Role of SagS signaling and regulatory events in biofilm formation and tolerance

 描述（由申请方提供）：铜绿假单胞菌在从手术部位、慢性和烧伤伤口分离的最常见人类病原体中排名第二。铜绿假单胞菌也是与囊性纤维化（CF）肺部感染相关的主要病原体之一，并导致这些患者的健康下降和预后不良。一旦建立，铜绿假单胞菌在生物膜中的生长使得很难根除生物体 通过抗菌处理。为了根除铜绿假单胞菌生物膜感染，应将努力集中在导致形成持久性和固有抗性生物膜的发育过程上。来自我们实验室的发现表明，双组分杂合SagS是铜绿假单胞菌生物膜形成和生物膜细胞转变为高度抗微生物剂抗性状态的关键调节剂。发现SagS通过两种独立的机制对这两种生物膜特异性调节回路做出贡献。生物膜的形成需要SagS和TCS BfiSR之间的分级的基于磷酸转移的信号传导，而生物膜耐受性被发现依赖于BrIR，但独立于磷酸转移、生物量积累、生物膜结构和生物膜成熟的后期阶段，从而表明SagS是两种生物膜特异性调节回路分叉的调节剂。然而，SagS如何有助于激活这两个不同的发展过程还没有很好的理解。该项目的目标是阐明SagS信号和调节事件有助于生物膜形成和生物膜细胞过渡到抗微生物剂耐受状态。该项目基于以下假设：SagS的HmsP结构域中存在的保守氨基酸（AA）残基有助于SagS促进生物膜特异性调节回路，从而实现生物膜发育和/或生物膜耐受性，并且通过丙氨酸取代干扰或阻断HmsP的感觉功能将导致受损的生物膜发育和/或生物膜耐受性。实验上，我们将在目标1中产生在位于HmsP（SagS的周质感觉结构域）中的保守AA残基中具有丙氨酸取代的SagS变体。随后在Aim 2中通过qRT-PCR分析表达所得SagS-HmsP突变构建体的SagS突变生物膜的brlR表达。然后使用抗生素敏感性和生物膜-MBC测定法分析显示改变或降低的brlR表达的突变生物膜的生物膜耐受性。负责SagS促进生物膜形成的残基将在目的3中通过分析突变细胞的附着、生物膜形成和SagS-HmsP突变与TCS BfiSR的相互作用来鉴定。这项详细研究的结果将有助于更完整地定义SagS激活生物膜发育和生物膜耐受性的机制以及如何操纵SagS功能。

 DESCRIPTION (provided by applicant): Pseudomonas aeruginosa ranks second among the most common human pathogens isolated from surgical sites, chronic and burn wounds. P. aeruginosa is also one of the principal pathogens associated with Cystic fibrosis (CF) pulmonary infection and responsible for a decline in health and poor prognosis for these patients. Once established, growth of P. aeruginosa in biofilms makes it very difficult to eradicate the organisms by antimicrobial treatment. In order to eradicate P. aeruginosa biofilm infections, efforts should be focused on the developmental process leading to the formation of persistent and inherently resistant biofilms. Findings from our laboratory suggest that the two-component hybrid SagS is a key regulator of P. aeruginosa biofilm formation and biofilm cells transitioning to a highly antimicrobial resistant state. SagS was found to contribute to these two biofilm-specific regulatory circuits via two independent mechanisms. Biofilm formation required the hierarchical phosphotransfer-based signaling between SagS and the TCS BfiSR while biofilm tolerance was found to be dependent on BrlR, but independent of phosphotransfer, biomass accumulation, biofilm architecture, and the later stages of biofilm maturation, thus indicating SagS to be the regulator at which the two biofilm-specific regulatory circuits diverge. However, how SagS contributes to the activation of the two distinct developmental processes is not well understood. The goal of this project is to elucidate SagS signaling and regulatory events contributing to biofilm formation and biofilm cells transitioning to an antimicrobial tolerant state. The project i founded on the hypotheses that conserved amino acid (AA) residues present in the HmsP domain of SagS contribute to SagS promoting biofilm-specific regulatory circuits enabling biofilm development and/or biofilm tolerance and that interfering with or blocking the sensory function(s) of HmsP, via alanine substitution, will result in impaired biofilm development and/or biofilm tolerance. Experimentally, we will generate in Aim 1 SagS variants harboring alanine substitutions in conserved AA residues located in HmsP, the periplasmic sensory domain of SagS. ¿sagS mutant biofilms expressing the resulting SagS-HmsPmutated constructs will be subsequently analyzed in Aim 2 by qRT-PCR for brlR expression. Mutant biofilms demonstrating altered or reduced brlR expression will then be analyzed for biofilm tolerance using antibiotic susceptibility and biofilm-MBC assays. Residues responsible for SagS contributing to biofilm development will be identified in Aim 3 by analyzing mutant cells for attachment, biofilm formation, and interactions of SagS- HmsPmutated with the TCS BfiSR. Findings from this detailed investigation will help to more completely define the mechanism by which SagS activates biofilm development and biofilm tolerance and how to manipulate SagS function(s).