The Role of Nuclease in Biofilm Development and Disease

核酸酶在生物膜发育和疾病中的作用

• 批准号: 8292121
• 负责人: KENNETH W. BAYLES
• 金额: $ 42.61万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8292121
• 关键词: Acquired Immunodeficiency Syndrome; Acute Disease; Address; Antibiotics; Applications Grants; Back; Biochemical; Cells; Chromatin; Chronic Disease; Collaborations; Communities; Community Healthcare; Cyclic Peptides; Cytoplasmic Granules; DNA; Development; Disease; Engineering; Equilibrium; Exposure to; Extracellular Structure; Foreign Bodies; Genome; Goals; Immune; Infection; Inflammatory Response; Instruction; Investigation; Laboratories; Life; Life Style; Link; Methods; Microbe; Microbial Biofilms; Modeling; Molecular; Mus; Pathogenesis; Peptide Library; Peptides; Process; Proteins; Reporting; Resistance; Role; Signal Transduction; Staphylococcus aureus; Streptococcus; Structure; Surface; Technology; Therapeutic; Virulence Factors; Work; base; chemotherapy; combat; extracellular; high throughput screening; intein; interest; killings; methicillin resistant Staphylococcus aureus; mouse model; mutant; neutrophil; new technology; novel strategies; nuclease; pathogen; quorum sensing; research study; small molecule; tool

Staphylococcus aureus is emerging as the most problemafic bacterial pathogen facing our community and healthcare settings. An effective strategy for S. aureus to survive in the host is to attach to a surface and develop into an encased community of cells called a biofilm. We recenfiy discovered that quorum-sensing can control the balance between a planktonic or biofilm lifestyle, suggesfing that modulafion of this dispersal mechanism could be an effective therapeutic strategy. In collaboration with Dr. Kenneth Bayles (the PI of this PPG), we demonstrated that a deletion of the S. aureus secreted nuclease (Nuc) caused an overall thickening of the biofilm and inhibited secondary structure formation, and we have confirmed a recent report that S. aureus possesses a second extracellular nuclease activity (Nuc2). Based on these findings, our central hypothesis is that control over extracellular nuclease activity is a critical determinant of biofilm maturafion and dispersal. To address this quesfion, in Specific Aim 1 we will (i) define the role of Nuc and Nuc2 in biofilm maturation; (ii) determine whether nuclease activity is important for biofilm dispersal; and (iii) modulate biofilm integrity with controlled exposure to nuclease. We further propose that S. aureus nuclease is an important virulence factor. To investigate the nuclease funcfion in disease, we will work with Dr. Tammy Kielian (Project 4 leader) and (i) examine the role of nuclease acfivity in evasion of neutrophil extracellular traps (NETs); (ii) define the significance of nuclease in mouse models of planktonic versus biofilm infecfion; and (iii) compare the host inflammatory response to nuclease in planktonic versus biofilm infection. Finally, we speculate that small-molecule inducers of nuclease activity could serve as anfi-biofilm therapeufics. Towards this end, in Specific Aim 3, we will employ new technology to generate cyclic peptide libraries in S. aureus that are amenable to high-throughput screening methods. More specifically, we will (i) screen for cyclic pepfides that induce nuclease expression through FACS; (ii) perform molecular and biochemical studies to identify pepfide targets; (iii) characterize the best candidates as dispersal agents in a biofilm infection model; and (iv) compare results to transposon mutants with increased nuclease activity. Overall, the goal of this Project is to understand how these S. aureus biofilm structures form and disassemble, the contribufion of extracellular DNA to this process, and the relevance in disease. RELEVANCE (See instructions):

金黄色葡萄球菌正在成为我们社区面临的最有问题的细菌病原体和 医疗保健设置。金黄色葡萄球菌在宿主中生存的有效策略是附着在表面上， 发展成一个称为生物膜的包含的细胞社区。我们恢复了群体感应 可以控制浮游生物或生物膜生活方式之间的平衡，并建议这种分散 机制可能是一种有效的治疗策略。与肯尼斯·贝尔斯（Kenneth Bayles）博士合作（ 这个ppg），我们证明了金黄色葡萄球菌分泌的核酸酶（NUC）的缺失导致了总体 生物膜增厚并抑制二级结构形成，我们已经确认了最近的报告 金黄色葡萄球菌具有第二个细胞外核酸酶活性（NUC2）。基于这些发现，我们的 中心假设是控制细胞外核酸酶活性是生物膜的关键决定因素 成熟和分散。为了解决此问题，在特定目标1中，我们将（i）定义nuc和 生物膜成熟的NUC2； （ii）确定核酸酶活性对于生物膜扩散是否重要； （iii） 调节生物膜完整性，以受控的核酸酶暴露。我们进一步提出了金黄色葡萄球菌核酸链蛋白酶 是重要的毒力因素。为了调查疾病的核酸酶功能，我们将与Dr. 塔米·基里安（Tammy Kielian）（项目4领导者）和（i）检查核酸酶囊性在逃避中性粒细胞中的作用 细胞外陷阱（网）； （ii）定义了核酸酶在浮游物与浮游生物模型中的重要性 生物膜的infimefion; （iii）比较宿主炎症反应对浮游生物与生物膜中核酸酶的炎症反应 感染。最后，我们推测核酸酶活性的小分子诱导剂可以用作Anfi-Biofilm Therapeufics。为此，在特定目标3中，我们将采用新技术来生成环状肽 金黄色葡萄球菌中的库可以适合高通量筛选方法。更具体地说，我们将（i） 筛选循环膜，可通过FACS诱导核酸酶表达； （ii）执行分子和 生化研究以鉴定偏爱靶标； （iii）将最佳候选人描述为一个分散剂 生物膜感染模型； （iv）将结果与核酸酶活性增加的转座子突变体进行比较。 总体而言，该项目的目标是了解这些金黄色葡萄球菌生物膜结构如何形成和 拆卸，细胞外DNA对这一过程的贡献以及与疾病的相关性。 相关性（请参阅说明）：