Innate Immunity to S. aureus biofilm

对金黄色葡萄球菌生物膜的先天免疫

• 批准号: 7750241
• 负责人: Tammy L Kielian
• 金额: $ 36.31万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7750241
• 关键词: Address; Anti-Bacterial Agents; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotics; Bacteria; Bacterial DNA; Bioluminescence; Body Surface; Cells; Clinical; Communities; Complex; Dendritic Cells; Development; Disease; Engineering; Enzymes; Exposure to; Foreign Bodies; Gene Expression; Genes; Growth; Image; Immune; Immune response; Immunity; In Vitro; Infection; Inflammatory; Inflammatory Response; Knowledge; Leukocytes; Luciferases; Mediator of activation protein; Medical Device; Metabolism; Microbial Biofilms; Modeling; Monitor; Mouse Strains; Mus; N-Acetylmuramoyl-L-alanine Amidase; Natural Immunity; Organism; Pathogenesis; Peptidoglycan; Phagocytes; Phenotype; Population; Process; Pseudomonas aeruginosa; Reporter; Respiratory Burst; Role; Staphylococcus aureus; TLR9 gene; Toll-Like Receptor 2; antimicrobial; bactericide; cell type; extracellular; human NOS2A protein; in vivo; macrophage; mutant; neutrophil; nitric oxide reductase; nuclease; peptidoglycan receptor; peripheral blood; programs; promoter; research study; response

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

图 4：对金黄色葡萄球菌生物膜的先天免疫反应 PI：Bayles，K.W. 具体目标 生物膜是临床上复杂基质中附着的细菌群落。 立场，身体表面或医疗设备上的生物膜感染是一个严峻的治疗挑战 尽管宿主对浮游生物有免疫反应，但生物体对传统抗生素有抵抗力。 细菌的特征相对较好（34、35、70、108、112），目前可用的信息很少 关于宿主对金黄色葡萄球菌生物膜的免疫以及生物体如何调节抗菌效应机制 迄今为止，大多数研究都是在这种保护性环境中进行的。 已对铜绿假单胞菌和表皮葡萄球菌进行了生物膜反应 (20, 51, 52, 73, 85, 114)， 其中中性粒细胞 (PMN) 已被证明可以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，只有一种。 已有出版物直接检查了白细胞对金黄色葡萄球菌生物膜的反应 (74)。 证明白细胞能够渗透金黄色葡萄球菌生物膜；然而，作者利用了 外周血白细胞的混合群体，因此，关于细胞类型的明确结论 因此，需要进行额外的机制研究来推进。 我们对金黄色葡萄球菌生物膜和宿主先天免疫之间相互作用的理解。 该提议的假设是金黄色葡萄球菌生物膜使宿主先天免疫反应偏离了 经典的促炎杀菌表型转向抗炎、促纤维化反应 我们将重点关注关键细菌识别分子的功能重要性（Toll¿ 类似受体 2 (TLR2) 和 TLR9) 以及抗微生物介质 (INOS)，通过研究其发病机制 使用异物感染模型 (17, 101) 的金黄色葡萄球菌生物膜，小鼠缺乏这些不同的 我们还将研究纯化的 PMN、巨噬细胞 (MO) 和树突状细胞 (DC) 对细胞的影响。 biofllm 存活以及这些免疫细胞是否通过转录诱导 biofllm 基因表达的变化 此外，我们将采用体内生物发光成像（IVIS）来监测免疫反应。 在整个生物膜发育过程中，利用经工程改造可表达荧光素酶的报告小鼠品系 启动子的控制对于宿主对革兰氏阳性细菌的免疫至关重要（即 iNOS、TLR2 和 NF-KB）。 我们将检查宿主对金黄色葡萄球菌胞壁蛋白水解酶（cidA 和 IrgAB）、核酸酶（nuc）、 和一氧化氮还原酶（nor）突变体来自该PPG项目1和3在小鼠异物感染中的作用 为了实现这些目标，将实现以下具体目标。 1. 比较宿主对金黄色葡萄球菌生物膜和浮游感染的先天免疫反应。 研究将 1) 定义当细菌在体外生长时吞噬细胞和金黄色葡萄球菌之间的相互作用 生物膜与浮游培养物的比较；2) 监测金黄色葡萄球菌生物膜中基因表达的变化 使用转录分析暴露于先天免疫细胞群 3) 表征先天免疫； 渗透到体内金黄色葡萄球菌生物膜及其激活曲线；4) 检查 NF-KB，金黄色葡萄球菌生物膜期间多种炎症基因的主要转录调节因子 使用 IVIS 进行增长。 2. 定义细胞外细菌DNA（eDNA）和肽聚糖（PGN）在调节宿主中的作用 对金黄色葡萄球菌生物膜的先天免疫这些实验将 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）缺陷的小鼠的生物膜定义了 使用 TLR2-荧光素酶报告小鼠和 IVIS 进行生物膜形成过程中 TLR2 激活的动力学；以及 3) 评估金黄色葡萄球菌核酸酶 (nuc) 和胞壁质水解酶 (cidA, IrgAB) 突变体在中形成生物膜的能力 分别是 TLR9 和 TLR2 KO 小鼠。 3. 检查导致金黄色葡萄球菌生物膜纤维化包膜的免疫机制。 研究将 1) 研究金黄色葡萄球菌生物膜是否通过促进宿主纤维化反应 MO从经典(M1)激活表型到替代(M2)激活表型的转变2)检查功能； INOS 在调节生物膜封装中的重要性；3) 追踪金黄色葡萄球菌的炎症反应 突变体被设计为缺乏参与细菌 NO 代谢（nor）的酶以及宿主来源的作用 在此过程中没有。 149