Release of Extracellular DNA during Biofilm Formation in Staphylococcus aureus

金黄色葡萄球菌生物膜形成过程中细胞外 DNA 的释放

基本信息

批准号：
9905483
负责人：
Daniel Kahne
金额：
$ 63.02万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-11 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9905483
关键词：
Anabolism Antibiotic Resistance Antibiotics Bacteria Biogenesis Biological Assay Biosensor Catheters Cell Wall Cells Cellular Structures Communities Congo Red Cytolysis Cytoplasmic Protein DNA Devices Drops Drug Targeting Dyes Electrostatics Enzymes Extracellular Matrix Genes Genetic Transcription Genus staphylococcus Glucose Goals Growth Impairment Individual Infection Label Measures Mediating Microbial Biofilms Modeling Molecular Genetics Mucous Membrane Mutation Nosocomial Infections Nucleotides Pathway interactions Peptidoglycan Periodicity Pharmaceutical Preparations Polymers Polysaccharides Proteins Resistance Role Second Messenger Systems Skin Source Staphylococcus aureus Surface Teichoic Acids Vancomycin-resistant S. aureus Work adenine phosphoribosyltransferase base cell envelope chronic wound combat drug development drug discovery experimental study extracellular gene function genetic approach genome-wide implantable device implantation lipoteichoic acid macromolecule methicillin resistant Staphylococcus aureus mutant new therapeutic target pathogenic bacteria phosphoric diester hydrolase small molecule tool transposon sequencing

项目摘要

Staphylococcus aureus is often the cause of nosocomial infections, particularly when these infections involve bacterial colonization of indwelling devices. The fact that these bacteria are frequently carried asymptomatically on the skin and mucosal surfaces of many individuals permits easy colonization of the pristine surfaces of such devices during implantation. Once biofilms grow on these surfaces, they are difficult to eradicate because the constituent cells become largely insensitive to the action of many commonly used antibiotics. These findings, combined with the fact that a large percentage of S. aureus isolates identified in infections are now resistant to numerous antibiotics makes it clear that new ways of combating staphylococcal biofilm-associated infections need to be developed. The goal of this project is to understand how methicillin-resistant S. aureus (MRSA) forms biofilms and thereby identify targets for the discovery of drugs that block or reverse the formation of surface-associated communities. To form a biofilm, S. aureus must produce an extracellular matrix. Major components of the matrix are proteins and DNA. Many of the proteins are cytoplasmic in origin and are thus moonlighting in their second role as components of the matrix. The extracellular DNA requires the matrix proteins in order to adhere to the cells, serving as an electrostatic net that holds the remaining cells together in the biofilm. Using a comprehensive molecular genetic approach, we identified genes needed for the release of extracellular DNA during biofilm formation, including the gene for a phosphodiesterase that controls the levels of a well-known second messenger, cyclic-di-AMP. We have shown that cyclic-di-AMP levels drop when cells enter the biofilm state. We propose to take advantage of a biosensor for measuring cyclic-di-AMP levels in cells to identify genes that control its levels as cells enter the biofilm state. We will take advantage of the discovery that mutants with altered levels of the second messenger are resistant to the dye Congo red to discover additional genes involved in controlling cyclic-di-AMP levels. We propose that the drop in cyclic-di-AMP levels weakens the cell envelope, causing some cells to lyse and liberate protein and DNA for incorporation into the matrix. We will investigate the hypothesis that low cyclic-di-AMP levels impair proper biosynthesis of teichoic acid, the cell envelope polymers needed for cell wall integrity. Finally, we exploit our fluorescent assay for c-di-AMP to carry out a cell-based screen for small molecules that cause levels of the second messenger to rise. These hits will be a starting point for the development of drugs that block biofilm formation.

金黄色葡萄球菌通常是医院感染的原因，特别是当这些感染涉及留置装置的细菌定植。事实是这些细菌经常在许多人的皮肤和粘膜表面无症状地携带允许在植入过程中在此类装置的原始表面上轻松定殖。一次生物膜生长在这些表面上，它们很难根除，因为组成细胞对许多常用抗生素的作用基本上变得不敏感。这些发现，再加上感染中发现的金黄色葡萄球菌分离株中很大一部分是现在对多种抗生素具有抗药性，这清楚地表明对抗葡萄球菌的新方法需要发展生物膜相关感染。该项目的目标是了解耐甲氧西林金黄色葡萄球菌 (MRSA) 如何形成生物膜，从而确定目标发现阻止或逆转表面相关群落形成的药物。为了形成生物膜，金黄色葡萄球菌必须产生细胞外基质。主要组成部分基质是蛋白质和DNA。许多蛋白质起源于细胞质，因此兼职作为矩阵的组成部分的第二个角色。细胞外DNA需要基质蛋白以粘附在细胞上，作为静电网来容纳剩余的细胞聚集在生物膜中。使用综合分子遗传学方法，我们确定了生物膜形成过程中释放细胞外 DNA 所需的基因，包括控制众所周知的第二信使水平的磷酸二酯酶基因，环-二-AMP。我们已经证明，当细胞进入生物膜状态时，环二磷酸腺苷水平会下降。我们建议利用生物传感器来测量细胞中的环二AMP水平识别当细胞进入生物膜状态时控制其水平的基因。我们将利用发现第二信使水平发生改变的突变体对染料具有抗性刚果红发现参与控制环二 AMP 水平的其他基因。我们认为，环二磷酸腺苷水平的下降会削弱细胞膜，导致一些细胞裂解并释放蛋白质和 DNA，以便掺入基质中。我们将研究低环二磷酸腺苷水平损害磷壁酸正常生物合成的假设酸，细胞壁完整性所需的细胞包膜聚合物。最后，我们利用 c-di-AMP 的荧光测定来进行基于细胞的筛选导致第二信使水平升高的小分子。这些热门歌曲将成为一个开始开发阻止生物膜形成的药物的关键点。