Investigating a novel contact-dependent killing system and its contribution to pathogen dominance in the urinary tract

项目总结 大肠埃希氏菌和奇异变形杆菌是重要的尿路病原体，也是引起尿路感染的最常见原因 导管相关性尿路感染(CAUTIs)。CAUTIs是最常见的医疗保健之一- 相关感染，导致留置尿管的死亡率几乎是非留置尿管患者的两倍 导尿管的病人。CAUTIs通常是多菌的，物种间的相互作用有助于 形成导管生物膜，增加脓毒症的风险。多菌环境是由两者共同塑造的 物种之间的合作和竞争互动。然而，不同的 目前对尿路定殖者的定义仍然不明确。竞争性相互作用的特征是 此外，由于大量竞争制度仍未被发现，这一事实也阻碍了这一进程。我们的整体 目的是研究竞争系统对肠杆菌科细菌在尿路中成功定植的作用。 一条小路。这项提议的具体目的是确定和进一步描述一种新的物种间竞争制度。 被奇异变形杆菌用来杀死竞争物种。我们发现临床和共生的奇异巴氏杆菌 菌株迅速而剧烈地(200万倍)降低了大肠杆菌和其他肠杆菌科细菌的活力 在共培养过程中。无细胞奇异假单胞菌培养上清液不会降低靶细胞的活力。杀戮 取而代之的是细胞之间的直接接触。然而，固定相中的一种热不稳定组分，即奇异拟青霉 上清液足以诱导杀戮，这表明该系统可能是通过群体感应来调节的。 杀死也发生在固体表面，在那里奇异肺炎杆菌能够穿透并杀死已建立的 大肠埃希菌的微菌落。这突显了杀死系统在混合生物膜中的潜在作用，这是一种 尿管定植和持久性的重要组成部分。唯一已知的接触依赖型 奇异P.mirabilis的竞争系统是6型分泌系统(T6SS)，但该系统缺乏突变体 仍然杀死了大肠杆菌。因此，我们推测奇异疟原虫使用一种新的接触依赖系统来杀死E. Coli细胞。本研究的目的是识别杀戮系统及其调控，明确致死机制。 作用，并阐明在多菌作用过程中奇异肺炎杆菌与大肠杆菌竞争的杀伤系统的作用 体内感染。在目标1中，我们将使用奇异P.mirabilis的转座子文库和基于生物发光的检测 筛选不能杀死大肠杆菌的奇异假单胞菌突变株，并鉴定编码效应分子、传递 系统和杀伤系统的调节分子。我们还将测试以下假设：仲裁感知 分子调节系统的活动，并研究大肠杆菌细胞在接触后死亡的机制。 与奇异P.mirabilis。在目标2中，我们将评估奇异P.mirabilis杀伤系统在多菌感染中的作用。 我们将首先测试奇异假单胞菌在导尿管上形成生物膜期间杀灭大肠杆菌的能力，然后 评价奇异肺炎杆菌和大肠埃希菌导管定植和向外周器官扩散情况 使用CAUTI小鼠模型的多微生物感染。

PROJECT SUMMARY Escherichia coli and Proteus mirabilis are important urinary tract pathogens and the most common causes of catheter-associated urinary tract infections (CAUTIs). CAUTIs are one of the most common health care- associated infections and contribute to nearly double the mortality rate of catheterized compared to non- catheterized patients. CAUTIs are frequently polymicrobial and inter-species interactions contribute to the development of catheter biofilms and increase the risk of sepsis. Polymicrobial environments are shaped by both cooperative and competitive interactions between species. However, specific interactions between different urinary tract colonizers currently remain poorly defined. Characterizations of competitive interactions is additionally hampered by the fact that a large number of competition systems remain undiscovered. Our overall goal is to study the role of competition systems for successful colonization of Enterobacteriaceae in the urinary tract. This proposal specifically aims to identify and further characterize a novel inter-species competition system employed by Proteus mirabilis to kill competitor species. We showed that clinical and commensal P. mirabilis strains quickly and drastically (2-million-fold) reduced viability of E. coli and other Enterobacteriaceae during co-culture. Cell-free P. mirabilis culture supernatant alone did not reduce viability of target cells. Killing instead required direct contact between cells. However, a heat-labile component in stationary phase P. mirabilis supernatant was sufficient to induce killing, suggesting that the system is likely regulated via quorum sensing. Killing also occurred on solid surfaces, where P. mirabilis was able to penetrate and kill all cells in established microcolonies of E. coli. This highlights a potential role of the killing system in mixed biofilms, which is an important component of urinary catheter colonization and persistence. The only known contact-dependent competition system in P. mirabilis is the Type 6 Secretion System (T6SS), but a mutant deficient in this system still killed E. coli. We thus hypothesize that P. mirabilis employs a novel contact-dependent system to kill E. coli cells. The objective of this study is to identify the killing system and its regulation, define the mechanism of action, and elucidate the role of the killing system for competition of P. mirabilis with E. coli during polymicrobial infections in vivo. In Aim 1, we will use a transposon library of P. mirabilis and a bioluminescence-based assay to screen for P. mirabilis mutants unable to kill E. coli and to identify genes coding for effector molecules, delivery system and regulatory molecules of the killing system. We will also test the hypothesis that quorum sensing molecules regulate the activity of the system and investigate the mechanism of how E. coli cells die after contact with P. mirabilis. In Aim 2 we will assess the role of the P. mirabilis killing system during polymicrobial infection. We will first test the ability of P. mirabilis to kill E. coli during biofilm formation on urinary catheters and then assess colonization of catheters and dissemination to peripheral organs for P. mirabilis and E. coli in single and polymicrobial infections using a CAUTI mouse model.