The Role of Nuclease in Biofilm Development and Disease

金黄色葡萄球菌正在成为我们社区面临的最具问题的细菌病原体， 医疗保健设置。一种有效的S.金黄色葡萄球菌在宿主中存活的方法是附着在表面上， 发展成一个被包围的细胞群落，称为生物膜。我们最近发现群体感应 可以控制一种营养或生物膜生活方式之间的平衡， 机制可能是一种有效的治疗策略。与Kenneth Bayles博士（PI）合作， 这个PPG），我们证明了S.金黄色葡萄球菌分泌的核酸酶（Nuc）引起整体 增厚的生物膜和抑制二级结构的形成，我们已经证实了最近的报告 那辆多金黄色葡萄球菌具有第二胞外核酸酶活性（Nuc 2）。根据这些发现，我们 中心假设是对细胞外核酸酶活性的控制是生物膜的关键决定因素 成熟与扩散为了解决这个问题，在具体目标1中，我们将（i）定义Nuc的作用， （ii）确定核酸酶活性对于生物膜分散是否重要;和（iii） 通过受控地暴露于核酸酶来调节生物膜完整性。我们进一步提出S.金黄色核酸酶 是重要的毒力因子。为了研究核酸酶在疾病中的作用，我们将与Dr。 Tammy Kielian（项目4负责人）和（i）检查核酸酶活性在中性粒细胞逃避中的作用 细胞外陷阱（NET）;（ii）定义核酸酶在小鼠模型中的意义， 生物膜感染;和（iii）比较宿主对促炎性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物可以作为抗真菌生物膜 治疗学为此，在具体目标3中，我们将采用新技术来生成环肽 图书馆在S。金黄色葡萄球菌，适合于高通量筛选方法。具体而言，我们将（i） 通过FACS筛选诱导核酸酶表达环肽;（ii）进行分子和 生物化学研究，以确定pepfide目标;（iii）表征最佳候选人作为分散剂， 生物膜感染模型;和（iv）将结果与具有增加的核酸酶活性的转座子突变体进行比较。 总的来说，这个项目的目标是了解这些S。金黄色葡萄球菌生物膜结构形成， 分解，细胞外DNA对这一过程的贡献，以及与疾病的相关性。 相关性（参见说明）：

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):