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

验证第 14 页中的目标。

• 批准号: 8636993
• 负责人: Donald T Moir
• 金额: $ 85.18万
• 依托单位: MICROBIOTIX, INC
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-03 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8636993
• 关键词: Address; Affect; Affinity; Affinity Labels; Animals; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Back; Bacteremia; Bacteria; Bacterial Toxins; Binding Sites; Biological Assay; Biology; Categories; Cessation of life; Chemical Structure; Chemicals; Clinical; Complex; Dependency; Development; Disease; Drug resistance; Engineering; Escape Mutant; Exhibits; Failure; Gene Targeting; Genes; Goals; Human; Immune; Immune response; Immune system; Individual; Infection; Intoxication; Kinetics; Label; Libraries; Mediating; Medical; Methods; Modeling; Molecular; Molecular Genetics; Molecular Target; Mutate; Mutation; Mutation Analysis; Organism; Outcome; Pathogenicity; Pathway interactions; Patients; Phagocytes; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pneumonia; Prevalence; Proteins; Pseudomonas aeruginosa; Publishing; Recovery; Regulation; Research; Role; Site; Structure; Toxin; Urinary tract infection; Ventilator; Virulence; affinity labeling; attributable mortality; cytotoxic; cytotoxicity; deep sequencing; drug discovery; experience; inhibitor/antagonist; multidisciplinary; mutant; pathogen; pathogenic bacteria; protective effect; resistant strain; screening; small molecule; tool; treatment strategy

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.

描述(申请人提供)：铜绿假单胞菌是呼吸机相关肺炎(VAP)的主要原因，目前的抗生素治疗策略显示失败率高达18%，即使在微生物对所用抗生素敏感的情况下也是如此。该项目的目标是通过验证III型分泌(T3SS)途径中容易受到小分子抑制的靶点，并确定它们在T3SS宿主-病原体相互作用中的作用，来满足这一关键的医学需求。T3SS是导致感染(如VAP、菌血症、尿路感染)的主要致病因子。细菌利用它来分泌毒素效应物并将其转移到宿主吞噬细胞中，从而削弱宿主的天然免疫防御。在动物感染模型中，功能性T3SS的存在与患者不良的临床结局和死亡显著相关，并显著降低存活率。该项目采用的策略是使用现有的化学探针来确定复杂T3SS机器的哪些组件对小分子化合物的抑制敏感。然后，将使用在探针靶标中携带突变的探针和菌株来定义那些易受攻击的成分在宿主-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、功能注释、可用药的靶点。已发表的四种化学结构无关的T3SS抑制剂在非细胞毒性浓度下对铜绿假单胞菌T3SS具有抑制作用，并被选为探针。其中两个探针对T3SS介导的效应性毒素的分泌和转位都有有效的抑制作用，而另外两个探针只抑制分泌或转位。因此，这些四种化学探针可能抑制至少三个不同的靶点或一个或多个MOE靶点内的不同区域。在目标1中，将使用两种平行的方法来确定这四个探针的分子靶标--(A)在探针上的允许位置添加光反应部分和分子手柄部分，应用光亲和探针修饰靶标(S)，回收和鉴定修饰的蛋白质；(B)应用分子遗传工具为探针富集物 逃逸突变体，对突变基因(S)进行深度测序鉴定。最后，将通过突变分析确认靶标的身份，并准备靶标基因突变体文库，以便于了解每个靶标在T3SS中的作用。在目标2中，探针和突变体文库将用于定义每个探针靶标在T3SS宿主-病原体相互作用中的作用。这些探针和突变体在调节、组装、分泌、转位和细胞毒性等广泛类别中对T3SS途径12个不同步骤的影响将被用来剖析靶标在T3SS机器和宿主-病原体相互作用中的作用。结果将为可用药的、与疾病相关的T3SS靶标提供特征逃逸突变，用于药物发现筛选。理想的靶标将被优先考虑为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角色。