Department of Microbiology and Immunology; Jacobs School of Medicine and Biomedical Sciences; State University of New York at Buffalo; Buffalo, NY, United States of America.
Department of Microbiology and Immunology; University of Michigan Medical School; Ann Arbor, MI, United States of America.
PLoS Pathog. 2019 Apr 22;15(4):e1007653. doi: 10.1371/journal.ppat.1007653. eCollection 2019 Apr.
The Gram-negative bacterium Proteus mirabilis is a common cause of catheter-associated urinary tract infections (CAUTI), which can progress to secondary bacteremia. While numerous studies have investigated experimental infection with P. mirabilis in the urinary tract, little is known about pathogenesis in the bloodstream. This study identifies the genes that are important for survival in the bloodstream using a whole-genome transposon insertion-site sequencing (Tn-Seq) approach. A library of 50,000 transposon mutants was utilized to assess the relative contribution of each non-essential gene in the P. mirabilis HI4320 genome to fitness in the livers and spleens of mice at 24 hours following tail vein inoculation compared to growth in RPMI, heat-inactivated (HI) naïve serum, and HI acute phase serum. 138 genes were identified as ex vivo fitness factors in serum, which were primarily involved in amino acid transport and metabolism, and 143 genes were identified as infection-specific in vivo fitness factors for both spleen and liver colonization. Infection-specific fitness factors included genes involved in twin arginine translocation, ammonia incorporation, and polyamine biosynthesis. Mutants in sixteen genes were constructed to validate both the ex vivo and in vivo results of the transposon screen, and 12/16 (75%) exhibited the predicted phenotype. Our studies indicate a role for the twin arginine translocation (tatAC) system in motility, translocation of potential virulence factors, and fitness within the bloodstream. We also demonstrate the interplay between two nitrogen assimilation pathways in the bloodstream, providing evidence that the GS-GOGAT system may be preferentially utilized. Furthermore, we show that a dual-function arginine decarboxylase (speA) is important for fitness within the bloodstream due to its role in putrescine biosynthesis rather than its contribution to maintenance of membrane potential. This study therefore provides insight into pathways needed for fitness within the bloodstream, which may guide strategies to reduce bacteremia-associated mortality.
革兰氏阴性菌变形杆菌是引起导管相关性尿路感染（CAUTI）的常见原因，这种感染可能会进一步发展为继发性菌血症。虽然有许多研究调查了在泌尿道中感染变形杆菌的实验，但对血液中的发病机制知之甚少。本研究使用全基因组转座子插入位点测序（Tn-Seq）方法确定了在血液中生存所必需的基因。利用一个包含 50000 个转座子突变体的文库，评估了 HI4320 基因组中非必需基因对在尾静脉接种后 24 小时小鼠肝脏和脾脏中的适应性相对于 RPMI、热失活（HI）新生血清和 HI 急性期血清中的生长的相对贡献。在血清中，有 138 个基因被鉴定为体外适应因子，主要涉及氨基酸转运和代谢，在脾脏和肝脏定植中，有 143 个基因被鉴定为感染特异性体内适应因子。感染特异性适应因子包括涉及双精氨酸易位、氨掺入和多胺生物合成的基因。构建了 16 个基因的突变体以验证转座子筛选的体外和体内结果，其中 12/16（75%）表现出预期的表型。我们的研究表明，双精氨酸易位（tatAC）系统在运动性、潜在毒力因子的易位以及血液中的适应性中起作用。我们还展示了血液中两种氮同化途径之间的相互作用，证明 GS-GOGAT 系统可能被优先利用。此外，我们表明，由于其在腐胺生物合成中的作用而不是在维持膜电位中的作用，双功能精氨酸脱羧酶（speA）对血液中的适应性很重要。因此，本研究提供了对血液中适应性所需途径的深入了解，这可能为减少与菌血症相关的死亡率提供策略。
