Identification of genes in an extraintestinal isolate of Escherichia coli with increased expression after exposure to human urine.

The identification of genes with increased expression in vivo may lead to the identification of novel or unrecognized virulence traits and/or recognition of environmental signals involved in modulating gene expression. Our laboratory is studying an extraintestinal isolate of Escherichia coli as a model pathogen. We had previously used human urine ex vivo to identify the unrecognized urovirulence genes guaA and argC and to establish that arginine and guanine (or derivatives) were limiting in this body fluid (T. A. Russo et al., Mol. Microbiol. 22:217-229, 1996). In this study, we have continued with this approach and identified three additional genes that have increased expression in human urine relative to Luria-Bertani (LB) medium. Expression of ure1 (urine-responsive element) is increased a mean of 47.6-fold in urine but completely suppressed by exogenous glucose. This finding suggests that ure1 is regulated by catabolite repression and that limiting glucose in urine is a regulatory signal. ure1 is present in the E. coli K-12 genome, but its function is unknown. Although disruption of ure1 results in diminished growth in human urine, limiting concentrations of amino acids, nucleosides, or iron (Fe), or changes in osmolarity or pH do not affect the expression of ure1. Therefore, Ure1 appears to have a role independent of the synthesis or uptake of these nutrients and does not appear to be involved in osmoprotection. iroN(E. coli) is a novel E. coli gene with 77% DNA homology to a catecholate siderophore receptor gene recently identified in Salmonella. Its expression is increased a mean of 27.2-fold in urine and is repressed by exogenous Fe and a urinary pH of 5.0. This finding supports the contention that Fe is a limiting element in urine and that alteration of pH can affect gene expression. It is linked to the P-pilus (prs) and F1C fimbrial (foc) gene clusters on a pathogenicity island and appears to have been acquired by IS1230-mediated horizontal transmission. The homologous iroN(E. coli) sequence is significantly more prevalent in urinary tract and blood isolates of E. coli compared to fecal isolates. Last, the expression of ArtJ, an arginine periplasmic binding protein, is increased a mean of 16.6-fold in urine. This finding implicates arginine concentrations as limited in urine and, in combination with previous data demonstrating that argC is important for urovirulence, suggests that the ability of E. coli to synthesize or acquire arginine is important for urovirulence. ure1, iroN(E. coli), and artJ all have increased expression in human blood and ascites relative to LB medium as well. The identification of these genes increases our understanding of regulatory signals present in human urine, blood, and ascites. Ure1, IroN(E. coli), and ArtJ also warrant further evaluation as virulence traits both within and outside the urinary tract.

Identification of two previously unrecognized genes (guaA and argC) important for uropathogenesis.

Our knowledge of the traits possessed by extraintestinal isolates of Escherichia coli, necessary for growth and survival in urine, is limited. To identify such determinants, transposon (TnphoA'1,4) mutant libraries of a clinical isolate (CP9) were generated and screened for derivatives exhibiting decreased growth in urine in vitro, and for mutants with active lacZ fusions that were induced in urine relative to laboratory medium. Using this approach we identified two genes, guaA (CPA24) and argC (CPI-1), which were previously unrecognized as being important for growth in human urine. Unexpectedly, not only does CPA24 (guaA) not grow in human urine in vitro, but it is sensitive to its effects, undergoing a 2-3 log loss of viability over 6 h. By contrast, CPA24 neither grows nor is killed in M9 minimal medium and artificial urine. Therefore, we postulate that lack of guanine or its derivatives in urine, and the inability of CPA24 to synthesize these compounds de novo, prevents CPA24 from synthesizing other guanine (or derivatives)-dependent products that are critical for growth and survival in urine. Although it seems logical that decreased growth in urine in vitro should correlate with diminished urovirulence, this concept was tested by challenging mice with CPA24 in vivo in a mouse model of urinary tract infection (UTI). Indeed, CPA24 was found to be significantly less virulent compared with its wild-type parent CP9. CPI-1(argC) was identified because of the significant induction of its argC::lacZ fusion in urine. Subsequent testing in urine demonstrated that its growth was significantly diminished in all urine samples tested (four females, three males). Polyamine synthesis is dependent upon, in part, the arginine biosynthetic pathway. Therefore, we tested whether the induction of argC in urine and/or the decreased growth of CPI-1 was a result of low levels of polyamines or arginine in urine. The results suggest that low levels of arginine, but not polyamines, in human urine are responsible. When tested in vivo in the mouse model of UTI, CPI-1 was also found to be significantly less virulent than CP9. In summary, we have established that guaA and argC are the first genes, which we are aware of, that have been shown to contribute to the growth of E. coli in urine in vitro and both have diminished urovirulence in vivo. These results support the concept that urine can be used in vitro as a screening tool to identify urovirulence traits.

The O4 specific antigen moiety of lipopolysaccharide but not the K54 group 2 capsule is important for urovirulence of an extraintestinal isolate of Escherichia coli.

Group 2 capsules and lipopolysaccharides are regarded as important virulence factors in extraintestinal isolates of Escherichia coli, but their specific contributions to bladder and renal infections, if any, are unknown. Proven isogenic derivatives deficient in the K54 antigen alone (CP9.137), the O4 antigen alone (CP921), or both the K54 and O4 antigens (CP923) were compared with their wild-type parent (CP9 [O4/K54JH5]) for growth in human urine in vitro and for virulence in vivo in a mouse model of ascending urinary tract infection (UTI). Growth of CP9.137 and CP921 was equivalent to that of CP9 in human urine. CP923 demonstrated a small but reproducible decrease in log-phase growth but achieved the same plateau density. In the mouse model of UTI, the isogenic mutant deficient in the 04 antigen alone (CP921) and, to a greater degree, the derivative deficient in both the K54 and O4 antigens (CP923) were significantly less virulent in nearly all parameters measured. In contrast, the K54 knockout derivative was as virulent as its parent, CP9, in causing bladder infection and nearly as virulent in causing renal infection. These results demonstrate an important role for the O4 antigen moiety of lipopolysaccharide in the pathogenesis of UTI. The possibility that the K54 antigen also plays a minor role cannot be excluded.