Exploring envelope stress response toxicity and regulation in gram-negative bacteria

探索革兰氏阴性菌的包膜应激反应毒性和调节

• 批准号: 10629505
• 负责人: Boo Shan Tseng
• 金额: $ 14.68万
• 依托单位: UNIVERSITY OF NEVADA LAS VEGAS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10629505
• 关键词: Address; Affect; Antibiotics; Bacteria; Base Pairing; Binding; Cells; Client; Codon Nucleotides; Complex; Data; Development; Escherichia coli; Future; Gammaproteobacteria; Gene Expression; Gene Proteins; Goals; Gram-Negative Bacteria; Health; Heat shock proteins; Heat-Shock Response; Homologous Gene; Infection; Knowledge; Literature; Methods; Microbe; Minority-Serving Institution; Mission; Modeling; Molecular Chaperones; Multi-Drug Resistance; Organism; Outcome; Predisposition; Production; Program Sustainability; Proteins; Pseudomonas aeruginosa; Public Health; Publishing; Recording of previous events; Regulation; Repression; Research; Research Personnel; Role; Sigma Factor; Stress; Suppressor Mutations; System; Testing; Toxic effect; United States National Institutes of Health; Vibrio cholerae; Western Blotting; Work; bactericide; biological adaptation to stress; cell envelope; genome sequencing; improved; infection burden; marginalized population; mutant; pathogen; repaired; response; student training; therapeutic target; whole genome

Critical to bacterial survival is the proper coordination and response to external stress. For example, the envelope stress response (ESR) allows bacteria to repair and defend against cell envelope damage, which is often sustained during antibiotic exposure. However, overactivation of the ESR is toxic in various microbes, suggesting that the ESR may be manipulated to kill bacteria. To exploit this vulnerability, how bacterial cells overcome this toxicity and regulate ESR overactivation needs to be understood. Preliminary work uncovered that the heat shock co-chaperone DnaJ regulates the sE-regulated ESR in Pseudomonas aeruginosa. The objective of this application is to uncover the mechanism of this regulation and characterize its extent. Although DnaJ, in complex with DnaK and GrpE, represses the heat shock response via degradation of this response’s alternative sigma factor, preliminary data suggest that DnaJ regulates the activity of the P. aeruginosa sE homolog AlgU via a different mechanism. The overarching hypothesis is that DnaJ does not regulate AlgU activity via changes in protein levels of known ESR regulators, that instead DnaJ regulates AlgU activity and the ESR via direct binding to this sigma factor, and that this role of DnaJ on the sE-dependent ESR may be conserved across gram-negative bacteria. This hypothesis will be tested via three specific aims. In Aim 1, the effect of DnaJ on gene expression and protein levels of AlgU-dependent ESR regulators will be determined via RT-qPCR and Western Blot under conditions of ESR activation. In Aim 2, DnaJ binding partners that affect the AlgU-dependent ESR will be identified. This Aim will examine which DnaJ domain is important for proper AlgU- dependent ESR activation, if DnaJ binds to AlgU, and if the effect of DnaJ on the ESR requires DnaK, a DnaJ- binding partner that is important for its functions in the heat shock response. In Aim 3, DnaJ-dependent activation of the sE-regulated ESR will be examined in two other, highly genetically tractable gram-negative bacteria, Escherichia coli and Vibrio cholerae, to examine if this mechanism is potentially conserved across Gammaproteobacteria. The outcomes of these Aims are expected to define the mechanistic effect of DnaJ on the ESR (Aims 1-2) and address the potential universality of this mechanism (Aim 3). Furthermore, this work will add to our long-term goal of understanding the mechanism(s) underlying AlgU toxicity in P. aeruginosa, which is important if therapeutics targeting the sE-dependent ESR are to be developed. These outcomes and their potential applications are expected to have a positive impact on the growing problem of multidrug- resistant infections. In addition, as DnaJ has been shown to affect multiple stress response systems in addition to the ESR, this proposal speculates that DnaJ may be a universal stress coordination hub across bacteria, emphasizing its importance in overall bacterial stress response. Finally, this work will benefit the research excellence of a minority-serving institution by sustaining the program of a PI with a strong history of training student researchers from marginalized groups.

对细菌的生存至关重要的是适当的协调和对外部压力的反应。例如， 信封应力反应（ESR）允许细菌修复和防御细胞信封损伤，即 在抗生素暴露期间经常维持。但是，ESR的过度激活在各种微生物中有毒， 表明可以操纵ESR以杀死细菌。为了利用这种脆弱性，细菌细胞如何 克服这种毒性并调节ESR过度活化需要了解。初步工作被发现 热休克伴侣DNAJ调节铜绿假单胞菌中的SE调节ESR。这 该应用的目的是揭示该调节的机制并表征其程度。虽然 DNAJ与DNAK和GRPE复杂，反映了通过此响应的降解的热冲击响应 替代Sigma因子，初步数据表明DNAJ调节铜绿假单胞菌的活性 同源物通过不同的机制。总体假设是DNAJ不调节Algu 通过已知ESR调节剂的蛋白质水平的变化而进行活性，而DNAJ调节了Algu活性和 ESR通过直接结合与该Sigma因子，并且DNAJ在SE依赖性ESR中的这种作用可能为 跨革兰氏阴性细菌保守。该假设将通过三个特定目的进行检验。在AIM 1中 DNAJ对ALGU依赖性ESR调节剂的基因表达和蛋白质水平的影响将通过 在ESR激活条件下，RT-QPCR和Western印迹。在AIM 2中，DNAJ约束伙伴影响了 将确定依赖ALGU的ESR。该目标将检查哪个DNAJ域对于适当的algu-很重要 依赖性ESR激活，如果DNAJ与ALGU结合，并且DNAJ对ESR的影响需要DNAK，则DNAJ- 结合伙伴对于其在热冲击响应中的功能很重要。在AIM 3中，DNAJ依赖性 将以另外两个高度一致的革兰氏阴性剂量检查SECHENCHED ESR的激活 细菌，大肠杆菌和弧菌霍乱，以检查这种机制是否有可能保守 伽马杆菌。这些目标的结果有望定义DNAJ的机械效应 ESR（目标1-2）并解决了该机制的潜在宇宙（AIM 3）。此外，这项工作 将增加我们理解铜绿假单胞菌中基础ALGU毒性的机制的长期目标， 如果要开发针对SE依赖性ESR的治疗，这一点很重要。这些结果和 预计他们的潜在应用将对多饮用的日益增长的问题产生积极影响 抗性感染。另外，由于DNAJ已被证明会影响多个应力响应系统。 对于ESR，该提案推测DNAJ可能是跨细菌的通用应力协调枢纽， 强调其在整体细菌应激反应中的重要性。最后，这项工作将使研究受益 通过维持PI计划具有悠久培训历史，少数派服务机构的卓越 边缘化群体的学生研究人员。