Quorum sensing-dependent interactions with biofilms and innate immunity defenses

描述(申请人提供)：金黄色葡萄球菌是社区和医院环境中最常见的急性和慢性感染原因之一。作为一种无害的共生菌，金黄色葡萄球菌在健康成年人群中定植了很大比例，主要分布在鼻道，但通常会转变为一种致命的病原体，传播并导致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，被认为在向侵袭性状态的转变中起着重要作用，但当金黄色葡萄球菌处于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。我们认为，群体感应控制着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到逃避宿主防御。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的激活使其能够从生物膜上脱离。此外，我们的初步结果发现，AGR调节子的上调先于脱离，其机制是由细胞外丝氨酸蛋白酶介导的。在这一应用中，我们建议定义生物膜分离机制。对于特定的目标1，我们将(I)进行微阵列实验，以确定在脱落之前上调和下调的基因；(Ii)鉴定介导生物膜脱落的丝氨酸蛋白酶；以及(Iii)鉴定已鉴定的脱落酶。在初步测试中，我们还获得了在人类呼吸道上皮细胞的气-液界面生长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从这些上皮细胞的附着和脱离。为了解决这个问题，在特定的目标2中，我们将(I)确定群体感应在呼吸道上皮细胞附着和生物膜形成中的作用；(Ii)确定上皮细胞的炎症反应；以及(Iii)调查从上皮细胞脱离。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逃逸。专业吞噬细胞，如多形核白细胞(PMN)是否存在类似的机制尚不清楚。我们假设群体感应激活将先于逃离PMN，我们已经进行了概念验证研究来开始解决这个问题。为此，在具体目标3中，我们将(I)研究PMN与群体感应活性和非活性菌株之间的相互作用；(Ii)研究群体感应金黄色葡萄球菌对PMN和IIA磷脂酶A2协同杀伤敏感性的影响；以及(Iii)分析PMN与已从呼吸道上皮细胞或逃逸的吞噬小体中脱落的金黄色葡萄球菌细胞的相互作用。在每个特定的目标中，我们还将比较实验室菌株和社区相关的甲氧西林耐药菌株(CA-MRSA)的转换机制，以深入了解群体感应对CA-MRSA特殊毒力的贡献。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。对这些转移机制的了解可能会导致创新的治疗方法，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内，没有发生任何事件，但当出现机会时，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到侵袭性病原体的转变的理解。

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.