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

细菌生存的关键是对外界压力的适当协调和反应。例如， 包膜应激反应(ESR)允许细菌修复和防御细胞包膜破坏，这是 通常在抗生素暴露期间持续存在。然而，ESR的过度激活对各种微生物都是有毒的， 这表明血沉可能被操纵以杀灭细菌。为了利用这个漏洞，细菌细胞是如何 克服这种毒性并调节ESR过度激活需要被理解。初步工作已揭开 热休克辅助伴侣DNAJ调节铜绿假单胞菌中Se调节的ESR。这个 这项应用的目的是揭示这一调节的机制并表征其程度。虽然 在DNAK和GRPE的复合体中，DNAJ通过降解热休克反应来抑制这种反应 替代Sigma因子，初步数据表明Dna J调节铜绿假单胞菌的活性 通过一种不同的机制同源阿尔古。最重要的假设是DNAJ不调节ALGU 通过改变已知的ESR调节器的蛋白质水平来调节活性，而不是DNAJ调节阿尔古活性和 ESR通过直接与这个Sigma因子结合，Dna J在Se依赖的ESR上的这种作用可能是 在革兰氏阴性细菌中保守。这一假设将通过三个具体目标进行检验。在目标1中， DNAJ对阿尔古依赖ESR调节剂基因表达和蛋白水平的影响将通过以下方式确定 ESR激活条件下的RT-qPCR和Western Blot。在AIM 2中，DNAJ结合了影响 将识别依赖于阿尔古的ESR。这个目标将检查哪个DNAJ结构域对于正确的算法是重要的- 依赖的ESR激活，如果DNAJ与ALGU结合，并且如果DNAJ对ESR的影响需要DNAK，则DNAJ- 结合伙伴，对其在热休克反应中的作用非常重要。在目标3中，依赖于DNAJ 硒调节的ESR的激活将在另外两个高度遗传易感性的革兰氏阴性菌中进行检测 细菌，大肠杆菌和霍乱弧菌，以检查这一机制是否在 变形革兰氏菌。这些目标的结果预计将定义DNAJ对 ESR(目标1-2)和这一机制的潜在普遍性(目标3)。此外，这项工作 这将增加我们理解铜绿假单胞菌阿尔古毒素潜在机制的长期目标(S)， 如果要开发针对Se依赖的ESR的治疗方法，这一点很重要。这些结果和 它们的潜在应用预计将对日益严重的多药问题产生积极影响。 抵抗力感染。此外，由于DNAJ已被证明影响多个应激反应系统，此外 根据ESR，这一建议推测DNAJ可能是跨细菌的通用压力协调中心， 强调其在整体细菌应激反应中的重要性。最后，本文的工作将对研究工作有所裨益。 通过支持具有良好培训历史的PI计划来卓越地服务于少数族裔服务机构 来自边缘群体的学生研究人员。

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.