Role of BdlA in biofilm dispersion and virulence properties of P. aeruginosa

描述（由申请人提供）：铜绿假单胞菌是与囊性纤维化（CF）肺部感染相关的主要病原体之一，在慢性和烧伤创面分离的最常见病原体中排名第二。一旦形成，铜绿假单胞菌生物膜很难通过抗菌治疗根除。虽然生物膜的临床意义已经确立，但对生物膜分散的机制知之甚少，这是生物膜发育的最后一步。我们假设生物膜的分散是通过调节c-diGMP水平、蛋白质相互作用和蛋白质磷酸化发生的。我们还假设生物膜分散改变了急性和慢性感染的铜绿假单胞菌的毒力。最近的研究结果表明，P. aeruginosa生物膜的分散与表型的转换和细胞内信号信使环二gmp （c- diGMP）的减少相一致。我们实验室的证据也表明，分散需要蛋白质磷酸化和合成。此外，在我们的实验室中，生物膜分散被至少7种蛋白质调节，包括BdlA、2种传感器蛋白质、营养诱导分散信号的潜在受体、参与相互调节c-diGMP水平的蛋白质和一个反应调节因子，我们认为这是一个不同于已知c-diGMP调节途径的新途径的一部分。我们的数据进一步表明，分散缺陷铜绿假单胞菌突变体毒性较弱。提出的研究的目的是表征参与调节生物膜分散的途径，并确定分散在急性和慢性感染中的作用。我们将使用特异性靶1体内和体外试验来确定哪些蛋白质具有c-diGMP调节活性，以及它们的酶活性是否依赖于BdlA。在特定目标2中，我们将在交联剂存在下使用V5- his融合和共免疫沉淀（“下拉”）试验来确定蛋白质-蛋白质相互作用是否在生物膜分散中发挥作用。蛋白质鉴定将与蛋白质相互作用的信息结合使用，以确定参与分散的直接、收敛或间接途径。在特定的Aim 3中，我们将使用生化、遗传和蛋白质组学方法确定磷酸化在c-diGMP酶活性的分散和调节中的作用。在Specific Aim 4中，我们将利用慢性和急性肺部感染模型，描述BdlA和其他三种蛋白在铜绿假单胞菌毒力和生物膜形成中的作用。该实验旨在确定铜绿假单胞菌突变体在生物膜分散受损是否在急性感染中毒性较低，但在慢性感染中毒性较强。评估将基于细菌计数、肺组织病理学和生物光子图像扫描，这允许细菌传播和生物膜形成的可视化。这项对生物膜分散过程的详细研究的结果有望导致基于抑制或调节生物膜分散来治疗和控制生物膜感染的更有效的治疗策略。公共卫生相关性：声明铜绿假单胞菌是医院获得性感染的常见原因，也是囊性纤维化患者死亡的主要原因。铜绿假单胞菌的特点之一是其对抗生素的高内在耐药性。因此，需要新的策略来对抗这种细菌。本研究旨在了解传感器蛋白BdlA在慢性和急性感染模型中铜绿假单胞菌毒力特性以及导致生物膜分散的途径中的作用。这项研究的结果有望产生基于抑制或调节参与生物膜分散的基因的创新和有效的治疗策略，以预防全身性感染和/或治疗和控制生物膜感染。

DESCRIPTION (provided by applicant): P. aeruginosa is one of the principal pathogens associated with Cystic fibrosis (CF) pulmonary infection and ranks 2nd among the most common pathogens isolated from chronic and burn wounds. Once established, P. aeruginosa biofilms are difficult to eradicate by antimicrobial treatment. While the clinical relevance of biofilms is well established, little is known about the mechanism of biofilm dispersion, the last step in biofilm development. We hypothesize that biofilm dispersion occurs via modulation of c-diGMP levels, protein-protein interactions, and protein phosphorylation. We also hypothesize that biofilm dispersion alters the virulence of P. aeruginosa in acute and chronic infections. Recent findings indicate that P. aeruginosa biofilm dispersion coincides with a switch in phenotype and a decrease in the intracellular signaling messenger cyclic-di-GMP (c- diGMP). Evidence from our lab also indicates that dispersion requires protein phosphorylation and synthesis. Furthermore, biofilm dispersion has been shown in our lab to be regulated by at least seven proteins including BdlA, 2 sensor proteins, potential receptors for nutrient-induced dispersion signals, proteins involved in reciprocally modulating c-diGMP levels, and a response regulator, that we believe form part of a novel pathway that is distinct from known c-diGMP-modulated pathways. Our data further indicate that dispersion-deficient P. aeruginosa mutants are less virulent. The goal of the proposed studies is to characterize the pathway involved in regulating biofilm dispersion, and to determine the role of dispersion in acute and chronic infections. We will use in Specific Aim 1 in vivo and in vitro assays to determine which of the proteins have c-diGMP modulating activity and whether their enzymatic activity is dependent on BdlA. In Specific aim 2, we will make use of V5- His-fusions and co-immunoprecipitation ("pull-down") assays in the presence of crosslinking agents to determine whether protein-protein interactions play a role in biofilm dispersion. Protein identifications will be used in combination with information on protein-protein interactions to determine direct, convergent or indirect pathways involved in dispersion. In specific Aim 3, we will determine the role of phosphorylation in dispersion and modulation of c-diGMP enzymatic activities using biochemical, genetic and proteomic approaches. In Specific Aim 4, we will characterize the role of BdlA and three other proteins in P. aeruginosa virulence and biofilm formation in vivo using chronic and acute lung infection models. The experiments are designed to determine whether P. aeruginosa mutants impaired in biofilm dispersion are less virulent in acute infections but more virulent in chronic infection. Assessments will be based on bacterial enumeration, lung histopathology, and biophotonic image scanning which allows for the visualization of bacterial dissemination and biofilm formation. Findings from this detailed investigation of the dispersion process are expected to lead to more effective treatment strategies based on inhibition or regulation of biofilm dispersion to treat and control biofilm infections. PUBLIC HEALTH RELEVANCE: Statement Pseudomonas aeruginosa is a common cause of hospital acquired infection and the leading cause of death in patients with cystic fibrosis. One of the hallmarks of P. aeruginosa is its high intrinsic resistance to antibiotics. New strategies are therefore needed to combat this bacterium. This proposal is aimed at understanding the role of the sensor protein BdlA in virulence properties of P. aeruginosa in chronic and acute infection models and in the pathway resulting in biofilm dispersion. Findings from this research are anticipated to result in innovative and effective treatment strategies based on inhibition or regulation of genes involved in biofilm dispersion to prevent systemic infections and/or to treat and control biofilm infections.