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: 7907757
• 负责人: Karin Sauer
• 金额: $ 30.17万
• 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/7907757
• 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; 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; 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.

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，在与慢性伤口和烧伤的最常见病原体中排名第二。一旦建立，铜绿假单胞菌生物膜就难以通过抗菌治疗消除。虽然生物膜的临床相关性已经很好地确定，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。我们假设生物膜分散剂是通过调节C-二酸水平，蛋白质 - 蛋白质相互作用和蛋白质磷酸化发生的。我们还假设生物膜分散体改变了铜绿假单胞菌在急性和慢性感染中的毒力。最近的发现表明，铜绿假单胞菌生物膜分散剂与表型的开关相吻合，细胞内信号传导Messenger Cyclic-Di-GMP（C- DIGMP）的减少。我们实验室的证据还表明，分散需要蛋白质磷酸化和合成。此外，在我们的实验室中已显示生物膜分散剂至少受到七种蛋白质的调节，包括BDLA，2种传感器蛋白，养分诱导的分散信号的潜在受体，与互相调节C-DIGMP水平相互调节的蛋白质以及一个新颖的路径的一部分是与c-Dig不同的一部分。我们的数据进一步表明，缺乏分散性铜绿假单胞菌突变体的毒力较少。拟议研究的目的是表征调节生物膜分散剂的途径，并确定分散体在急性和慢性感染中的作用。我们将在体内和体外测定中使用特定的目标1来确定哪些蛋白质具有C-DIGMP调节活性，以及​​它们的酶活性是否取决于BDLA。在特定的目标2中，我们将利用V5-His fusions和共免疫沉淀（“下拉”）测定法，在存在交联剂的情况下，以确定蛋白质 - 蛋白质相互作用是否在生物膜分散体中起作用。蛋白质鉴定将与有关蛋白质 - 蛋白质相互作用的信息结合使用，以确定涉及分散体的直接，收敛或间接途径。在特定的目标3中，我们将使用生化，遗传和蛋白质组学方法来确定磷酸化在分散和调节中的作用。在特定的目标4中，我们将使用慢性和急性肺部感染模型在体内表征BDLA和其他三种蛋白质在体内的铜绿假单胞菌毒力和生物膜形成中的作用。该实验旨在确定生物膜分散剂受损的铜绿假单胞菌突变体是否在急性感染中毒性较小，但在慢性感染中更具毒性。评估将基于细菌枚举，肺部组织病理学和生物图像扫描，该扫描允许可视化细菌传播和生物膜形成。对分散过程的详细研究的结果有望根据抑制或调节生物膜分散剂来治疗和控制生物膜感染。公共卫生相关性：铜绿假单胞菌是囊性纤维化患者的医院后感染和死亡的主要原因的常见原因。铜绿假单胞菌的标志之一是其对抗生素的固有耐药性。因此，需要新的策略来对抗这种细菌。该建议旨在理解传感器蛋白BDLA在铜绿假单胞菌在慢性和急性感染模型中以及导致生物膜分散的途径中的作用。预计这项研究的结果将基于对生物膜分散涉及的基因的抑制或调节，以防止全身感染和/或治疗和控制生物膜感染。