The Role of Sigma Factor B in Staphylococcus aureus biofilm development

描述（由申请人提供）：金黄色葡萄球菌是社区和医院环境中急性和慢性感染的最常见原因之一。金黄色葡萄球菌粘附宿主组织或医疗植入物并发展成生物膜的能力是慢性感染的关键决定因素。生物膜面临的挑战是它对宿主防御和抗菌治疗的非凡抵抗力。我们最近证明了金黄色葡萄球菌的细胞间通讯系统，也被称为群体感应或agr系统，控制着浮游和生物膜状态之间的切换。我们推测，其他调控途径也参与了这一控制机制。我们已经开始筛选水手转座子文库，并发现在rsbUVW-sigB位点上的插入消除了金黄色葡萄球菌形成生物膜的能力。表型是由于缺乏Sigma B （SigB）活性，并扩展到社区相关的耐甲氧西林金黄色葡萄球菌（CA-MRSA）菌株。我们的中心假设是细胞外酶水平的增加阻碍了SigB缺陷菌株的生物膜形成。对于Specific Aim 1，我们将(i)定义SigB缺陷菌株的细胞外蛋白质组；（ii）鉴定鉴定的蛋白在SigB生物膜表型中的作用；（iii）研究SigB在生物膜扩散中的作用。我们之前证明了agr表达的增加具有抗生物膜特性，并且从我们的研究和已发表的报告中可以明显看出，SigB系统中的缺陷上调agr表达。为了支持这一发现，我们发现agr系统的突变可以恢复sigb缺陷菌株的生物膜形成。我们假设SigB系统控制着一种影响agr的未知蛋白的表达。为了解决这些调控级联的相互联系，在Specific Aim 2中，我们将(i)确定ArlRS双组分系统是否可能是未知蛋白；（ii）采用蛋白质组学方法鉴定与agr启动子结合的蛋白质；（iii）鉴定鉴定的蛋白质。在这两个特定的目标中，我们将在CA-MRSA菌株中重复实验，以深入了解SigB对这些分离株的异常毒性的贡献。总之，拟议的实验将确定SigB在生物膜形成中的作用以及与agr群体感应系统的互连。公共卫生相关性：金黄色葡萄球菌是慢性细菌性疾病的主要原因。我们的初步研究表明，甲氧西林敏感和耐甲氧西林金黄色葡萄球菌（MRSA）的Sigma B调控级联对于生物膜的形成至关重要，这是已知的慢性感染的决定因素。对西格玛B在生物膜形成中的作用的进一步了解有助于开发消除金黄色葡萄球菌生物膜的创新治疗方案。

DESCRIPTION (provided by applicant): Staphylococcus aureus is one of the most common causes of acute and chronic infections in both community and hospital settings. The ability of S. aureus to adhere to host tissues or medical implants and develop into biofilms is a critical determinant of chronic infections. The challenge presented by a biofilm is its extraordinary resistance to both host defenses and antimicrobial therapies. We recently demonstrated that the S. aureus cell-to-cell communication system, also called quorum-sensing or the agr system, controls the switch between the planktonic and biofilm state. We speculate that other regulatory pathways feed into this control mechanism. We have begun screening a mariner transposon library and discovered that insertions in the rsbUVW-sigB locus eliminated the ability of S aureus to form a biofilm. The phenotype was due to lack of Sigma B (SigB) activity and extended to community-associated methicillin-resistant S. aureus (CA-MRSA) strains. Our central hypothesis is that an increased level of extracellular enzymes blocks biofilm formation in SigB defective strains. For Specific Aim 1, we will (i) define the extracellular proteome of SigB defective strains; (ii) characterize the role of identified protein(s) in the SigB biofilm phenotype; and (iii) investigate the role of SigB in biofilm dispersal. We previously demonstrated that increased agr expression has anti-biofilm properties, and it is evident from our studies and published reports that defects in the SigB system upregulate agr expression. In support of this finding, we discovered that mutations in the agr system restore biofilm formation in SigB-defective strains. We hypothesize that the SigB system is controlling the expression of an unknown protein that affects agr. To address the interconnection of these regulatory cascades, in Specific Aim 2, we will (i) determine whether the ArlRS two-component system could be the unknown protein; (ii) employ a proteomic approach to identify proteins bound to the agr promoter; and (iii) characterize the identified protein(s). In both specific aims, we will repeat experiments in a CA-MRSA strain to gain insight on the contribution of SigB to the exceptionally virulent nature of these isolates. Altogether, the proposed experiments will define the role of SigB in biofilm formation and the interconnection with the agr quorum-sensing system. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus is a leading cause of chronic bacterial disease. Our preliminary studies have demonstrated that the Sigma B regulatory cascade in methicillin-susceptible and methicillin-resistant S. aureus (MRSA) is essential for biofilm formation, a known determinant of chronic infections. An improved understanding of role of Sigma B in biofilm formation could aid the development of innovative treatment options for eliminating S. aureus biofilms.