Intracellular persistence factors in urinary tract infections

持续性感染通常是由在体外表现出抗生素敏感性的细菌病原体引起的，这表明在宿主复杂的环境中抗生素治疗的内在局限性。虽然可变的患者因素对抗生素的疗效有贡献，但抗生素持久性的基本原则已经确立。在体外，抗生素持久性是在理想的生长条件下观察到的，这些休眠的亚群由于抗生素靶标的不活跃而耐受抗生素。在体内，细胞内感染阶段提供了对免疫系统许多组成部分的保护，而宿主病原体的相互作用可以促进抗生素的持久性。在我的博士后研究中，我和我的同事们研究了耐多药肺炎克雷伯菌的适应性进化，发现了尿路感染中持久性的全球进化，这涉及到肺炎克雷伯菌固有的能力，即持续存在于膀胱上皮细胞LAMP1阳性空泡中，并耐受致命浓度的抗生素。对肺炎克雷伯菌细胞内抗生素持久性的后续调查发现，与细胞外环境相比，休眠的多药耐药细菌在细胞内环境中出现的频率更高，这与越来越多的证据将宿主病原体相互作用与抗生素耐药性联系在一起。为了针对细胞内细菌感染，我们开发了细胞内存活的高通量筛选试验，并进行了化合物筛选，导致发现了一种宿主靶向化合物，该化合物可诱导膀胱细胞内的抗菌活性，并在潜伏性尿路感染的小鼠模型中显示体内疗效。我建议进一步研究肺炎克雷伯菌在膀胱上皮细胞LAMP1阳性空泡中持续存在的内在能力，以更好地了解持续性尿路感染的分子基础。细胞内持久性因子将在排列的转座子突变体库中确定。对对细胞内存活影响最大的基因进行验证实验和表型表征将导致更详细的机制调查，重点是已确定的持久性因素之一。蛋白质相互作用实验将有助于阐明细胞内持续存在的途径，这可能导致发现促进细胞内生存的宿主病原体相互作用。此外，将在尿路感染的小鼠模型中研究破坏细胞内生存的突变的影响，这将为研究细胞内感染阶段在导致持续感染中的作用提供机会。

Persistent infections are often caused by bacterial pathogens that display in vitro antibiotic susceptibility, illustrating the intrinsic limitations of antibiotic therapy in the complex environment of the host. While variable patient factors contribute to antibiotic efficacy, fundamental principles of antibiotic persistence have been established. In vitro, antibiotic persistence is observed under ideal growth conditions in dormant subpopulations that tolerate antibiotics due to inactivity of antibiotic targets. In vivo, intracellular infection stages provide protection against many components of the immune system, while host pathogen interactions can promote antibiotic persistence. In my postdoctoral studies, my colleagues and I studied the adaptive evolution of multidrug- resistant Klebsiella pneumoniae and discovered global evolution of persistence in urinary tract infections, which involved an intrinsic ability of K. pneumoniae to persist in LAMP1 positive vacuoles of bladder epithelial cells and tolerate lethal concentrations of antibiotics. A follow-up investigation of intracellular antibiotic persistence in K. pneumoniae found that dormant, multidrug-tolerant bacteria occurred at higher frequency in the intracellular environment compared to the extracellular environment, in line with an increasing body of evidence linking host pathogen interactions to antibiotic tolerance. To target intracellular bacterial infections, we developed a high-throughput screening assay of intracellular survival and conducted a compound screen that led to the discovery of a host-targeting compound that induces intracellular antimicrobial activity in bladder cells and displays in vivo efficacy in a mouse model of latent UTI. I propose to further investigate the intrinsic ability of K. pneumoniae to persist intracellularly in LAMP1 positive vacuoles of bladder epithelial cells to gain a better understanding of the molecular basis of persistent urinary tract infections. Intracellular persistence factors will be identified in an arrayed transposon mutant library. Validation experiments and phenotypic characterization of genes with the strongest impact on intracellular survival will lead to a more detailed mechanistic investigation that focuses on one of the identified persistence factors. Protein interaction experiments will help to elucidate a pathway of intracellular persistence which could lead to the discovery of host pathogen interactions that promote intracellular survival. Moreover, the impact of a mutation that disrupts intracellular survival will be investigated in a mouse model of urinary tract infection which will provide an opportunity to study the role of intracellular infection stages in causing persistent infections.