The repeat-in-toxin family member TosA mediates adherence of uropathogenic Escherichia coli and survival during bacteremia

Abstract

Uropathogenic Escherichia coli (UPEC) is responsible for the majority of uncomplicated urinary tract infections (UTI) and represents the most common bacterial infection in adults. UPEC utilizes a wide range of virulence factors to colonize the host, including the novel repeat-in-toxin (RTX) protein TosA, which is specifically expressed in the host urinary tract and contributes significantly to the virulence and survival of UPEC. tosA, found in strains within the B2 phylogenetic subgroup of E. coli, serves as a marker for strains that also contain a large number of well-characterized UPEC virulence factors. The presence of tosA in an E. coli isolate predicts successful colonization of the murine model of ascending UTI, regardless of the source of the isolate. Here, a detailed analysis of the function of tosA revealed that this gene is transcriptionally linked to genes encoding a conserved type 1 secretion system similar to other RTX family members. TosA localized to the cell surface and was found to mediate (i) adherence to host cells derived from the upper urinary tract and (ii) survival in disseminated infections and (iii) to enhance lethality during sepsis (as assessed in two different animal models of infection). An experimental vaccine, using purified TosA, protected vaccinated animals against urosepsis. From this work, it was concluded that TosA belongs to a novel group of RTX proteins that mediate adherence and host damage during UTI and urosepsis and could be a novel target for the development of therapeutics to treat ascending UTIs.

Fig 1

In vitro TosA expression constructs and putative tos operon structure. (A) Schematic illustrating construction of CFT073 araB_P-tosC. A chimeric PCR construct consisting of two plasmid fragments generated from pKD4 and pKD46, containing a Kan^r-conferring gene and an arabinose-inducible promoter, respectively, were amplified with primers that contain homology to the region between tosR and tosC (HR). A second set of primers, one the reverse complement of the other, were used as linking primers to combine the two PCR products together in a third PCR (LP). A schematic shows each ORF in the 15-kb region of tosA. The area denoted by brackets below genes, marked P1 to P4, illustrates amplicons that span adjacent genes. (B) Western blot of TosA induction. Cultures of CFT073 and CFT073 araB_P-tosC were induced with 0.1 mM arabinose. At the indicated times, glucose was added to the cultures to give a final concentration of ca. 0.2%. Cultures were harvested at 3 h and processed for Western blot analysis. Proteins were reacted with TosA antiserum. Overexpression of TosA at 30, 60, and 90 min obscures the upper portion of these lanes. (C) RT-PCR of amplicons spanning the region between the two genes denote below each section. gDNA, genomic CFT073 DNA; (−) no RT, no-reverse-transcriptase control; cDNA, cDNA prepared from RNA from arabinose-induced bacteria. (D) qPCR results using RNA isolated from bacteria collected either from five mice transurethrally infected with wild-type CFT073 or from LB culture of wild-type CFT073 incubated at 37°C with aeration. Three replicates were normalized to the expression of gapA. In vivo, tosA expression was comparable to gapA expression (that is, well expressed), whereas in vitro expression of tos genes represented only a fraction of gapA expression. (E) TosA purified from the cytoplasmic fraction of arabinose-induced CFT073(pBAD-tosA) by gel filtration chromatography. A 0.8-μg portion of protein was loaded. M, protein standard markers; P, purified TosA. (F) Western blot with polyclonal TosA antiserum of wild-type (CFT073) and CFT073 araB_P-tosC cells cultured under arabinose-inducing conditions. A total of 25 μl of late-exponential culture was loaded per lane; TosA marks the position just above the 250-kDa size standard. (G) Predicted molecular weights and amino acid identity to homologs for tos operon-encoded proteins. Homologs are as follows: PapB of Edwardsiella tarda (45% over 73 amino acids [aa]), TolC of Proteus mirabilis (44% over 392 amino acids [aa]), LssB family member of Neisseria sicca (62% over 707 aa), HlyD-like P. mirabilis (77% over 405 aa), outer membrane adhesin-like Shewanella woodyi protein (28% over 1,795 aa), LuxR/Sigma 70 family member of Citrobacter koseri (32% over 153 aa), and LuxR/UhpA family member of Vibrio campbelli (22% over 73 aa).

Fig 2

TosA is expressed in infected bladder, kidney, spleen, and liver. Female C57/Bl6 mice inoculated transurethrally (A and B) or via tail vein injection (C and D) with CFT073(pGENmut3.1). At 48 h postinoculation, the kidneys (A) and spleens (B) and at 24 h postinfection the spleens (C) and livers (D) were removed and processed for immunofluorescence microscopy. GFP antiserum staining is shown in green, TosA antiserum in red, phalloidin staining in white, and DAPI in blue. Measurements of individual bacteria are denoted by scale bars.

Fig 3

TosA localizes to the outer membrane. (A to C) Immunogold-TEM micrographs of TosA antiserum-stained bacteria. Bacteria were cultured under arabinose-inducing conditions. (A) CFT073 ΔtosA show no immunogold particles. (B) CFT073 araB_P-tosC marked with numerous immunogold particles. (C) TOP10(pBAD-tosA) cells show no immunogold staining. All images were acquired at ×64,000 magnification; scale bars denote 100 nm.

Fig 4

Cellular localization and protease susceptibility of TosA. (A) The indicated bacterial constructs were induced or not with arabinose and fractionated as described in Materials and Methods. Samples were subjected to SDS-PAGE and Western blotting with TosA antiserum. Fractions: Cyt, cytosol; IM, inner membrane; OM, outer membrane; Sup, supernatant. (B) The indicated constructs were induced with arabinose as described in Materials and Methods. Whole bacterial cells in suspension were incubated for 1 h with the indicated amount of proteinase K (Protease). Bacteria were treated with SDS-gel sample buffer, electrophoresed, and subjected to Western blotting with TosA antiserum. Blank lanes in the lower right panel contained no sample.

Fig 5

TosA mediates adherence to epithelial cells of the upper urinary tract. Wild-type CFT073 (WT) and CFT073 araB_P-tosC (TosA), cultured under arabinose-inducing conditions, were used to test adherence to confluent monolayers of epithelial cells in vitro at an MOI averaging 0.6. After incubation and washing, the cells were lifted off and plated for CFU determination. The data are expressed as the percentage of the inoculum that was adherent to the monolayer. (A) MM55.K cell adherence assay conducted with wild type (WT) and TosA (white bars) and repeated after cells were pretreated for 5 min with TosA antiserum (black bars). (B) Adherence assay conducted with seven distinct cell lines derived from throughout the urinary tract and expressed as the percentage of inoculum that was adherent to the monolayer. Raw data are supplied in Table S1 in the supplemental material.*, P < 0.05 as calculated by a Student t test.

Fig 6

TosA enhances fitness during bacteremia. A murine model of bacteremia was used to compete wild-type CFT073 against CFT073 ΔtosA. An equal mixture of both strains was diluted to deliver 10⁶ CFU in 100 μl, which was injected in the tail vein of female C57BL/6 mice. At 24 h postinoculation, liver and spleen tissue was removed, and the CFU of each strain were determined. The log₁₀ competitive index (mutant CFU/wild-type CFU) is shown, with lines representing median values. P values are indicated on the graph.

Fig 7

TosA enhances lethality in a zebrafish model of ExPEC pathogenesis. At 48 h postfertilization zebrafish embryos were microinjected with bacteria into either the pericardial cavity around the developing heart or into the circulation valley to initiate bacteremia and sepsis. (A) Wild-type (WT) CFT073 and CFT073 ΔtosA were mixed in equal proportions and inoculated into the pericardial cavity (PC) or the circulation valley (blood) with 1,000 CFU in 1 nl. At 16 h postinoculation, embryos were homogenized, and the CFU load of each strain was determined. The data are expressed as competitive indexes (mutant CFU/wild-type CFU), which were not significantly different. (B) Survival curve of zebrafish embryos injected in the circulation valley with 1,000 CFU of either wild-type CFT073 or CFT073 ΔtosA. (C) Immunofluorescence microscopy of the pericardial cavity (PC) or tails (blood) of embryos injected with wild-type CFT073 or araB_P-tosC bacteria cultured under arabinose-inducing conditions. Fish were maintained in water supplemented with arabinose and, 16 h postinoculation, fixed and processed for imaging. Anti-E. coli antibody staining marks bacteria red and TosA antiserum staining is displayed in green. The merged files in the right panels appear green (and not yellow) due to the far more intense anti-TosA staining than the anti-E. coli staining. Scale bars, 100 μm. (D) Survival curves of fish infected with CFT073 araB_P-tosC, incubated with or without arabinose induction. Mock-infected embryos were incubated in the presence of arabinose. n = 40 embryos for each survival curve.

Fig 8

TosA vaccination protects against urosepsis. (A) Purified TosA was utilized in an experimental vaccine model according to the depicted schedule: female C57/Bl6 mice were vaccinated with 100 μg of protein and then boosted one and 2 weeks later with 25 μg. Three weeks after primary vaccination the mice were challenged with wild-type CFT073 via transurethral infection (B) or tail vein injection (C). The CFU/g of tissue was determined for each organ site at 48 h postinfection (B) or 24 h postinfection (C) and compared to mice mock-vaccinated with PBS. *, P = 0.004.

Fig 9

TosA contains characteristic RTX repeats and a novel repeat structure. (A) DNA sequences of hlyA and tosA from the CFT073 genome were searched for the RTX repeat structure GGXGXD using PATTINPROT software program set to find regions with 75% identity to the consensus RTX sequence. The repeats were aligned and used to create sequence logos using WebLogo software (http://weblogo.berkeley.edu/logo.cgi). The height of the letters at each position indicates the level of conservation at that amino acid position (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_pattinprot.html). HlyA, top panel; TosA, bottom panel. Twelve repeats were found for hlyA, and nine were found for tosA. (B) The CFT073 genome annotation originally described tosA (first annotated as upxA) as a 5,325-nucleotide ORF encoding a putative protein 1610 amino acids long (predicted molecular mass, 163,819 Da). Sequence analysis identified two tandem repeats in the nucleotide sequence, depicted in panel as gray bars underneath a schematic of tosA, which shows the repeats relative location proximal to the 5′ of the gene and N terminus of the predicted protein. (C) Cloning and expression of tosA indicate the gene is actually ∼8,000 nucleotides in length and encodes a protein slightly larger than 250,000 Da (see Fig. 1A). Restriction enzyme mapping of full tosA PCR products is consistent with the presence of five of these repeats in a tandem array (data not shown). (D) Amino acid sequence of the tandem repeats is 335 residues long.