Innate Immunity to S. aureus biofilm

对金黄色葡萄球菌生物膜的先天免疫

基本信息

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
项目类别：
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

项目摘要

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

4：对金黄色葡萄球菌生物膜PI的先天免疫反应：K.W。Bayles 具体目标生物膜是复杂基质中包含的细菌群落。来自临床角度，身体表面或医疗设备上的生物膜感染代表了严重的治疗挑战由于生物是对常规抗生素的顽固性。虽然宿主对浮游生物的免疫反应细菌的特征性很好（34、35、70、108、112），目前几乎没有信息有关宿主对金黄色葡萄球菌的免疫力以及生物如何调节抗细菌效应器机制在这个受保护的环境中组织时。迄今为止，大多数研究对先天性免疫的研究对生物膜的反应已经用铜绿假单胞菌和表皮链球菌（20、51、52、73、85、114），嗜中性粒细胞（PMN）已被证明可以吞噬与生物膜相关的细菌并产生氧化爆发，尽管与浮游细菌相比降低了水平。据我们所知，只有一个存在对白细胞对金黄色葡萄球菌生物膜的反应的公共属性（74）。这项研究证明白细胞能够渗入金黄色葡萄球菌生物膜。但是，作者利用了外周血白细胞的混合种群，因此，有关细胞类型的定义结论无法与生物膜互动。因此，需要其他机械研究来进步我们对金黄色葡萄球菌生物膜和宿主先天免疫之间的串扰的理解。该提议的假设是，金黄色葡萄球菌生物福利偏向于宿主先天免疫反应经典的促炎细菌表型，抗炎的促脑膜化反应细菌持久性。我们将重点关注关键细菌识别分子的功能重要性（TOLL¿ 像受体2（TLR2）和TLR9）以及抗微生物介质（INOS）一样 S.金黄色葡萄球菌使用外国体内感染模型（17，101），其小鼠特有的小鼠分子。我们还将研究纯化的PMN，巨噬细胞（MO）和树突状细胞（DC）对 Biofllm存活以及这些免疫细胞是否会通过转录来影响生物福利基因表达的变化分析。此外，我们将采用体内生物发光成像（IVI）监测免疫反应通过生物膜开发利用记者的小鼠菌株，以表达荧光素酶控制宿主免疫的启动子对革兰氏良好的细菌（即INOS，TLR2和NF-KB）的控制。最后，我们将检查宿主对金黄色葡萄球菌水解酶（CIDA和IRGAB）的免疫反应，核酸酶{nuc），和一氧化氮减少了来自小鼠异物感染中该PPG项目1和3的{NOR）突变体模型。为了解决这些目标，将执行以下特定目标。 1。将宿主的先天免疫反应与金黄色葡萄球菌生物膜与浮游感染相比。这些研究将1）定义吞噬细胞与金黄色葡萄球菌在体外的相互作用，当生物膜与浮游文化； 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谱分析暴露于先天免疫细胞的人群； 3）特征先天免疫浸入体内金黄色葡萄球菌生物膜及其活化谱； 4）检查的暂时激活 NF-KB是金黄色葡萄球菌生物膜期间的主要转录调节剂，用于广泛的炎症基因阵列使用IVI的增长。 2。定义细胞外细菌DNA（EDNA）和辣椒（PGN）在调节宿主中的作用对金黄色葡萄球菌的先天免疫。这些实验将1）评估宿主对金黄色葡萄球菌的响应使用小鼠的生物膜适合TLR9（细菌DNA的受体）和TLR2（PGN受体）； 2）Deflne the 使用TLR2-荧光酶报告基因小鼠和IVIS生物膜发育过程中TLR2 Activafion的动力学； 3）评估金黄色葡萄球菌（NUC）和murein水解酶（CIDA，IRGAB）突变体在中形成生物膜的能力 TLR9和TLR2 KO小鼠，分别分别。 3。检查导致金黄色葡萄球菌生物膜的纤维化封装的免疫力学。这些研究将1）调查金黄色葡萄球菌生物膜是否通过促进宿主纤维反应编程。 MO从经典（ML）转变为替代（M2）活化表型； 2）检查功能 iNOS在控制生物膜封装方面的重要性； 3）跟踪对金黄色葡萄球菌的炎症反应突变体设计为缺乏细菌涉及的酶无代谢{nor）和宿主衍生的作用在此过程中没有。 149