protein is ∼140 kDa and is processed during translocation to release a 111.8-kDa passenger domain into the extracellular milieu. Vat shares 78% identity to the APEC-associated temperature-sensitive hemagglutinin (Tsh), which is almost identical (>99% amino acid identity) to the SPATE hemoglobin binding protein (Hbp) (51, 52). Hbp has been analyzed extensively in the E. coli intra-abdominal clinical strain EB1, and its crystal structure has been solved (53, 54). Tsh/Hbp possess dual proteolytic and adhesive properties (55–57). Unlike Tsh/Hbp, Vat is unable to digest casein at 37°C (45, 46).
Despite these functional differences, the high protein sequence identity shared between Tsh/Hbp and Vat has led to confusion in the annotation of vat genes within E. coli genomes available in the NCBI database. For example, the CFT073 vat gene (c0393) has been annotated as hbp (58) and even referred to as tsh due to its temperature-dependent regulation (59). In addition, the vat gene from UPEC strain 536 is annotated as sepA, which encodes the Shigella extracellular protein A (45).
In this study, we have examined the sequence conservation of vat genes from available E. coli genomes and compared their genomic locations, with the aim to correct existing annotation errors and vat nomenclature. We also examined the roles of the putative MarR regulator identified downstream of the vat gene and the histone-like nucleoid structuring protein (H-NS) in regulation of the vat gene. Finally, we examined the prevalence, expression, and secretion of Vat in a collection of UPEC urosepsis isolates and investigated its immunogenicity by examining plasma from urosepsis patients.
MATERIALS AND METHODS
Ethics statement.
This study was performed in accordance with the ethical standards of the University of Queensland, Princess Alexandra Hospital, Gold Coast Hospital, Queensland Health, Griffith University, and the Helsinki Declaration. The study was approved, and the need for informed consent was waived by the institutional review boards of the Princess Alexandra Hospital (2008/264), Queensland Health, and Griffith University (MSC/18/10/HREC).
Bacterial strains and growth conditions.
E. coli strains CFT073 (60), IHE3034 (61), 536 (62), MG1655 (63), and BL21 (64), as well as the E. coli reference (ECOR) collection (65), were described previously. The 45 urosepsis UPEC strains were isolated from blood from patients presenting with urosepsis at the Princess Alexandra Hospital (Brisbane, Australia). A matching urine sample from each patient was also cultured; in all cases, the blood and urine isolates were identical, as determined by virulence gene profiling. Unless otherwise stated, strains used in this study were routinely grown at 37°C on solid or in liquid lysogeny broth (LB) supplemented with antibiotics: kanamycin (100 μg/ml), ampicillin (100 μg/ml), or chloramphenicol (30 μg/ml). Supplementation of the growth medium with l-arabinose (0.2% [wt/vol]) or isopropyl-β-d-thiogalactopyranoside (IPTG [1 mM]) was used to induce plasmid-mediated gene expression.
Bioinformatic analysis.
The presence of the vat gene was determined in 77 complete E. coli genomes (listed in Table S1 in the supplemental material) available from the National Center for Biotechnology Information (NCBI) database by BLAST analysis using the vat gene (c0393) from the CFT073 genome (GenBank accession no. AE014075.1 [58]) as a search tool. The cutoff was set at >85% amino acid identity of the encoded protein sequence. The genomic location surrounding the vat gene in each of the vat-positive strains was investigated in Artemis (66). All vat genes identified were located on a PAI defined by the proA and yagU genes. The nucleotide sequence of each vat-associated PAI was compared in EasyFig (67).
A comparative protein analysis of the MarR family of transcriptional regulators (see Table S2 in the supplemental material) was performed to analyze their phylogenetic relationship relative to VatX. The MarR data set was compiled using an iterative approach that involved BLAST analysis against the 77 complete NCBI E. coli genomes listed in Table S1 in the supplemental material. Representative protein sequences, underlined in Table S2, were chosen for each MarR-type regulator based on previous characterization in the literature. These sequences included MarR from MG1655 (b1530), MprA (EmrR) from MG1655 (b2684), HosA from E2348/69 (E2348C_3010), HpcR/HpaR from strain W (WFL_22965), SlyA from MG1655 (b1642), and PapX from CFT073 (c3582). Each of the representative sequences were used in a BLAST search against the 77 complete E. coli genomes, and 330 homologous protein sequences were identified (E < 0.001). The evolutionary relationships among VatX and other representative MarR regulators, as well as the protein sequences listed in Table S2, were inferred using Clustal Ω (68, 69) and visualized through FigTree (70).
DNA manipulation and genetic techniques.
DNA techniques were performed as previously described (71). Isolation of plasmid DNA was performed using the QIAprep spin column miniprep kit (Qiagen). PCRs were performed using the specified primers, which were sourced from Integrated DNA Technologies (Singapore). PCR products were amplified using Taq DNA polymerase, according to the manufacturer's instructions (New England BioLabs). Sequencing reactions were performed using the BigDye Terminator version 3.1 cycle DNA sequencing kit, as per the manufacturer's specifications (Applied Biosystems), and analyzed by the Australian Equine Genome Research Centre. Cloning reactions involving restriction endonucleases were performed as per the manufacturer's instructions (New England BioLabs).
MLST and PCR screening.
The prevalence of the vat gene was assessed by PCR using primers 2020 (5′-GTATATGGGGGGCAACATAC-3′) and 2021 (5′-GTGTCAGAACGGAATTGTCG-3′), which were designed based on the sequence of the vat gene from CFT073 (c0393). The vat gene sequences from 10 of the 31 vat-positive UPEC urosepsis strains were determined and deposited in the NCBI database. The sequence type of the UPEC urosepsis strains was determined using a seven-gene multilocus sequence typing (MLST) scheme (http://mlst.ucc.ie/mlst/dbs/Ecoli) (72). PCR was performed as follows: initial denaturation at 94°C for 5 min, 25 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s, followed by a final extension at 72°C for 7 min.
Construction of deletion mutants.
The vat (c0393), vatX (c0392), and hns (c1701) genes were mutated in CFT073 using λ Red-mediated homologous recombination (73). Briefly, the kanamycin gene from pKD4 or the chloramphenicol gene from pKD3 was amplified using PCR primers containing 50-bp flanking regions homologous to the target genes vat (3353, 5′-TCGTAATGAACACAGTTCATCTGATCTCCACACACCAAGACTTGATAAGCTCACGTCTTGAGCGATTGTGTAGG-3′, and 3354, 5′-GAAACCACCACCCCATGATTTTGTTTTACCGCTGTACAGGCCTGCTGACGCGACATGGGAATTAGCCATGGTCC-3′), vatX (5232, 5′-TTCACGATACTTCATGTAACACTCAGGTTGAGTAATCTTCGTGTAGGCTGGAGCTGCTTC-3′, and 5233, 5′-AGAATACATTGTAAGAAGATGACTGTTAGTATGTTTTAACACATATGAATATCCTCCTTA-3′), and hns (1583, 5′-TCGTGCGCAGGCAAGAGAATGTACACTTGAAACGCTGGAAGAAATGCTGGGTGTAGGCTGGAGCTGCTTC-3′, and 1584, 5′-TTGATTACAGCTGGAGTACGGCCCTGGCCAGTCCAGGTTTTAGTTTCGCCCATATGAATATCCTCCTTAG-3′). Amplified fragments were transformed into CFT073(pKD56) expressing the λ Red recombinase in order to facilitate homologous recombination for inactivation deletion of the target gene. Removal of the antibiotic resistance gene cassette was performed using plasmid pCP20, as previously described, enabling the construction of the CFT073 vatX hns double mutant.
Construction of plasmids.
A segment of the vat gene corresponding to amino acid residues 63 to 465 of the passenger domain was amplified from CFT073 using primers 1491 (5′-TACTTCCAATCCAATGCTCCTTACCAGACATACCGCG-3′) and 1494 (5′-TTATCCACTTCCAATGTTACCCCGCATATTGATCATTGCC-3′) and cloned into the pLicA vector using ligation-dependent cloning to generate pVat403, expressing a truncated Vat protein (Vat403) with a six-histidine N-terminal fusion. The full-length vat gene (c0393) and the downstream vatX gene (c0392) were PCR amplified from CFT073 using the following primer pairs: vat, 1524 (5′-CGCGCTCGAGATAATAAGGAATTACTATGAATAAAATATACGCTC-3′) and 1525 (5′-CGCGCAAGCTTCAAAGCAATAGTCCCTTTGC-3′), and vatX, 5244 (5′-CGCGCTCGAGATAATAAGGAATCTTCATGAGTTTTCTTTTGCCGTGTGG-3′) and 5245 (5′-CCCGGAAGCTTTCAATTAACATTAAGGTTTGATA-3′). The PCR products were purified and cloned into XhoI-HindIII-digested pSU2718 to generate the plasmids pVat and pVatX. Transcription of the vat and vatX genes in these plasmids was regulated by the lac promoter (74).
Comparative qRT-PCR.
Comparative quantitative reverse transcriptase PCR (qRT-PCR) was performed essentially as previously described (47). Briefly, strains CFT073, CFT073 vatX, and CFT073 vatX(pVatX) were grown in LB broth (supplemented with IPTG) until exponential-growth phase. The total RNA from each strain was extracted using the RNeasy minikit, as per the manufacturer's instruction (Qiagen). Samples were subjected to RNase-free DNA digestion, and first-strand cDNA synthesis was performed using SuperScript III (Invitrogen Life Technologies) with random hexamer (50 ng/μl) primers (Invitrogen Life Technologies). Residual RNA was digested by RNase H, and samples were repurified, as recommended by the manufacturer (Qiagen). The ViiA 7 instrument and software (version 1.2.1) were used to carry out RT-PCRs (95°C for 10 s and then 95°C for 15 s, 60°C for 15 s, and 72°C for 30 s for 40 cycles). Primers specific to the vat gene (5470, 5′-TACCGTAACCAGCTCATCAACAG-3′, and 5471, 5′-CATACCCACCTGTTACCCAATGT-3′) and gapA (control; 820, 5′-GGTGCGAAGAAAGTGGTTATGAC-3′, and 821, 5′-GGCCAGCATATTTGTCGAAGTTAG-3′) were used to amplify transcripts with Sybr Green I (5 μl) master mix (Applied Biosystems). Each reaction was performed in triplicate, and a subsequent melting curve was generated for validation of the results (95°C for 15 s, 60°C for 1 min, and 95°C for 10 s). Cycle threshold (CT) values obtained were normalized to the endogenous control, and the 2−ΔΔCT method (75) was applied for the comparative analysis. The resulting ratios were statistically analyzed using a one-way analysis of variance (ANOVA). All experiments were performed in triplicate.
5′ RACE and Virtual Footprint analysis.
The transcriptional start site for vat was determined using the 5′ rapid amplification of cDNA ends (RACE) system (version 2.0; Invitrogen Life Technologies), according to the manufacturer's specifications. Two gene-specific primers (5863, 5′-ATGCAGATAGTGCCAGAG-3′, and 5864, 5′-CTCTGCGGGTACTCCCTTTAC-3′) were used. Putative DNA binding motifs in the vat promoter region were identified using Virtual Footprint software (76).
EMSA.
An electrophoretic mobility shift assay (EMSA) was performed essentially as described previously (77) but with minor adaptations. Briefly, four individual fragments (152 bp, 218 bp, 312 bp, and 479 bp) were PCR amplified from the plasmid pBR322 with the following primers: 152 bp, 5′-CATTGGACCGCTGATCGT-3′ and 5′-CTTCCATTCAGGTCGAGGT-3′; 218 bp, 5′-AATATTATTGAAGCATTTATCAGGGTTA-3′ and 5′-ATGATAAGCTGTCAAACATGAGA-3′; 312 bp, 5′-TATCGACTACGCGATCATGG-3′ and 5′-TCTCCCTTATGCGACTCCTG-3′; and 479 bp, 5′-GACCGATGCCCTTGAGAG-3′ and 5′-GATCGAAGTTAGGCTGGTAAGA-3′. The 218-bp fragment containing the H-NS-repressed bla gene promoter was included in the assay as a positive control, while the remaining three fragments do not bind H-NS. The vat gene promoter region (252 bp) encompassing all three of the identified putative H-NS binding sites was also PCR amplified (primers: 6103, 5′-CCTGAGAAAAAGCAAACAACA-3′, and 6104, 5′-TTTTAGAGCGTATATTTTATTCAT-3′) from the genomic DNA of CFT073. This 252-bp fragment was added in an equimolar ratio with the control fragments (7.5 nM per fragment [∼100 ng]). Purified native H-NS protein was added to each reaction mixture in increasing concentrations (0 μM, 0.1 μM, 0.5 μM, and 1.0 μM). Reaction mixtures were incubated at room temperature (15 min in H-NS binding buffer) to allow for protein-DNA complex formation. Samples were examined by high-resolution agarose gel electrophoresis (using 3% Lonza MetaPhor [50 V at 4°C]) and viewed under UV light after staining with ethidium bromide (0.5 μg/ml). Invitrogen's 1-kbp+ ladder was used as a molecular marker.
Preparation of supernatant proteins.
Bacterial cultures (10 ml) were standardized to an optical density at 600 nm (OD600) of 1.0 and centrifuged (2,057 × g), and the supernatant was collected and filtered (0.22 μm). Proteins were precipitated by the addition of 10% trichloroacetic acid (TCA) overnight at 4°C. Following precipitation, supernatant fractions were concentrated by centrifugation (12,100 × g) and washed twice with 80% acetone to remove residual TCA. Proteins were resuspended in a final volume of 0.1 ml (100-fold concentration).
Purification of denatured His-tagged Vat protein.
A bacterial culture (200 ml) of E. coli BL21(λDE3) expressing the truncated Vat403 protein encoded on plasmid pVat403 was grown in LB. Bacterial cells were pelleted by centrifugation (2,057 × g) and lysed (7 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl [pH 8.0]). The recombinant Vat403 protein was purified under denaturing conditions using Qiagen's nickel-nitrilotriacetic acid (Ni-NTA) spin column kit. The cleared lysate was passed through a preequilibrated column via centrifugation (270 × g) to allow for the 6×His-tagged Vat protein to bind. The column was washed (0.1 M NaH2PO4, 0.01 M Tris-HCl [pH 6.3]), and the bound Vat protein was eluted (0.1 M NaH2PO4, 0.01 M Tris-HCl [pH 4.5]) by centrifugation (890 × g). Protein concentrations were determined using the bicinchoninic acid protein assay kit, as per the manufacturer's instructions (Thermo Scientific Pierce Biotechnology). The purity of the eluted protein was validated by sodium dodecyl disulfide-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (12% polyacrylamide gel) and Coomassie staining.
Immunoblotting.
The purified His-tagged recombinant Vat protein was used to generate a Vat-specific polyclonal antibody, according to a standard protocol (Institute of Medical and Veterinary Science, South Australia, Australia). Concentrated supernatant proteins were resuspended in 50 μl of SDS loading buffer (100 mM Tris-HCl, 4% [wt/vol] SDS, 20% [wt/vol] glycerol, 0.2% [wt/vol] bromophenol blue [pH 6.8]), and a 10-μl sample was boiled for 10 min prior to SDS-PAGE. SDS-PAGE and transfer of proteins to a polyvinylidene difluoride (PVDF) membrane for Western blot analysis were performed as previously described (78). Anti-Vat polyclonal antibodies were used as the primary antibody, and alkaline phosphatase-conjugated anti-rabbit antibodies (Sigma-Aldrich) were used as the secondary antibody. Sigmafast BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate–Nitro Blue Tetrazolium; Sigma-Aldrich) was used as the substrate for detection.
Human plasma samples and measurement of Vat immunogenicity.
Blood plasma (collected within 4 days postadmission) and matching clinical isolates were obtained from 45 urosepsis patients admitted to the Princess Alexandra Hospital (Brisbane, Australia). The clinical strains isolated from each urosepsis patient were grouped as Vat positive (Vat+) and Vat negative (Vat−), according to the prevalence of the vat gene, as determined by PCR screening using vat-specific primers. A negative-control group of plasma samples was independently obtained from 42 healthy volunteers with no recent history of UTI. The enzyme-linked immunosorbent assay (ELISA) was performed using Nunc MaxiSorp flat-bottom 96-well microtiter plates (Thermo Scientific). Each well was coated with recombinant Vat protein (10 μg/ml) using carbonate coating buffer (18 mM Na2CO3, 450 mM NaHCO3 [pH 9.3] at 4°C overnight). The plates were washed twice with 0.05% (vol/vol) Tween 20-PBS (PBST) and blocked with 5% (wt/vol) skim milk in PBST (150 μl) for 90 min at 37°C. Each well was then washed four times with PBST prior to incubation (90 min at 37°C) with individual plasma samples (1:10 dilution). Unbound antibodies were removed by washing with PBST. Peroxidase-conjugated anti-human IgG (1:30,000 dilution in 5% skim milk) was applied as a secondary antibody for detection (incubated at 37°C for 90 min). Plates were washed four times with PBST, and bound anti-human IgG was detected using 3,3′,5,5′-tetramethylbenzidine as the substrate. The reactions were stopped with 1 M HCl. The absorbance of each well was measured at 450 nm using the SpectraMax Plus 384 plate reader via the SoftMax Prov5 program. The data obtained were analyzed using the GraphPad Prism 5 software, and a one-way ANOVA was performed.
Nucleotide sequence accession numbers.
The vat gene sequences from 10 of the 31 vat-positive UPEC urosepsis strains were deposited in GenBank under the following accession numbers: PA11B vat, KR094926; PA15B vat, KR094927; PA32B vat, KR094928; PA38B vat, KR094929; PA42B vat, KR094930; PA48B vat, KR094931; PA56B vat, KR094932; PA57B vat, KR094933; PA60B vat, KR094934; and PA66B vat, KR094935.
RESULTS
The vat gene is located on a pathogenicity island at a conserved genomic location.
The prevalence of vat was assessed in 77 complete E. coli genomes available in the NCBI database (see Table S1 in the supplemental material). The vat gene was identified in 14 of these strains; these included the previously characterized vat-positive UPEC strains CFT073 and 536, as well as 12 additional strains from which vat has not been characterized (APEC O1, NRG 857C, LF82, IHE3034, S88, 83972, PMV-1, clone D i2, clone D i14, ATCC 25922, Nissle 1917, and UM146). In all 12 strains, the vat gene was part of a pathogenicity island (PAI) flanked by the proA and yagU genes relative to the E. coli K-12 MG1655 chromosome. This genomic location is consistent with the original identification of vat in APEC strain Ec222 (46). Closer examination of the genomic context of vat revealed that the upstream region (i.e., the yagU end) is highly conserved. In contrast, the region downstream of vat (i.e., the proA end) exhibits extensive variation, with a range of different DNA segments inserted at various positions of the PAI in strains APEC O1, 83972, UM146, 536, and Ec222 (Fig. 1A).
FIG 1
Vat is secreted by several genome-sequenced UPEC strains.
The secretion of Vat following growth in LB broth at 37°C was assessed from a selection of the vat-positive UPEC strains described above (i.e., CFT073, IHE3034, and 536). As a positive control, the vat gene from CFT073 was amplified by PCR, cloned into the low-copy-number expression vector pSU2718 to generate the plasmid pVat, and transformed into E. coli MG1655 to generate the recombinant strain MG1655(pVat). Western blot analysis using a Vat-specific antibody detected a single band of ∼110 kDa that corresponded to the predicted size of the secreted passenger domain of Vat in the supernatant of MG1655(pVat) but not the vector control strain MG1655(pSU2718) (Fig. 1B). The vat gene was also mutated in CFT073 to generate null mutant strain CFT073 vat. SDS-PAGE and Western blot analysis of the supernatant fraction obtained from CFT073 and CFT073 vatX using our Vat-specific antibody identified the secreted Vat passenger domain from CFT073 but not CFT073 vat (Fig. 1B). Finally, we also detected bands corresponding to the Vat passenger domain in the supernatant fraction prepared from strains IHE3034 and 536. Taken together, our data demonstrate that Vat is expressed and secreted by the genome-sequenced strains CFT073, IHE3034, and 536.
A marR-like gene is located immediately downstream of the vat gene.
We were interested to study the regulation of Vat and noted a small open reading frame located directly downstream of the vat gene in all vat-positive strains (Fig. 1A). This gene, which we have termed vatX, corresponds to c0392 in CFT073 (58) and ORF26 in the VAT-PAI from Ec222 (46). The VatX protein sequence is highly conserved (99% amino acid identity in the 14 vat-positive completely sequenced strains described above) and shares 44% amino acid identity with the CFT073 P pilus-associated transcriptional regulator PapX. Further analysis of VatX revealed that it contains a MarR PFAM domain (PF01047) and a helix-turn-helix motif characteristic of DNA binding proteins. To examine the relationship between VatX and other regulator proteins, we generated a data set comprising previously characterized E. coli MarR-type regulators (see Table S2 in the supplemental material) (47, 79–82). A multiple-sequence alignment using representative regulator protein sequences (Fig. 2) and a more-detailed phylogenetic analysis of all MarR-like sequences identified in the 77 complete E. coli genomes described above (see Fig. S1 in the supplemental material) revealed that VatX forms a distinct clade within the MarR regulator family and is most closely related to the PapX, SfaX, and FocX fimbria-associated regulators (47, 80, 83, 84).
FIG 2
Expression of the vat gene is upregulated by VatX.
The proximity, orientation, and conserved genetic organization of the vat and vatX genes led us to examine whether VatX contributes to the regulation of the vat gene. In order to study this, we generated a CFT073 vatX mutant (CFT073 vatX) and examined the transcription of vat in CFT073 and CFT073 vatX using comparative qRT-PCR. In addition, the vatX gene from CFT073 was PCR amplified and cloned into the pSU2718 expression vector (to generate the plasmid pVatX) and used to complement the CFT073 vatX mutant. No significant difference was observed in the level of vat mRNA transcribed in CFT073 and CFT073 vatX following growth in LB broth at 37°C (Fig. 3A). However, the overexpression of VatX in CFT073 vatX(pVatX) resulted in an approximately 3-fold increase in the level of vat mRNA transcription compared to that in the wild type (WT) CFT073 (Fig. 3A). To explore further the effect of VatX on Vat expression, we compared the levels of Vat secreted into the supernatant fraction by CFT073, CFT073 vatX, and CFT073 vatX(pVatX) by Western blot analysis (Fig. 3B). Consistent with our transcriptional data, the overexpression of VatX in CFT073 vatX(pVatX) resulted in a significantly increased level of Vat in the culture supernatant, while no difference in the level of secreted Vat was observed in CFT073 and CFT073 vatX. A similar increase in secreted Vat was also observed when WT CFT073 was transformed with plasmid pVatX (i.e., in strain CFT073[pVatX]) (Fig. 3B). Taken together, our results demonstrate that while the deletion of vatX does not alter the level of Vat secretion in LB broth, its overexpression significantly enhances Vat expression.
FIG 3
Transcription of the vat gene is directly repressed by H-NS.
Given the regulatory effect exhibited by VatX on vat transcription, we investigated the promoter region of the vat gene to identify putative binding sites for other transcription factors. The transcriptional start site for vat was determined using 5′ RACE and was mapped to a position 80 bp upstream of the Vat ATG start codon. Consensus −35 (5′-ATCACA-3′) and −10 (5′-ATTAAT-3′) promoter sequence elements, separated by an 18-bp spacer region, were identified upstream of this site (Fig. 4A). The Virtual Footprint software was used to analyze the vat promoter region for putative regulatory binding sites. From this in silico analysis, two putative H-NS nucleation sites were identified on the anti-sense strand overlapping the 18-bp spacer region and the 5′ end of the −35 element. A third H-NS nucleation site was determined on the direct strand 10-bp downstream of the transcriptional start site.
FIG 4
The global transcriptional regulator H-NS is known to bind to curved and AT-rich DNA sequences upstream of several defined UPEC virulence genes (85), including genes encoding toxins (86–89) and autotransporter proteins (8, 10, 90). To investigate the effect of H-NS on vat transcription, the levels of Vat expression were compared by Western blot analysis of supernatant fractions prepared from WT CFT073, CFT073 vat, CFT073 vatX, CFT073 hns, and a CFT073 vatX hns double mutant (Fig. 4B). The amount of Vat secreted by CFT073 hns and CFT073 vatX hns was markedly increased compared to the amount secreted by WT CFT073. Consistent with previous results, the level of Vat detected in the supernatant fraction of CFT073 vatX was similar to that detected in the supernatant fraction of WT CFT073.
H-NS binds to the vat promoter region.
To further investigate the role of H-NS in the repression of vat transcription, an EMSA was performed using increasing concentrations of native H-NS protein and the 252-bp PCR-amplified region of the vat gene promoter possessing the three potential H-NS binding sites (Fig. 4C). As a positive control, the bla gene promoter from the cloning vector pBR322 was also PCR amplified and included in the assay; H-NS is known to bind to this DNA fragment (91). Three additional fragments amplified from regions of pBR322 known not to bind H-NS were included in the assay as negative controls. In our experiment, H-NS bound with strong affinity to the DNA fragment corresponding to the vat gene promoter. Indeed, this binding affinity was stronger than that observed for the DNA fragment containing the control bla gene promoter. No binding of H-NS to the negative-control DNA fragments was observed, demonstrating the specificity of H-NS binding in this assay.
vatX is cotranscribed with vat.
H-NS regulates the transcription of several UPEC genes by competing for binding to their promoter element with a MarR-type regulatory protein; this includes SfaX binding to the sfa2 fimbrial promoter (80), PapX binding to the flhDC flagellum master regulator promoter (92), and SlyA binding to the type 1 fimbria fimB recombinase promoter (93). The SfaX and PapX regulator genes are cotranscribed as part of their respective upstream fimbrial operon (encoding S- and P-type fimbriae, respectively [47, 80]). Taking this into consideration, we employed RT-PCR analysis to test for transcription of the vat and vatX genes as a single mRNA in CFT073. Due to the increased amount of Vat protein secreted by the CFT073 hns mutant strain (as shown by Western blotting), total RNA was extracted from this strain, converted to cDNA, and screened for a vat-vatX nucleic acid fragment using internal primers specific for both genes by RT-PCR (Fig. 4D). For comparison, an additional set of primers was used to amplify the vatX gene alone. Bands corresponding to the predicted sizes determined for the vatX and the vat-vatX transcripts were amplified from CFT073 hns cDNA. Thus, while we cannot rule out the presence of an independent promoter upstream of vatX, our results demonstrate that the vat-vatX genes are cotranscribed in the absence of H-NS.
Vat is prevalent, highly conserved, and secreted by UPEC urosepsis isolates.
The vat gene has been shown to be most prevalent in E. coli strains from the B2 phylogenetic group, with similar distributions observed among cystitis, pyelonephritis, prostatitis, and bloodstream isolates (45). Based on the observation that vat is required for UPEC fitness in a mouse model of systemic infection (50), we screened a collection of urosepsis strains for the vat gene using PCR. The vat gene was identified in 68% (31/45) of the urosepsis strains. MLST analysis revealed that strains from ST73, ST12, and ST95 were most predominant in this collection (Fig. 5). Furthermore, supernatant fractions produced by these strains were examined by Western blotting to analyze the expression and secretion of Vat following growth in LB at 37°C. For all strains, a band corresponding to the Vat passenger domain hybridized with the Vat-specific polyclonal antibody. The sequence of the vat gene was determined from vat-positive strains representing each ST and found to be highly conserved (≥97% amino acid identity [Fig. 5]). Minor sequence variations occurred at six locations within the passenger domain of the protein. These residues were located within two regions in the Vat passenger domain (Fig. 5), both of which are distal to the serine protease catalytic motif based on a structural model built using the Hbp passenger domain (see Fig. S2 in the supplemental material).
FIG 5
Presence of vat is associated with increased anti-Vat IgG produced during infection.
The high prevalence of vat in the UPEC urosepsis strains examined in this study, in combination with its secretion during in vitro growth, prompted us to examine if an immunological response against Vat was elicited during infection. To test this, an ELISA was performed using blood plasma samples collected from the same urosepsis patients from which the urosepsis strains were collected (Fig. 6). The blood plasma samples were examined for the presence of Vat-specific IgG antibodies using purified recombinant Vat protein. The urosepsis patients were divided into two groups: those infected with a vat-positive UPEC strain (n = 31) and those infected with a vat-negative UPEC strain (n = 14). As an additional control, 42 plasma samples collected from age- and sex-matched healthy individuals were also examined for an immunological response against the Vat protein. In this assay, we observed a significant difference (P < 0.05) in the anti-Vat IgG plasma titer in patients infected with a vat-positive strain compared to those infected with a vat-negative strain or healthy individuals. Taken together, these data suggest that Vat is a highly conserved immunogenic protein that is expressed by many UPEC isolates during infection.
FIG 6
DISCUSSION
UPEC strains possess an array of virulence factors that are critical for their ability to cause disease in extraintestinal niches, such as the urinary tract and the bloodstream (94, 95). Vat is a member of the SPATEs, which contribute to the fitness of E. coli during systemic infection (46, 50). In this study, we performed a comprehensive bioinformatic and molecular analysis of the vat gene. We defined the transcriptional regulation of vat and demonstrated its immunogenicity using plasma samples from urosepsis patients.
The genomic location of the vat gene was examined in all vat-positive completely sequenced E. coli strains available in the NCBI database. The vat gene was shown to reside within the thrW-PAI, downstream of proA, and upstream of yagU relative to the E. coli MG1655 chromosome. This is consistent with a previous report that examined the presence of vat in UPEC strains CFT073 and 536 and the neonatal meningitis strain RS218 (45). The gene content of the vat-containing thrW PAI was conserved in the majority of strains examined, although some differences were noted in strains Ec222, APEC O1, 83972, UM146, and 536. Overall, our bioinformatic analysis revealed that the vat gene (and the colocated vatX regulator gene) is present in a range of different E. coli pathotypes.
Several studies have previously assessed the prevalence of the vat gene in E. coli. A study conducted by Parham et al. (45) reported a high prevalence of vat in group B2 phylogenetic strains of the ECOR collection. A high frequency of the vat gene has also been observed in B2 strains associated with cystitis, pyelonephritis, and prostatitis (45, 59), and vat has been strongly associated with avian pathogenic E. coli (APEC) (96). Our analysis identified the vat gene in 68% of urosepsis isolates (n = 45). We also demonstrated that the sequence of vat is highly conserved within a selection of strains representative of each of the 10 different sequence types identified in our collection. At the amino acid level, minor sequence variations were located within two regions (VR1, S520 to K529, and VR2, E783 to V823) of the Vat passenger domain. However, the canonical serine protease domain that is important for the catalytic function of SPATEs was conserved in all 10 of the Vat sequences analyzed. Western blotting was also performed to examine Vat expression and revealed that Vat is expressed and secreted by all of the urosepsis strains examined when grown at human core body temperature. Further investigation is required to determine whether the minor sequence changes observed in Vat are associated with corresponding differences in its cytotoxic properties.
Bioinformatic analysis identified a gene encoding a putative MarR-like transcriptional regulator immediately downstream of vat (i.e., vatX). Although mutation of vatX did not result in a detectable change in vat transcription or translation, overexpression of VatX via the introduction of a plasmid containing the vatX gene (pVatX) was shown to positively regulate vat, resulting in a 3-fold increase in vat transcription and a significant increase in the level of secreted Vat protein. These data were suggestive of more complex regulatory control of the vat gene. We therefore mapped the promoter of vat and identified several putative H-NS binding sites proximal to this region. H-NS is a histone-like DNA binding protein that shows affinity for AT-rich and bent nucleation sites on DNA (97). In E. coli, H-NS has been shown to regulate multiple genes, including genes associated with virulence, pH, osmoregulation, and temperature sensing (98–101). Our EMSA data revealed a strong interaction between H-NS and a 252-bp region of the vat promoter that contains three putative H-NS binding sites. A role for H-NS in vat regulation was subsequently demonstrated through the examination of a CFT073 hns mutant, which secreted a significantly higher level of Vat than the parent CFT073 strain. Taken together, these results demonstrate that the regulation of vat is coordinated by both VatX and H-NS and further highlight the role of H-NS in the regulation of UPEC virulence factors (8, 9).
The MarR family of transcriptional regulators controls the expression of multiple different genes, including virulence factors, often in response to environmental stress (reviewed in references 102 and 103). Bioinformatic analysis of MarR-type regulators from 77 completely sequenced E. coli genomes revealed a high level of amino acid sequence conservation for proteins in each clade but significant variation between MarR regulators from different clades. VatX clustered as a separate clade and is most closely related to PapX. Interestingly, other fimbria-associated MarR-type regulators were also found within the PapX clade (see Fig. S1 in the supplemental material). Despite their association with different fimbriae, these regulatory proteins are highly conserved (≥97% amino acid identity). Some strains, such as E. coli 536, 83972, and Nissle 1917, possess three or more chromosomal copies of papX (see Table S2 in the supplemental material). PapX regulates UPEC motility by repressing transcription of the flhDC master regulator genes (47). We investigated the potential for VatX to repress flagellum-mediated motility of CFT073. However, no significant difference in motility was observed among WT CFT073, CFT073 vatX, and the complemented CFT073 vatX(pVatX) strain after growth at 28°C and 37°C (data not shown). The FliC major flagellin subunit was also produced at similar levels in all three strains, as determined by immunoblotting (data not shown). Taken together, our data have identified VatX as a new member of the MarR-type family that appears to regulate vat in concert with H-NS. Further work is now required to map the direct binding of VatX to the vat gene promoter and to examine the competitive interplay between VatX and H-NS in the regulation of vat transcription.
In a recent study using high-throughput transposon mutagenesis screening (50), the vat gene was shown to contribute to survival of the UPEC strain CFT073 in the bloodstream of mice. This, together with the observation that many urosepsis strains secrete Vat, prompted us to examine the immunoreactivity of Vat in urosepsis patients. We detected a significant increase in the Vat-specific IgG titer in the plasma of urosepsis patients infected with vat-positive UPEC strains compared to that in plasma from patients infected with vat-negative strains and healthy controls. Although we cannot rule out that the responses we detected are in part due to previous or ongoing infection that culminated in sepsis, overall, the data are consistent with the notion that Vat is expressed during infection and elicits a strong immune response in some patients. Further work is now required to understand the role of Vat during human infection and its cytotoxicity profile.
ACKNOWLEDGMENTS
We thank David Looke, Joan Faoagali, and other members of the microbiology lab, Princess Alexandra Hospital, for the collection of urosepsis strains and plasma samples and Barbara Johnson for the collection of patient clinical data.
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Information & Contributors
Information
Published In
Journal of Bacteriology
Volume 198 • Number 10 • 15 May 2016
Pages: 1487 - 1498
Editor: V. J. DiRita
PubMed: 26858103
Copyright
© 2016 American Society for Microbiology.
History
Received: 6 October 2015
Accepted: 4 February 2016
Published online: 28 April 2016
Contributors
Editor
V. J. DiRita
Editor
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Citation
2016.
Molecular Characterization of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli. J Bacteriol
198:.
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