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: 7750239
• 负责人: Tammy L Kielian
• 金额: $ 42.33万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7750239
• 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 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并 发育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 可以控制浮游或生物膜生活方式之间的平衡，表明这种分散的调节 机制可能是一种有效的治疗策略。与 Kenneth Bayles 博士（PI 这个PPG），我们证明金黄色葡萄球菌分泌核酸酶（Nuc）的缺失导致了整体 生物膜增厚并抑制二级结构形成，我们已经证实了最近的一份报告 金黄色葡萄球菌具有第二种细胞外核酸酶活性（Nuc2）。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 成熟和扩散。为了解决这个问题，在具体目标 1 中，我们将 (i) 定义 Nuc 的角色和 Nuc2 参与生物膜成熟； (ii) 确定核酸酶活性对于生物膜分散是否重要； (三) 通过控制核酸酶的暴露来调节生物膜的完整性。我们进一步提出金黄色葡萄球菌核酸酶 是重要的毒力因子。为了研究疾病中的核酸酶功能，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) 定义核酸酶在浮游小鼠模型与 生物膜感染； (iii) 比较浮游生物与生物膜中宿主对核酸酶的炎症反应 感染。最后，我们推测核酸酶活性的小分子诱导剂可以作为抗生物膜 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 金黄色葡萄球菌中适合高通量筛选方法的文库。更具体地说，我们将 (i) 通过FACS筛选诱导核酸酶表达的环肽； (ii) 进行分子和 确定肽目标的生化研究； (iii) 将最佳候选者描述为分散剂 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 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）：