Quorum sensing-dependent interactions with biofilms and innate immunity defenses

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。作为一种无害的共生菌，金黄色葡萄球菌在很大比例的健康成年人中定植，主要在鼻腔通道中，但经常转变为毒性病原体，传播并引起严重和毁灭性的疾病。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，被认为在切换到侵入状态中很重要，但是当金黄色葡萄球菌在生物膜或中性粒细胞内时，这种转变是如何发生的尚不清楚。我们提出群体感应控制了一种普遍的扩散机制，允许金黄色葡萄球菌从生物膜状态过渡并逃避宿主防御。为了支持这一假设，我们开发了一种新的方法来合成群体感应信号，并发现Agr激活可以使生物膜脱离。此外，我们的初步结果揭示了Agr调控子的上调先于分离，其机制是由细胞外丝氨酸蛋白酶介导的。在这个应用中，我们建议定义生物膜脱离机制。对于Specific Aim 1，我们将(i)进行微阵列实验，以鉴定分离前上调和下调的基因；（ii）鉴定介导生物膜脱离的丝氨酸蛋白酶；(3)鉴定分离酶的性质。在初步试验中，我们还获得了在人气道上皮细胞的气液界面培养金黄色葡萄球菌的专业知识，我们假设群体感应激活将调节这些上皮细胞的附着和脱离。为了解决这个问题，在专项目标2中，我们将(i)定义群体感应在气道上皮附着和生物膜形成中的作用；（ii）确定上皮炎症反应；（iii）研究上皮细胞的脱离。最后，已知群体感应的激活先于金黄色葡萄球菌从非吞噬细胞中逃逸。是否类似的机制发生在专业吞噬细胞，如多形核白细胞（PMN），尚不清楚。我们假设群体感应激活将先于PMN的逃逸，并且我们已经进行了概念验证研究来开始解决这个问题。为此，在Specific Aim 3中，我们将(i)检查PMN与群体感应活性菌株和非活性菌株之间的相互作用；（ii）研究金黄色葡萄球菌群体感应对PMN和IIA组磷脂酶A2协同杀伤敏感性的影响；（iii）分析PMN与从气道上皮分离或逃离吞噬体的金黄色葡萄球菌细胞的相互作用。在每个特定目标中，我们还将比较实验室菌株与社区相关的耐甲氧西林菌株（CA-MRSA）的过渡机制，以便深入了解群体感应对CA-MRSA异常毒力的贡献。总之，这些研究的结果将扩大我们对金黄色葡萄球菌从殖民者转变为入侵者的机制的理解。了解这些过渡机制可能会导致创新疗法，阻止这种致命的侵袭性病原体的传播。公共卫生相关性：金黄色葡萄球菌通常生活在人类鼻道中而不会发生意外，然而，当出现机会时，这种病原体是社区和医院环境中细菌感染的最常见原因之一。我们的研究将促进对金黄色葡萄球菌如何使用细胞间通信系统来控制从各种生活方式状态转变为侵袭性病原体的理解。

DESCRIPTION (provided by applicant): Staphylococcus aureus is one of the most common causes of acute and chronic infections in both community and hospital settings. As an innocuous commensal, S. aureus colonizes a large percentage of the healthy adult population, predominantly in the nasal passages, but often transitions into a virulent pathogen, disseminating and causing severe and devastating disease. The S. aureus cell-to-cell communication system, also called quorum-sensing or the Agr system, is thought to be important in the switch to an invasive state, but how this transition occurs when S. aureus is in a biofilm or inside a neutrophil is not clear. We propose that quorum-sensing controls a universal dispersal mechanism allowing S. aureus to transition out of a biofilm state and evade host defenses. In support of this hypothesis, we developed a novel method to synthesize the quorum-sensing signal and found that Agr activation enables detachment from biofilms. Further, our preliminary results have uncovered that up- regulation of the Agr regulon precedes detachment and that the mechanism is mediated by extracellular serine proteases. In this application, we propose to define the biofilm detachment mechanism. For Specific Aim 1, we will (i) perform microarray experiments to identify genes up and down-regulated prior to detachment; (ii) identify the serine proteases mediating biofilm detachment; and (iii) characterize the identified detachment enzymes. In preliminary tests, we have also gained expertise in growing S. aureus at the air-liquid interface of human airway epithelial cells, and we hypothesize that quorum-sensing activation will modulate attachment and detachment from these epithelial cells. To address this question, in Specific Aim 2, we will (i) define the role of quorum- sensing in attachment and biofilm formation on airway epithelia; (ii) determine the epithelia inflammatory responses; and (iii) investigate detachment from epithelial cells. Finally, activation of quorum-sensing is known to precede S. aureus escape from non-phagocytic cells. Whether a similar mechanism occurs with professional phagocytes, such as polymorphonuclear leukocytes (PMN), is not clear. We hypothesize that quorum-sensing activation will precede escape from PMN, and we have performed proof-of-concept studies to begin addressing this question. Towards this end, in Specific Aim 3, we will (i) examine interactions between PMN and quorum-sensing active and inactive strains; (ii) investigate the impact of S. aureus quorum-sensing in susceptibility to synergistic killing by PMN and group IIA phospholipase A2; and (iii) analyze PMN interactions with S. aureus cells that have detached from airway epithelia or escaped phagosomes. In each specific aim, we will also compare the transition mechanisms of laboratory strains with the community-associated methicillin resistant strains (CA-MRSA), in order to gain insight into the contribution of quorum-sensing to the exceptional virulence of CA-MRSA. Altogether, the results of these studies will expand our understanding of the S. aureus mechanisms used to transition from a colonizer into an invader. Knowledge of these transition mechanisms could lead to innovative therapies that block the spread of this virulent, invasive pathogen. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus normally lives in the human nasal passages without incident, and yet when presented with an opportunity, this pathogen is one of the most common causes of bacterial infections in both community and hospital settings. Our studies will advance the understanding of how S. aureus uses a cell-to-cell communication system to control the transition from a variety of lifestyle states into an invasive pathogen.