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

 描述(申请人提供)：铜绿假单胞菌在从手术部位、慢性创面和烧伤创面分离的最常见的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，并导致这些患者健康状况下降和预后不良。一旦铜绿假单胞菌在生物膜中生长，就很难根除这种微生物。 通过抗菌治疗。为了根除铜绿假单胞菌生物被膜感染，应将重点放在导致形成持久性和内在耐药生物被膜的发育过程上。我们实验室的研究结果表明，双组分杂交凹陷是铜绿假单胞菌生物膜形成和生物膜细胞向高度抗菌素耐药状态过渡的关键调节因子。SAGS被发现通过两个独立的机制对这两个生物膜特异的调节电路做出贡献。生物膜的形成需要SAGS和TCS BfiSR之间基于磷转移的分级信号，而生物膜的耐受性取决于BrlR，但与磷转移、生物量积累、生物膜构筑和生物膜成熟的后期阶段无关，因此表明SAGS是两个生物膜特异调控电路分歧的调节器。然而，SAGS如何促进这两个不同的发育过程的激活还不是很清楚。该项目的目标是阐明SAGS信号和调节事件有助于生物膜的形成和生物膜细胞过渡到抗菌素耐受状态。我的项目建立在这样的假设之上，即存在于SAG的HmsP结构域中的保守氨基酸(AA)残基有助于SAG促进生物膜特异的调节电路，使生物膜发育和/或生物膜耐受性得以实现，并且通过丙氨酸替代干扰或阻断HmsP的感觉功能(S)，将导致生物膜发育和/或生物膜耐受性受损。在实验上，我们将在目标1中产生SAGS变体，该变体在位于SAGS的周质感受域HmsP中的保守AA残基中含有丙氨酸取代。表达所得到的SAGS-HmsP突变构建体的SAGS突变生物膜随后将在AIM 2中通过qRT-PCR分析brlR的表达。表现为brlR表达改变或降低的突变生物膜将使用抗生素敏感性和生物膜-MBC分析来分析生物膜的耐受性。在目标3中，将通过分析突变细胞的附着、生物膜形成以及突变的SAGS-HmsP与TCS BfiSR的相互作用来确定导致生物膜发育的SAGS的残基。这一详细调查的结果将有助于更全面地确定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(S)。

 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).