Innate Immunity to S. aureus biofilm

4：对金黄色葡萄球菌生物膜的先天免疫反应 具体目标 生物膜是包含在复杂基质中的细菌附着群落。从一家诊所 从观点来看，人体表面或医疗器械上的生物被膜感染是一个严重的治疗挑战 因为生物体对常规抗生素是顽固的。尽管宿主对浮游生物的免疫反应 细菌已经被很好地描述了(34，35，70,108,112)，目前几乎没有信息 关于宿主对金黄色葡萄球菌生物被膜的免疫以及生物如何调节抗菌效应机制 当在这种保护性的环境中组织时。到目前为止，大多数研究都涉及先天免疫 铜绿假单胞菌和表皮葡萄球菌对生物膜的反应(20，51，52，73，85,114)， 中性粒细胞(PMN)吞噬生物膜相关细菌并产生氧化 爆发，尽管与浮游细菌相比水平较低。据我们所知，只有一个 在白细胞对金黄色葡萄球菌生物被膜的反应已被直接检测的情况下，存在公共问题(74)。本研究 证明了白细胞能够渗透金黄色葡萄球菌生物膜；然而，作者利用一种 外周血白细胞的混合群体，并由此确定了关于细胞类型的五个结论(S) 不能与生物膜相互作用。因此，需要更多的机制研究来推进 我们对金黄色葡萄球菌生物膜和宿主先天免疫之间的相互作用的理解。 这一建议的假设是金黄色葡萄球菌生物膜扭曲了宿主的先天免疫反应 经典的促炎杀菌表型对有利的抗炎、促凝血反应 细菌的持久性。我们将重点介绍关键细菌识别分子(Toll？)的功能重要性 如受体2(TLR2和TLR9)以及抗微生物介体(INOS) 使用异物感染模型(17,101)的小鼠缺乏这些不同种类的金黄色葡萄球菌生物被膜 分子。我们还将研究纯化的PMN、巨噬细胞(MO)和树突状细胞(DC)对 生物膜存活和这些免疫细胞是否通过转录诱导生物膜基因表达的变化 侧写。此外，我们将使用体内生物发光成像(IVIS)来监测免疫反应 在整个生物膜的开发过程中，利用经工程设计表达荧光素酶的报告鼠品系 启动子的控制在宿主对革兰氏阳性菌的免疫中起关键作用(即iNOS、TLR2和NF-KB)。最后， 我们将检测宿主对金黄色葡萄球菌毛霉素水解酶(CIDA和IrgAB)、核酸酶(Nuc)、 和来自该PPG项目1和3的一氧化氮还原酶{NOR)突变体在小鼠异物感染中的作用 模特。为实现这些目标，将实现以下具体目标。 1.比较宿主对金黄色葡萄球菌生物被膜和浮游生物感染的天然免疫反应。这些 研究将1)明确吞噬细胞和金黄色葡萄球菌在体外的相互作用，当细菌在 生物膜与浮游培养的比较；2)监测金黄色葡萄球菌生物膜中基因表达的变化 利用转录谱分析暴露于天然免疫细胞群体；3)表征天然免疫 体内金黄色葡萄球菌生物膜的渗入及其激活情况；以及4)检测 金黄色葡萄球菌生物被膜过程中广泛炎症基因的主要转录调控因子--核因子-KB 使用静脉注射成形器进行生长。 2.明确细胞外细菌DNA(EDNA)和肽聚糖(PGN)在调节宿主中的作用 对金黄色葡萄球菌的天然免疫力。这些实验将1)评估宿主对金黄色葡萄球菌的反应 使用TLR9(细菌DNA受体)和TLR2(PGN受体)的小鼠生物膜；2)引导 利用TLR2-荧光素酶报告小鼠和IVIS研究生物膜发育过程中TLR2激活的动力学； 金黄色葡萄球菌核酸酶(Nuc)和毛霉素水解酶(CIDA，IrgAB)突变体在体内形成生物膜的能力 TLR9和TLR2 KO小鼠。 3.检测导致金黄色葡萄球菌生物膜纤维化包膜的免疫机制。这些 研究将1)调查金黄色葡萄球菌生物膜是否通过促进 MO从经典的(M1)表型向交替的(M2)激活表型的转变；2)检查功能 INOS在调节生物被膜包裹中的重要性；以及3)追踪金黄色葡萄球菌的炎症反应 缺乏参与细菌NO代谢的酶和宿主衍生的作用的突变体 在这个过程中没有。 149

4: Innate Immune Response to S. aureus Biofilm PI: Bayles, K.W. SPECIFIC AIMS Biofilms are adherent communities of bacteria contained within a complex matrix. From a clinical standpoint, biofilm infections on body surfaces or medical devices represent a serious therapeufic challenge since organisms are recalcitrant to conventional antibiotics. Although host immune responses to planktonic bacteria have been relafively well characterized (34, 35, 70, 108, 112), little information is currently available regarding host immunity to S. aureus biofilms and how organisms modulate anti-bacterial effector mechanisms when organized in this protective milieu. To date, the majority of studies invesfigafing the innate immune response to biofilms have been performed with P. aeruginosa and S. epidermidis (20, 51, 52, 73, 85, 114), where neutrophils (PMNs) have been shown to phagocytize biofilm-associated bacteria and produce oxidative bursts, albeit at reduced levels compared to planktonic bacteria. To the best of our knowledge, only one publicafion exists where leukocyte responses to S. aureus biofilm have been direcfiy examined (74). This study demonstrated that leukocytes are capable of infiltrating S. aureus biofilms; however, the authors utilized a mixed population of peripheral blood leukocytes and as such, definifive conclusions regarding the cell type(s) interacting with the biofilm cannot be made. Therefore, additional mechanistic studies are needed to advance our understanding of the cross-talk between S. aureus biofilm and host innate immunity. The hypothesis ot this proposal is that S. aureus biofllm skews the host innate immune response from a classical pro-inflammatory bactericidal phenotype towards an anti-inflammatory, pro-flbrotic response to favor bacterial persistence. We will focus on the funcfional importance of key bacterial recognifion molecules (Toll¿ like receptor 2 (TLR2) and TLR9) as well as anti-microbial mediators (INOS) by studying the pathogenesis of S. aureus biofilm using a foreign body infection model (17, 101) with mice deficient for these various molecules. We will also investigate the effects of purified PMN, macrophages (MO), and dendritic cells (DC) on biofllm survival and whether these immune cells induce changes in biofllm gene expression by transcriptional profiling. In addifion, we will employ in vivo bioluminescence imaging (IVIS) to monitor the immune response throughout biofilm development utilizing reporter mouse strains engineered to express luciferase under the control of promoters pivotal in host immunity to gram-posifive bacteria (i.e. iNOS, TLR2, and NF-KB). Finally, we will examine the host immune response to S. aureus murein hydrolase {cidA and IrgAB), nuclease {nuc), and nitric oxide reductase {nor) mutants from Projects 1 and 3 of this PPG in the mouse foreign body infection model. To address these objectives, the following specific aims will be performed. 1. Compare host innate Immune responses to S. aureus biofilm versus planktonic infection. These studies will 1) define the interactions between phagocytes and S. aureus in vitro when bacteria are grown in biofilm versus planktonic culture; 2) monitor changes in gene expression in S. aureus biofilms following exposure to innate immune cell populafions using transcripfional profiling; 3) characterize innate immune infiltrates into S. aureus biofilm in vivo and their activation profiles; and 4) examine the temporal activation of NF-KB, a major transcriptional regulator for a wide-array of inflammatory genes, during S. aureus biofilm growth using IVIS. 2. Define the role of extracellular bacterial DNA (eDNA) and peptidoglycan (PGN) in modulating host innate immunity to S. aureus biofllm. These experiments will 1) evaluate the host response to S. aureus biofilms using mice deflcient for TLR9 (receptor for bacterial DNA) and TLR2 (receptor for PGN); 2) deflne the kinefics of TLR2 activafion during biofilm development using TLR2-luciferase reporter mice and IVIS; and 3) evaluate the ability of S. aureus nuclease {nuc) and murein hydrolase {cidA, IrgAB) mutants to form biofilms in TLR9 and TLR2 KO mice, respecfively. 3. Examine the immune mechanisms leading to fibrotic encapsulation of S. aureus biofilms. These studies will 1) investigate whether S. aureus biofilm programs a host fibrofic response by facilitating the transition of MO from a classically (Ml) to an alternatively (M2) activated phenotype; 2) examine the funcfional importance of INOS in regulating biofilm encapsulation; and 3) track the inflammatory response to a S. aureus mutant engineered to lack an enzyme involved in bacterial NO metabolism {nor) and the role of host-derived NO in this process. 149