Identification of protease and rpoN-associated genes of uropathogenic Proteus mirabilis by negative selection in a mouse model of ascending urinary tract infection

Abstract

Proteus rnirabilis, a motile Gram-negative bacterium, is a principal cause of urinary tract infections in patients with functional or anatomical abnormalities of the urinary tract or those with urinary catheters in place. Thus far, virulence factors including urease, flagella, haemolysin, various fimbriae, IgA protease and a deaminase have been characterized based on the phenotypic traits conferred by these proteins. In this study, an attempt was made to identify new virulence genes of P. rnirabilis that may not have identifiable phenotypes using the recently described technique of signature-tagged mutagenesis. A pool of chromosomal transposon mutants was made through conjugation and kanamycin/tetracycl ine selection; random insertion was confirmed by Southern blotting of chromosomal DNA isolated from 16 mutants using the aphA gene as a probe. From the total pool, 2.3% (9/397) auxotrophic mutants and 3.5% (14/397) swarming mutants were identified by screening on minimal salts agar and Luria agar plates, respectively. T h i r t y per cent of the mutants, found to have either no tag or an unamplifiable tag, were removed from the input pool. Then lo7 c.f.u. from a 96-mutant pool (-lo5 c.f.u. of each mutant) were used as an input pool to transurethrally inoculate seven CBA mice. After 2 d infection, bacteria were recovered from the bladders and kidneys and yielded about l o 5 c.f.u. as an output pool. Dot blot analysis showed that two of the 96 mutants, designated B2 and B5, could not be hybridized by signature tags amplified from the bladder output pool. Interrupted genes from these two mutants were cloned and sequenced. The interrupted gene in B2 predicts a polypeptide of 37-3 kDa that shares amino acid similarity with a putative protease or collagenase precursor. The gene in B5 predicts a polypeptide of 32-6 kDa that is very similar to that encoded by ORF284 of the rpoN operon controlling expression of nitrogen-regulated genes from several bacterial species. The virulence of the two mutants was tested further by co-challenging CBA mice with each mutant and the parental strain. After 1 week of infection, the B2 and B5 mutants were recovered in numbers 100-fold and 1000-fold less than the parental strain, respectively. Using an in vitro assay, it was shown that the B2 mutant had significantly less (P = O*OOOl) extracellular protease activity than the wild-type strain. These findings demonstrate that signaturetagged mutagenesis is a viable approach to identify bacterial genes associated with the ability to infect the urinary tract.

Characterisation of an acapsular mutant of Burkholderia pseudomallei identified by signature tagged mutagenesis

“…Signature tagged mutagenesis (STM) has been used successfully in several bacterial pathogens to identify virulence determinants in vivo [25][26][27][28][29][30][31][32][33]. Briefly, a pool of transposons is assembled, each harbouring a unique oligonucleotide tag.…”

TheyrpABoperon ofYersinia ruckeriencoding two putative U32 peptidases is involved in virulence and induced under microaerobic conditions

“…11 However, YrpA shows only a 27% identity to PrtC, whereas it shares 85% identity with a protease of P. mirabilis belonging to the U32 family. 13 These differences in identity indicate heterogeneity among the members of this family of proteases which are also present in gram-positive bacteria such as Streptococcus mutants 19 and Streptococcus agalactiae. 20 However, these two enzymes, YrpA and YrpB, exhibit a high degree of identity with other bacterial proteases from the enterobacteria group.…”

“…1). Both domains are present in the STM proteaseA of P. mirabilis 13 and the PrtC collagenase of P. gingivalis. 11,12 Based on these identities the loci were named yrpA and yrpB (Yersinia ruckeri protease A and B, respectively).…”

“…88%), and S. enterica (EHC45316, 88%) and with the protein STMproteaseA of P. mirabilis (AAC64577, 85%). 13 The second ORF is an 879 bp locus which codes for a 292-amino-acid protein that shares a high sequence identity with hypothetical proteins of Yersinia pseudotuberculosis (YP_001722436, 84%), Yersinia pestis (EIS91064, 84%), Serratia plymuthica (EKF66684, 80%), Dickeya dadantii (YP_003332207, 79%), and Pectobacterium carotovorum (YP_003016184, 79%). Multiple alignments of amino acid sequences of the homologous Y. ruckeri YrpA and YrpB peptidases with human pathogenic yersiniae (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) showed a high percentage identity ranging from 92-93% for the first ORF to 84-86% for the second one.…”

“…11 This is a calcium-dependent metalloprotease, capable of degrading type I collagen but not gelatin or synthetic collagen substrates. 12 The U32 peptidases have been described in other pathogenic bacteria such as Proteus mirabilis, 13 Helicobacter pylori, 14 Salmonella enterica, 15 and Aeromonas veronii. 16 All of them are putative collagenases and it is generally accepted that they contribute to bacterial infection.…”