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

细菌生存的关键是对外部压力的适当协调和反应。比如说 包膜应激反应（ESR）允许细菌修复和防御细胞包膜损伤，这是 通常在抗生素暴露期间持续。然而，ESR的过度激活在各种微生物中是有毒的， 这表明ESR可能被操纵以杀死细菌。为了利用这种脆弱性，细菌细胞如何 克服这种毒性和调节ESR过度激活需要被理解。前期工作未完成 热休克辅助分子伴侣DnaJ调节铜绿假单胞菌中sE调节的ESR。的 本申请的目的是揭示这种调节的机制并表征其程度。虽然 DnaJ，与DnaK和GrpE复合，通过降解热休克反应，抑制热休克反应。 作为替代σ因子，初步数据表明DnaJ调节铜绿假单胞菌sE的活性， AlgU通过不同的机制。总体假设是DnaJ不调节AlgU 活性通过已知ESR调节剂的蛋白质水平的变化，而不是DnaJ调节AlgU活性， DnaJ通过直接结合到这种σ因子上而影响ESR，而DnaJ对依赖于sE的ESR的这种作用可能是 在革兰氏阴性菌中是保守的。这一假设将通过三个具体目标进行检验。在目标1中， DnaJ对AlgU依赖性ESR调节因子的基因表达和蛋白水平的影响将通过以下方法确定： 在ESR活化条件下的RT-qPCR和蛋白质印迹。在目标2中，DnaJ结合配偶体，影响 将确定AlgU依赖性ESR。这个目标将检查DnaJ结构域对正确的AlgU是重要的。 如果DnaJ与AlgU结合，并且如果DnaJ对ESR的作用需要DnaK，则DnaJ- 结合伴侣，这是重要的，其功能在热休克反应。在目标3中，DnaJ依赖性 将在另外两个高度遗传易处理的革兰氏阴性细胞中检查sE调节的ESR的激活。 细菌，大肠杆菌和霍乱弧菌，以检查这种机制是否可能是保守的跨越 γ-变形菌这些目标的结果预期将定义DnaJ对以下方面的机制作用： （目标1-2），并探讨这一机制的潜在普遍性（目标3）。此外，这项工作 将增加我们了解铜绿假单胞菌中AlgU毒性的潜在机制的长期目标， 如果要开发靶向sE依赖性ESR的治疗剂，这是重要的。这些成果和 它们的潜在应用预计将对日益严重的多药滥用问题产生积极影响， 耐药性感染此外，由于DnaJ已被证明会影响多种应激反应系统， 对于ESR，该提议推测DnaJ可能是跨细菌的通用应力协调中心， 强调其在整体细菌应激反应中的重要性。最后，本文的工作将有助于研究 通过维持具有强大培训历史的PI计划，实现少数民族服务机构的卓越 来自边缘化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.