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

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.革兰氏阴性奇异变形杆菌（Proteus mirabilis）是导尿管相关尿路感染（catheter-associated urinary tract infections, CAUTIs）的主要致病菌之一，此类感染通常为多微生物混合感染。此前诸多研究已在上行性尿路感染小鼠模型中揭示了奇异变形杆菌致病的毒力因子，但关于其在导尿管相关尿路感染或混合感染中的致病机制，目前仍所知甚少。本研究利用五组各含10000株转座子突变体的文库，并借助转座子插入位点测序（transposon insertion-site sequencing, Tn-Seq）技术，鉴定了奇异变形杆菌HI4320在单菌种导尿管相关尿路感染，以及与斯图亚特普罗威登斯菌（Providencia stuartii）BE2467混合感染的导尿管相关尿路感染中的全套适应因子。输入突变体文库中的436个基因未检测到转座子插入，因此被认定为奇异变形杆菌在丰富培养基中生长所必需的基因。在单菌种导尿管相关尿路感染模型中，共鉴定出629个奇异变形杆菌适应因子。与斯图亚特普罗威登斯菌共感染的转座子插入位点测序结果显示，其中217/629（35%）的基因与单菌种测序鉴定出的适应因子重合，另有1353个额外的适应因子仅在混合感染定植过程中发挥作用。我们针对8个候选目标基因构建了突变体以验证初始筛选结果：其中7/8（88%）的突变体在单菌种导尿管相关尿路感染模型中表现出预期表型，3/3（100%）的突变体验证了混合感染导尿管相关尿路感染的预期表型。本研究不仅验证了此前在上行性尿路感染模型中报道的诸多奇异变形杆菌适应决定因子，还发现了仅与导尿管相关尿路感染相关的新型适应决定因子，并严格评估了混合感染如何影响细菌的适应需求。例如，我们发现由于斯图亚特普罗威登斯菌对亮氨酸的高亲和力摄取，奇异变形杆菌在混合感染过程中需要支链氨基酸生物合成途径。进一步研究在单菌种与混合感染导尿管相关尿路感染中均能赋予竞争优势的基因与通路，有望为降低奇异变形杆菌导尿管相关尿路感染的发病率与严重程度提供可靠的治疗干预靶点。