Validating targets in p. aeruginosa type III secretion using chemical probes

描述（由申请人提供）：铜绿假单胞菌是呼吸机相关性肺炎（VAP）的主要原因，目前的抗生素治疗策略显示失败率高达18%，即使生物体对所施用的抗生素敏感。该项目的目标是通过验证易受小分子抑制的III型分泌（T3SS）途径中的靶点，并确定它们在T3SS宿主-病原体相互作用中的作用，来解决这一关键的医疗需求。T3SS是铜绿假单胞菌主要的毒力决定因素，有助于感染的建立和传播（如VAP、菌血症、尿路感染）。细菌利用它分泌毒素效应物并将其转运到宿主吞噬细胞中，从而削弱宿主的先天免疫防御。在动物感染模型中，功能性T3SS的存在与不良临床结果和患者死亡显著相关，并显著降低生存率。本项目采用的策略是使用现有的化学探针来确定复杂T3SS机器的哪些成分容易受到小分子化合物的抑制。然后，携带探针靶点突变的探针和菌株将被用来确定这些易感成分在宿主-病原体相互作用中的作用。结果将在T3SS宿主-病原体相互作用中提供多达四个经过良好验证的、功能注释的、可药物靶点。四种已发表的T3SS抑制剂具有不相关的化学结构，在非细胞毒性浓度下对铜绿假单胞菌T3SS有抑制作用。其中两个探针是t3ss介导的分泌和效应毒素易位的有效抑制剂，而另外两个探针仅抑制分泌或易位。因此，这四种化学探针可能抑制至少三个不同的靶标或一个或多个靶标内的不同区域。在目标1中，将使用两种平行的方法来鉴定这四种探针的分子靶标——(a)在探针上允许的位点上添加光反应性和分子柄部分，应用光亲和探针来修饰靶标，回收和鉴定修饰的蛋白质；(b)应用分子遗传学工具富集探针

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is the leading cause of ventilator-associated pneumonia (VAP), and current antibiotic treatment strategies exhibit failure rates as high as 18%, even when the organism is susceptible to the antibiotic being administered. The goal of this project is to address this critical medical need by validating targets in the type III secretion (T3SS) pathway that are susceptible to inhibition by small molecules and determining their roles in the T3SS host-pathogen interaction. T3SS is the major P. aeruginosa virulence determinant contributing to the establishment and dissemination of infections (e.g. VAP, bacteremia, urinary tract infections). It is utilized by the bacterium to secrete and translocate toxin effectors into host phagocytes, thereby weakening the host's innate immune defenses. The presence of a functional T3SS is significantly associated with poor clinical outcomes and death in patients and markedly reduces survival in animal infection models. The strategy employed in this project is to use existing chemical probes to determine which components of the complex T3SS machine are susceptible to inhibition by small molecule compounds. Then, probes and strains carrying mutations in the probe targets will be used to define the roles of those vulnerable components in the host-pathogen interaction. Results will provide up to four well-validated, functionally annotated, druggable targets in the T3SS host-pathogen interaction. Four published T3SS inhibitors with unrelated chemical structures that are inhibitory to P. aeruginosa T3SS at non-cytotoxic concentrations have been selected as probes. Two of the probes are potent inhibitors of both T3SS-mediated secretion and translocation of effector toxins while the other two probes inhibit only secretion or translocation. Thus, these fou chemical probes likely inhibit at least three distinct targets or distinct regions within one or moe targets. In Aim 1, two parallel approaches will be used to identify the molecular targets of these four probes - (a) addition of photo-reactive and molecular handle moieties to permissible sites on the probes, application of photo-affinity probes to modify the target(s), recovery and identification of modified proteins; (b) application of molecular genetic tools to enrich for probe escape mutants followed by identification of the mutated gene(s) by deep sequencing. Finally, target identity will be confirmed by mutation analysis, and target gene mutant libraries will be prepared to facilitate understanding the role of each target in T3SS. In Aim 2, the probes and mutant libraries will be used to define the role of each probe target in the T3SS host-pathogen interaction. Effects of the probes and mutants on twelve distinct steps in the T3SS pathway within the broad categories of regulation, assembly, secretion, translocation, and cytotoxicity wil be used to dissect the roles of the targets in the T3SS machine and in host-pathogen interactions. Results will provide druggable, disease-relevant T3SS targets with characterized escape mutants for use in drug-discovery screening. Ideal targets will be prioritized as highly sensitive to rapid inhibition by probes and involved in critical roles in host-pathogen interaction. PUBLIC HEALTH RELEVANCE: The increasing prevalence antibiotic-resistant strains of bacterial pathogens represents an unmet medical need. Type-three secretion is a mechanism used by many pathogenic bacteria to increase their virulence in human infections. Bacterial toxins secreted by this method reduce the protective effect of the infected individual's own innate immune system. Results of this project will provide detailed information on the identity and function of components of the type-three secretion apparatus that are vulnerable to attack by drugs, and thus, facilitate new drug discovery.