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

BdlA 在铜绿假单胞菌生物膜分散和毒力特性中的作用

• 批准号: 8115835
• 负责人: Karin Sauer
• 金额: $ 25.88万
• 依托单位: STATE UNIVERSITY OF NY,BINGHAMTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-08 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8115835
• 关键词: Acute; Amino Acid Sequence Homology; Antibiotic Resistance; Area; Bacteria; Biochemical Genetics; Biological Assay; Biophotonics; Burn injury; Cause of Death; Chronic; Co-Immunoprecipitations; Data; Development; Enzymes; Gene Expression Regulation; Goals; Health; Histopathology; Homologous Gene; Image; Imagery; Infection; Investigation; Lead; Lung; Microbial Biofilms; Modeling; Mus; Nosocomial Infections; Nutrient; Pathway interactions; Phenotype; Phosphorylation; Play; Post-Translational Protein Processing; Process; Property; Protein Biosynthesis; Proteins; Proteomics; Pseudomonas aeruginosa; Publishing; Pulmonary Cystic Fibrosis; Regulation; Research; Role; Scanning; Signal Transduction; Signaling Protein; Site; Systemic infection; Testing; Tissues; Virulence; Virulent; antimicrobial; base; clinically relevant; combat; crosslink; cystic fibrosis patients; design; diguanylate cyclase; effective therapy; in vitro Assay; in vivo; innovation; mutant; novel; pathogen; prevent; protein protein interaction; public health relevance; receptor; research study; response; sensor; treatment strategy

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.

简介(申请人提供)：铜绿假单胞菌是囊性纤维化(CF)肺部感染的主要病原体之一，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，就很难通过抗菌治疗来根除。虽然生物膜的临床相关性已得到很好的证实，但对生物膜发展的最后一步--生物膜分散的机制知之甚少。我们假设生物膜的分散是通过调节c-diGMP水平、蛋白质-蛋白质相互作用和蛋白质磷酸化发生的。我们还假设，在急性和慢性感染中，生物膜分散改变了铜绿假单胞菌的毒力。最近的研究结果表明，铜绿假单胞菌生物被膜的分散与表型的转换和细胞内信号信使c-diGMP(c-diGMP)的减少相一致。我们实验室的证据还表明，分散需要蛋白质的磷酸化和合成。此外，我们的实验室已经证明，生物膜的分散至少受7种蛋白质的调节，包括BdlA、2个传感器蛋白、营养诱导分散信号的潜在受体、参与相互调节c-diGMP水平的蛋白质以及反应调节因子，我们认为这些蛋白质构成了不同于已知的c-diGMP调节途径的新途径的一部分。我们的数据进一步表明，分散缺陷的铜绿假单胞菌突变株的毒力较低。拟议研究的目标是确定参与调节生物被膜扩散的途径，并确定扩散在急性和慢性感染中的作用。我们将在体内和体外实验中使用特定目标1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。在具体目标2中，我们将利用V5-His-融合和在交联剂存在下的免疫共沉淀(“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-蛋白质相互作用的信息结合使用，以确定参与分散的直接、汇聚或间接途径。在具体目标3中，我们将使用生化、遗传学和蛋白质组学方法来确定磷酸化在c-diGMP酶活性分散和调节中的作用。在特定的目标4中，我们将利用慢性和急性肺部感染模型研究BdlA和其他三种蛋白在体内铜绿假单胞菌毒力和生物被膜形成中的作用。这些实验旨在确定在生物被膜分散中受损的铜绿假单胞菌突变株在急性感染中的毒力是否较低，但在慢性感染中是否更强。评估将基于细菌计数、肺组织病理学和生物光子图像扫描，从而实现细菌传播和生物膜形成的可视化。对分散过程的这一详细调查结果预计将导致基于抑制或调节生物膜分散的更有效的治疗策略，以治疗和控制生物膜感染。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对抗生素具有高度的内在耐药性。因此，需要新的战略来对抗这种细菌。本研究旨在了解感应蛋白BdlA在铜绿假单胞菌在慢性和急性感染模型中的毒力特性中的作用，以及在导致生物被膜扩散的途径中的作用。这项研究的结果预计将导致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，以预防系统性感染和/或治疗和控制生物膜感染。