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

描述（由申请方提供）：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%，即使该微生物对所给予的抗生素敏感。该项目的目标是通过验证易受小分子抑制的III型分泌（T3 SS）途径中的靶标并确定其在T3 SS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。T3 SS是导致感染（例如VAP、菌血症、尿路感染）形成和传播的主要铜绿假单胞菌毒力决定因素。它被细菌利用来分泌毒素效应物并将其转移到宿主吞噬细胞中，从而削弱宿主的先天免疫防御。功能性T3 SS的存在与患者的不良临床结局和死亡显著相关，并显著降低动物感染模型的存活率。该项目采用的策略是使用现有的化学探针来确定复杂的T3 SS机器的哪些组件容易受到小分子化合物的抑制。然后，将使用在探针靶标中携带突变的探针和菌株来确定那些脆弱组分在宿主-病原体相互作用中的作用。结果将在T3 SS宿主-病原体相互作用中提供多达四个经过充分验证的、功能注释的可药用靶标。已选择四种具有不相关化学结构的已发表T3 SS抑制剂作为探针，这些抑制剂在非细胞毒性浓度下对铜绿假单胞菌T3 SS具有抑制作用。其中两个探针是T3 SS介导的效应毒素分泌和易位的有效抑制剂，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种不同的靶标或一种或莫伊靶标内的不同区域。在目的1中，将使用两种平行的方法来鉴定这四种探针的分子靶点-（a）在探针的允许位点上添加光反应和分子手柄部分，应用光亲和探针修饰靶点，回收和鉴定修饰的蛋白质;（B）应用分子遗传工具富集探针， 逃逸突变体，然后通过深度测序鉴定突变基因。最后，将通过突变分析确认靶标身份，并制备靶标基因突变体文库，以便于理解每个靶标在T3 SS中的作用。在目的2中，探针和突变体文库将用于定义每个探针靶标在T3 SS宿主-病原体相互作用中的作用。探针和突变体对调节、组装、分泌、易位和细胞毒性的广泛类别内的T3 SS途径中的12个不同步骤的影响将用于剖析靶标在T3 SS机器和宿主-病原体相互作用中的作用。结果将提供可药用的，疾病相关的T3 SS靶点与特征的逃逸突变体用于药物发现筛选。理想的靶标将优先考虑对探针的快速抑制高度敏感，并参与宿主-病原体相互作用的关键作用。 公共卫生相关性：细菌病原体的耐药菌株的流行率不断增加，代表了一种未满足的医疗需求。第三型分泌是许多病原菌用于增加其在人类感染中的毒力的机制。通过这种方法分泌的细菌毒素降低了受感染个体自身先天免疫系统的保护作用。该项目的结果将提供关于易受药物攻击的第三类分泌器官的成分的身份和功能的详细信息，从而促进新药的发现。

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.