Lipid A & Innate Immune Receptors in Neisseria Infection

描述（由申请人提供）： 控制脑膜炎奈瑟菌和N.由于抗生素耐药性的增加和缺乏淋病奈瑟氏球菌的疫苗，淋病感染是世界范围内的一个主要公共卫生问题。脑膜炎血清群B和脑膜炎奈瑟氏菌。淋病我们的长期目标是确定奈瑟氏球菌致病性的分子决定因素，以开发新的治疗方法，允许快速评估细菌毒力，识别需要积极治疗的患者，并促进疫苗设计。在公认的奈瑟球菌毒力因子中，脂寡糖（LOS）是对生物体的促炎细胞因子应答的主要诱导物。我们的工作和其他人的工作已经表明，奈瑟球菌LOS的脂质A（LA）部分与先天免疫受体toll样受体4（TLR 4）接合，以启动信号级联，导致各种细胞类型（如单核细胞、嗜中性粒细胞和粘膜上皮细胞）表达细胞因子。TLR 4通路是保护宿主免受细菌感染的有效免疫应答所必需的，但其活化也可诱导导致败血性休克和死亡的促炎状态。我们已经表明，来自不同奈瑟球菌菌株的天然LOS在LA的酰化和磷酸化方面具有可变性，这与其在体外诱导脑膜炎球菌感染患者中发现的相同炎性细胞因子的效力相关。我们还证明了来自N.脑膜炎菌株89 I在人单核细胞中诱导TNF-1的能力不同。我们最近证实，奈瑟球菌LOS通过NF-：B和IFN调节因子3（IRF-3）转录因子激活MyD 88依赖性和TRIF依赖性途径，据我们所知，将是第一个报告NF-：B的直接激活与更多数量的磷酰基取代基正相关的。我们还发现，MIP-11和MIP-12在用含有具有最多磷酰基取代的LA的LOS处理的细胞中更高。这些数据支持我们的总体假设，即LA的炎症潜力随着磷酰基取代基数量的增加而增加，并且不同奈瑟球菌菌株LOS内LA结构的变化是先天免疫系统对奈瑟球菌感染应答程度的主要决定因素。基于TLR 4在LOS识别中的关键作用，很明显，LOS对TLR 4的不适当信号传导可能在奈瑟球菌感染期间产生重要后果，导致过度反应，如脑膜炎球菌败血症和淋球菌盆腔炎性疾病。因此，在本文提出的研究中，我们试图使用脑膜炎球菌菌株89 I的遗传缺失和可变表达突变体来定义确定炎症信号传导的LA结构元件，以产生具有磷酸化和/或酰化状态不同的单一不变LA部分的LOS。具体目标是：（1）确定在LA结构变异的背景下调节对奈瑟球菌LOS的TLR 4依赖性先天免疫应答的诱导的分子机制。我们将构建遗传变量表达LA突变体，并通过确定突变LOS通过TLR 4发出信号、成熟树突状细胞和CD 4 T细胞、诱导microRNA、结合TLR 4-MD-2复合物以及使用计算机模拟诱导与TLR 4-MD-2的分子相互作用差异的潜力，开发LA生物活性的预测模型。(2)确定LA结构和功能的预测模型与致病性和非致病性奈瑟氏球菌菌株表达的LA的生物活性和相对丰度之间的相关性。我们将相关的致病性和非致病性菌株的LA分子的表达，代表TLR 4诱导的分子决定因素与其潜在的诱导炎症细胞因子。我们的研究有望建立炎症信号传导的LA结构决定因素，确定开发治疗奈瑟球菌感染的抗生素的新靶点，使新方法能够识别高毒力和高炎症菌株，并有助于LOS疫苗设计。

DESCRIPTION (provided by applicant): Control of Neisseria meningitidis and N. gonorrhoeae infections represents a major public health problem around the world due to increased antibiotic resistance and the lack of vaccines for N. meningitidis serogroup B and N. gonorrhoeae. Our long-term goal is to define molecular determinants of Neisserial pathogenicity to enable development of new therapeutics, to permit rapid assessment of bacterial virulence, to identify patients requiring aggressive treatment, and to facilitate vaccine design. Among the recognized Neisserial virulence factors, lipooligosaccharide (LOS) is a major inducer of the proinflammatory cytokine response to the organisms. Our work and that of others has shown that the lipid A (LA) portion of Neisserial LOS engages innate immune receptor toll-like receptor 4 (TLR4) to initiate a signaling cascade leading to cytokine expression by various cell types such as monocytes, neutrophils, and mucosal epithelial cells. The TLR4 pathway is required for an efficient immune response that protects the host from bacterial infection, but its activation can also induce a proinflammatory state leading to septic shock and death. We have shown that native LOS from different Neisserial strains has variability in both acylation and phosphorylation of LA which is correlated with its potency to induce the same inflammatory cytokines in vitro that are found in patients with meningococcal infection. We also have demonstrated that specific phosphoryl and acyl variants of LA from N. meningitidis strain 89I have differential capacities to induce TNF-1 in human monocytes. We recently confirmed that Neisserial LOS activates both MyD88-dependent and TRIF-dependent pathways through NF-:B and IFN regulatory factor 3 (IRF-3) transcription factors, and to our knowledge, will be the first to report that direct activation of NF-:B is positively correlated with a greater number of phosphoryl substituents. We also have found that MIP-11 and MIP-12 were higher in cells treated with LOS containing LA that had the most phosphoryl substitutions. These data support our overall hypotheses that the inflammatory potential of LA increases with increasing number of phosphoryl substituents, and that variation in the LA structure within the LOS of different Neisserial strains is the major determinant of the degree to which the innate immune system responds to Neisserial infection. Based on the key role of TLR4 in the recognition of LOS, it is apparent that inappropriate signaling of TLR4 by LOS could have important consequences during Neisserial infections, leading to exaggerated responses such as meningococcal sepsis and gonococcal pelvic inflammatory disease. Thus, in the studies proposed herein, we seek to define the LA structural elements that determine inflammatory signaling using genetic deletion and variable expression mutants of meningococcal strain 89I to produce LOS with a single invariant LA moiety that differs in the state of phosphorylation and/or acylation. The Specific Aims are: (1) To determine the molecular mechanisms that regulate the induction of TLR4-dependent innate immune responses to Neisserial LOS in the context of LA structural variation. We will construct genetic variable expression LA mutants and develop a predictive model of LA bioactivity by determining the potential of the mutant LOS to signal through TLR4, to mature dendritic cells and polarize T cells, to induce microRNA, to bind to the TLR4-MD-2 complex, and to induce differences in molecular interactions with TLR4-MD-2 using in silico modeling. (2) To determine the correlation between the predictive model of LA structure and function with the bioactivity and relative abundance of LA expressed by disease-causing and non- pathogenic Neisserial strains. We will correlate the expression by disease-causing and non-pathogenic strains of LA molecules that represent molecular determinants for TLR4 induction with their potential to induce inflammatory cytokines. Our studies are expected to establish the LA structural determinants of inflammatory signaling, to identify new targets for development of antibiotics to treat Neisserial infections, to enable new methods to identify hypervirulent and highly inflammatory strains, and to aid in LOS vaccine design.