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

验证第 14 页中的目标。

• 批准号: 8283415
• 负责人: Donald T Moir
• 金额: $ 86.29万
• 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/8283415
• 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; Screening procedure; Site; Structure; Toxin; Urinary tract infection; Ventilator; Virulence; affinity labeling; attributable mortality; cytotoxic; cytotoxicity; drug discovery; experience; inhibitor/antagonist; multidisciplinary; mutant; pathogen; pathogenic bacteria; protective effect; resistant strain; 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. 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.

描述（由申请人提供）：铜绿假单胞菌是呼吸机相关肺炎（VAP）的主要原因，并且当前的抗生素治疗策略也显示出高达18％的衰竭率，即使生物体对抗生素的敏感也易于给予抗生素。该项目的目的是通过验证III型分泌（T3SS）途径中的靶标，这些靶标易于小分子抑制，并确定其在T3SS宿主 - 病原体相互作用中的作用。 T3SS是有助于建立和传播感染（例如VAP，菌血症，尿路感染）的主要铜绿假单胞菌毒力决定因素。它被细菌用于分泌和转运毒素效应子到宿主的吞噬细胞中，从而削弱了宿主的先天免疫防御能力。功能性T3SS的存在与患者的临床结局和死亡差明显相关，并且在动物感染模型中显着降低了生存率。该项目中采用的策略是使用现有的化学探针来确定复杂T3SS机器的哪些组件容易受到小分子化合物抑制的影响。然后，将使用探针靶标中携带突变的探针和菌株来定义这些脆弱组件在宿主 - 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验证的，功能化注释的，可药的靶标。已经选择了四个具有无关化学结构的T3S抑制剂，这些抑制剂以非周期毒性浓度抑制对铜绿假单胞菌T3SS的抑制作用已被选为探针。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，而其他两个探针仅抑制分泌或易位。因此，这些FOU化学探针可能会抑制一个或MOE目标内的至少三个不同的目标或不同的区域。在AIM 1中，将使用两种平行方法来识别这四个探针的分子靶标 - （a）在探针上添加光反应性和分子手柄部分，并在探针上添加光接附属探针以修改靶标的，以修改靶标的，恢复和鉴定改良的蛋白质； （b）应用分子遗传工具以丰富探针 逃生突变体，然后通过深度测序鉴定突变基因。最后，目标身份将通过突变分析确认，目标基因突变库将准备促进了解每个目标在T3S中的作用。在AIM 2中，将使用探针和突变库来定义每个探针靶标在T3SS宿主 - 病原体相互作用中的作用。探针和突变体对在调节，组装，分泌，易位和细胞毒性的广泛类别中T3SS途径中十二个不同步骤的影响，将用于剖析目标在T3SS机器中和宿主 - 托管相互作用中的作用。结果将提供可药物疾病的T3SS目标，其特征性的逃生突变体用于药物发现筛查。理想目标将优先考虑探针对快速抑制，并参与宿主 - 病原体相互作用的关键作用。 公共卫生相关性：细菌病原体的抗生素抗生素菌株的增加代表了未满足的医疗需求。三型分泌是许多致病细菌使用的机制，以增加其在人类感染中的毒力。通过这种方法分泌的细菌毒素降低了受感染者自身先天免疫系统的保护作用。该项目的结果将提供有关易于受到药物攻击的第三型分泌仪的身份和功能的详细信息，从而有助于新药发现。