LeuX tRNA-dependent and -independent mechanisms of Escherichia coli pathogenesis in acute cystitis - PubMed (original) (raw)

LeuX tRNA-dependent and -independent mechanisms of Escherichia coli pathogenesis in acute cystitis

Thomas J Hannan et al. Mol Microbiol. 2008 Jan.

Abstract

Uropathogenic Escherichia coli (UPEC) contain multiple horizontally acquired pathogenicity-associated islands (PAI) implicated in the pathogenesis of urinary tract infection. In a murine model of cystitis, type 1 pili-mediated bladder epithelial invasion and intracellular proliferation are key events associated with UPEC virulence. In this study, we examined the mechanisms by which a conserved PAI contributes to UPEC pathogenesis in acute cystitis. In the human UPEC strain UTI89, spontaneous excision of PAI II(UTI89) disrupts the adjacent leuX tRNA locus. Loss of wild-type leuX-encoded tRNA(5)(Leu) significantly delayed, but did not eliminate, FimB recombinase-mediated phase variation of type 1 pili. FimX, an additional FimB-like, leuX-independent recombinase, was also found to mediate type 1 pili phase variation. However, whereas FimX activity is relatively slow in vitro, it is rapid in vivo as a non-piliated strain lacking the other fim recombinases rapidly expressed type 1 pili upon experimental infection. Finally, we found that disruption of leuX, but not loss of PAI II(UTI89) genes, reduced bladder epithelial invasion and intracellular proliferation, independent of type 1 piliation. These findings indicate that the predominant mechanism for preservation of PAI II(UTI89) during the establishment of acute cystitis is maintenance of wild-type leuX, and not PAI II(UTI89) gene content.

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Figures

Fig. 1

Overview of the genomic organization of PAI IIUTI89, the fim operon (‘Type 1’) and the fimX locus (‘FimX’). Open reading frames shown in the PAI IIUTI89 diagram are depicted either above or below the line, representing forward or reverse strand orientation of the genes, respectively. The direct repeat locations (arrows) are shown flanking the PAI IIUTI89 region, and the repeat sequences are shown below (underlined text). The small triangles flanking the direct repeat sequence text indicate the nucleotides deleted in the leuX gene of either UTI89 or 536 after spontaneous excision of each respective PAI II by recombination of the direct repeats. Recombinase genes in Type 1 and FimX loci are indicated in grey and ‘hypo’ indicates that the gene was annotated as a hypothetical protein. Inset: The relative chromosomal positions of these loci (‘II’, PAI IIUTI89; ‘T’, fim operon; ‘X’, fimX locus) and other UTI89 PAI are shown on the circular map at the lower right. ‘_ibeA_’ indicates PAI containing the invasion of brain endothelium virulence gene (Johnson et al., 2007). ‘Type II’ indicates type II secretion system island (Chen et al., 2006). PAI containing IcmF-associated homologous proteins are denoted IAHP (Das and Chaudhuri, 2003) followed by the tRNA insertion site. Roman numerals indicate similarity to PAI numbered by convention in pyelonephritis strain 536 (Dobrindt et al., 2002; Brzuszkiewicz et al., 2006).

Fig. 2

Mutation of leuX due to spontaneous excision of PAI IIUTI89 delays type 1 pili expression due to a complete loss of tRNA5Leu function. A–C. Type 1 pili expression and fimS phase switch orientation in static broth cultures of wt UTI89 (wt) and derivatives grown at 37°C. A. Mannose-sensitive haemagglutination (MSHA) titres (8 and 24 h) and representative phase assays of wt, TJH1 (leuX-, PAI II-) and TJH1 COMP (leuX+, PAI II-). B. MSHA titres (24 and 48 h) of wt and UTI89 leuX_Δ_G80, carrying either pBAD33 (VC) or pBAD-leuX (leuX) in trans. C. MSHA titres (8 and 24 h) and representative phase assays of wt or isogenic leuX mutants. All trials in (A), (B) and (C) were performed in duplicate in three independent experiments. Error bars represent standard deviations. D. Relative quantity (RQ) of leuX tRNA in wt or isogenic leuX mutants compared with UTI89 Δ_leuX_ after 24 h of static growth in broth culture at 37°C. Horizontal bars represent mean values and vertical bars show RQ minimum and maximum range. qRT-PCR was performed on cDNA derived from each strain grown in three independent cultures in two independent experiments. qRT-PCR reactions were performed in duplicate.

Fig. 3

Roles of FimB and FimX recombinases in type 1 pili phase switching in vitro in TJH1. A–C. Type 1 pili expression and fimS phase switch orientation in static broth cultures of TJH1 (leuX-, PAI II-) and derivatives grown at 37°C. A. MSHA titres (48 h) of TJH1 and derivatives ΔB (fimB-), ΔX (_fim_X-) or ΔXB (fimX-fimB-). The asterisk (*) indicates each strain carried pTrc99a as a vector control. Representative phase assays and FimA immunoblot results are shown for each sample. B. MSHA titres (24 h) of TJH1ΔXB (leuX-, PAI IIUT189-, fimX-, fimB-) carrying two of the following plasmids in trans: (i) control vectors (V), (ii) inducible fimB (B) and/or (iii) inducible fimX vector (X). Representative phase assays are shown for each sample. C. MSHA titres (24 h) of TJH1 and derivatives with either pBAD33 (VC) or leuX in trans. All trials in (A), (B) and (C) were performed in duplicate in three independent experiments. Error bars represent standard deviations.

Fig. 4

FimX-dependent phase variation of type 1 pili in vivo. A and B. Ex vivo gentamicin protection assays were performed to distinguish extracellular and intracellular bacteria in infected C3H/HeN female mice at 6 hpi (as per Experimental procedures). Luminal contents (Lumen) are obtained from PBS washes of the removed bladder and represent relative luminal colonization. Bladder shows the cfu per ml of intracellular bacteria in the gentamicin-treated bladder homogenate. A. Mice were transurethrally infected with 107 cfu of UTI89 with control plasmid (wt/VC), a UTI89_fimB-fimE_– strain phase OFF in vitro with either the control plasmid (Δ_fimBE_ OFF/VC) or a plasmid carrying the fimB and fimE genes (Δ_fimBE_ OFF/fimBE), or a Δ_fimBE_ strain phase ON in vitro carrying the control plasmid (Δ_fimBE_ ON/VC). B. Mice were transurethrally infected with 107 cfu of wt/VC or UTI89_fimB-fimE-fimX_– strains phase OFF in vitro carrying either control (Δ_fimBEX_ OFF/VC) or fimX (δ_fimBEX_ OFF/fimX) vectors in trans. C. C3H/HeN female mice were transurethrally infected with an equal ratio of UTI89 attλ::PSSH10-1 (wt, SpecR) and either fimB or fimX null bacteria (KanR) for competitive infections. In the wt versus fimB null competitive experiment, each strain carried a plasmid with fimX under the ara promoter and grown in the presence of

-arabinose to induce phase OFF to ON changes, normally requiring native fimB in vitro. Colony-forming units (cfu) and competitive indices were scored for each strain as indicated in Experimental procedures. A–C. Bars represent the median value for each group and _P_-values were calculated using the Mann–Whitney _U_-test.

Fig. 5

Wild-type leuX, not PAI IIUTI89 gene content, is necessary for full UPEC virulence during early acute events in experimental cystitis. A–C. The individual roles of the _leuX_-encoded tRNA5Leu and PAI II content in early events in acute cystitis were tested in an experimental cystitis model comparing the isogenic mutants UTI89 _leuX_ΔG80 (‘_leuX_–’, leuX-, PAI IIUTI89+), TJH1 (leuX-, PAI IIUTI89-) and TJH2 (leuX+, PAI IIUTI89-) with wt UTI89. A. Immunoblot detecting FimA and MSHA in the same inocula of UTI89 (wt) and derivatives used in the experiments displayed in (B) and (C), which were performed on the same day. The asterisks (*) indicate strains carried pBAD-fimX, in trans, and were grown statically in 0.1% arabinose in LB/Cm20 for 18 h, thereby overexpressing fimX and driving type 1 piliation prior to inoculation into mice. B. In vivo invasion of UTI89 (wt) and derivatives overexpressing fimX, measured by ex vivo gentamicin protection at 1 hpi. Luminal contents (Lumen) demonstrate the relative presence of each strain in the bladder lumen. Bladder shows the cfu present after gentamicin treatment of each bladder. These data are from a single experiment representative of three independent experiments, except for UTI89 leuX_Δ_G80 (n = 2). C. IBC formation by strains UTI89 and derivatives, all overexpressing fimX, at 6 hpi measured by β-galactosidase staining. These data are from a single experiment representative of two independent experiments, except for UTI89 leuX_Δ_G80 (n = 1). B and C: Bars represent the median value for each group and _P_-values were calculated using the Mann–Whitney _U_-test.

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LeuX tRNA-dependent and -independent mechanisms of Escherichia coli pathogenesis in acute cystitis - PubMed (original) (raw)