Genome-wide transposon mutagenesis of Proteus mirabilis: Essential genes, fitness factors for catheter-associated urinary tract infection, and the impact of polymicrobial infection on fitness requirements.

Alexandra O Johnson,Weisheng Wu,Andreas J Baumler,Harry L T Mobley,Lili Zhao,Sara N Smith,Chelsie E Armbruster,Valerie Forsyth-Deornellas

doi:10.1371/journal.ppat.1006434

Abstract

The Gram-negative bacterium Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Numerous prior studies have uncovered virulence factors for P. mirabilis pathogenicity in a murine model of ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. In this study, we utilized five pools of 10,000 transposon mutants each and transposon insertion-site sequencing (Tn-Seq) to identify the full arsenal of P. mirabilis HI4320 fitness factors for single-species versus polymicrobial CAUTI with Providencia stuartii BE2467. 436 genes in the input pools lacked transposon insertions and were therefore concluded to be essential for P. mirabilis growth in rich medium. 629 genes were identified as P. mirabilis fitness factors during single-species CAUTI. Tn-Seq from coinfection with P. stuartii revealed 217/629 (35%) of the same genes as identified by single-species Tn-Seq, and 1353 additional factors that specifically contribute to colonization during coinfection. Mutants were constructed in eight genes of interest to validate the initial screen: 7/8 (88%) mutants exhibited the expected phenotypes for single-species CAUTI, and 3/3 (100%) validated the expected phenotypes for polymicrobial CAUTI. This approach provided validation of numerous previously described P. mirabilis fitness determinants from an ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, we describe a requirement for branched-chain amino acid biosynthesis by P. mirabilis during coinfection due to high-affinity import of leucine by P. stuartii. Further investigation of genes and pathways that provide a competitive advantage during both single-species and polymicrobial CAUTI will likely provide robust targets for therapeutic intervention to reduce P. mirabilis CAUTI incidence and severity.

Highlights

The Gram-negative bacterium Proteus mirabilis thrives in a wide variety of environments, including soil, water sources, sewage, and as a commensal in the intestinal tract of humans and animals [1, 2]
The combination of genome-saturating transposon mutant libraries and transposon insertion-site sequencing (Tn-Seq) has allowed for the first global estimation of P. mirabilis essential genes, validation of numerous P. mirabilis virulence factors and fitness determinants from decades of studies using the ascending model of urinary tract infections (UTIs), the discovery of novel fitness determinants for catheter-associated urinary tract infections (CAUTIs), and a stringent assessment of how polymicrobial infection influences fitness requirements
Branched chain amino acid (BCAA) biosynthesis is an intriguing target for reducing bacterial colonization, but our results indicate that this pathway is only important for P. mirabilis during polymicrobial infection and would not be a suitable target for single-species infection by P. mirabilis

Summary

Introduction

The Gram-negative bacterium Proteus mirabilis thrives in a wide variety of environments, including soil, water sources, sewage, and as a commensal in the intestinal tract of humans and animals [1, 2]. P. mirabilis has been identified as the causative agent of numerous human illnesses including cystitis, pyelonephritis, prostatitis, as well as intra-abdominal, wound, eye, and burn infections [2]. While it is capable of causing uncomplicated urinary tract infections (UTIs), this organism is a much more common cause of catheter-associated UTI (CAUTI) [3,4,5]. UTIs and CAUTIs involving P. mirabilis are typically complicated by the formation of bladder and kidney stones (urolithiasis) and permanent renal damage [9,10,11], and may progress to bacteremia [12, 13] Despite these potentially severe complications of P. mirabilis infection, there are no currently licensed vaccines available for this organism and multidrug-resistant isolates are increasingly common [14, 15]

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Conclusion