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

4：对S.金黄色葡萄球菌生物膜PI：Bayles，K.W. 具体目标 生物膜是包含在复杂基质中的粘附细菌群落。从临床 从另一个角度来看，体表或医疗器械上的生物膜感染代表了严重的治疗挑战 因为生物体对常规抗生素是不敏感的。尽管宿主对抗肿瘤药物的免疫反应 细菌的特征相对较好（34，35，70，108，112），但目前可获得的信息很少 关于宿主对S.金黄色葡萄球菌生物膜和生物体如何调节抗菌效应机制 在这种保护性的环境下，到目前为止，大多数研究都是关于先天免疫的， 已经用铜绿假单胞菌和S.表皮（20，51，52，73，85，114）， 其中嗜中性粒细胞（PMN）已显示吞噬生物膜相关细菌并产生氧化性 爆发，尽管与嗜热细菌相比水平降低。据我们所知，只有一个 白细胞对S.金黄色葡萄球菌生物膜已被直接检查（74）。本研究 表明白细胞能够浸润S.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 不能与生物膜相互作用。因此，需要进行额外的机制研究， 我们对S.金黄色葡萄球菌生物膜和宿主先天免疫。 这一建议的假设是S。金黄色葡萄球菌生物膜使宿主的先天免疫反应从 经典的促炎杀菌表型朝向抗炎、促纤维化反应， 细菌持久性。我们将重点关注关键细菌趋化分子的功能重要性（Toll？ 类受体2（TLR2）和TLR9）以及抗微生物介质（INOS）的发病机制进行了研究。 S.金黄色葡萄球菌生物膜使用异物感染模型（17，101），小鼠缺乏这些各种 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 生物膜存活以及这些免疫细胞是否通过转录调节诱导生物膜基因表达的变化 侧写此外，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，利用经工程改造以在生物膜下表达荧光素酶的报告小鼠品系， 控制宿主对革兰氏阳性细菌的免疫中关键的启动子（即iNOS、TLR 2和NF-κ B）。最后， 我们将检测宿主对S.金黄色葡萄球菌胞壁蛋白水解酶（nuc），核酸酶（nuc）， 和一氧化氮还原酶（nor）突变体，来自本PPG的项目1和3，在小鼠异物感染中 模型为了实现这些目标，将实现以下具体目标。 1.比较宿主对S.金黄色葡萄球菌生物膜与嗜酸性感染。这些 研究将1）确定吞噬细胞和S.当细菌在体外生长时， 生物膜与渗透压培养; 2）监测S.金黄色葡萄球菌生物膜 使用转录谱分析暴露于先天性免疫细胞群体; 3）表征先天性免疫细胞群体; 渗入S.金黄色葡萄球菌生物膜在体内和它们的活化概况;和4）检查 NF-κ B是一系列炎症基因的主要转录调节因子，在S.金黄色葡萄球菌生物膜 使用IVIS进行生长。 2.确定胞外细菌DNA（eDNA）和肽聚糖（PGN）在调节宿主 对S.金黄色生物膜这些实验将1）评估宿主对S.金黄色 使用TLR9（细菌DNA受体）和TLR2（PGN受体）缺陷小鼠的生物膜; 2）定义 使用TLR2-荧光素酶报告小鼠和IVIS在生物膜发育期间TLR2活化的动力学;和3） 评价S.金黄色葡萄球菌核酸酶（nuc）和胞壁蛋白水解酶（murein hydrolase，IrgAB）突变体，以在 TLR9和TLR2 KO小鼠。 3.研究导致S.金黄色葡萄球菌生物膜。这些 研究将1）调查S.金黄色葡萄球菌生物膜通过促进 MO从经典（M1）到交替（M2）活化表型的转变; 2）检查MO的功能性 INOS在调节生物膜包裹中的重要性;和3）追踪对S.金黄色 突变体被工程化以缺乏参与细菌NO代谢的酶（nor）和宿主来源的 在这个过程中没有。 149