Lipid A & Innate Immune Receptors in Neisseria Infection

描述(由申请人提供)： 脑膜炎奈瑟菌和淋病奈瑟菌感染的控制是世界各地的一个主要公共卫生问题，原因是抗生素耐药性增加以及B群脑膜炎奈瑟菌和淋病奈瑟菌疫苗的缺乏。我们的长期目标是定义NeisSeries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。在已知的NeisSeries毒力因子中，脂低聚糖(LOS)是对生物体的促炎细胞因子反应的主要诱导者。我们和其他人的工作表明，NeisSerial LOS的脂质A(LA)部分与先天免疫受体Toll样受体4(TLR4)结合，启动信号级联反应，导致单核细胞、中性粒细胞和粘膜上皮细胞等多种细胞类型表达细胞因子。TLR4途径是有效的免疫反应所必需的，以保护宿主免受细菌感染，但它的激活也可以诱导促炎状态，导致感染性休克和死亡。我们已经证明，来自不同NeisSeries菌株的天然LOS在LA的酰化和磷酸化方面都存在差异，这与其在体外诱导与脑膜炎双球菌感染患者相同的炎性细胞因子的能力有关。我们还证明了脑膜炎奈瑟氏菌89I株LA的特异性磷酸化和酰化变异体具有诱导人单核细胞产生肿瘤坏死因子-1的不同能力。我们最近证实NeisSerial LOS通过核因子-：B和干扰素调节因子3(IRF-3)转录因子激活MyD88依赖和TRIF-依赖的途径，并且据我们所知，将第一个报道直接激活核因子-：B与更多的磷酸取代基呈正相关。我们还发现，在含有磷酸基取代最多的LA的LOS处理的细胞中，MIP-11和MIP-12的水平更高。这些数据支持我们的总体假设，即LA的炎症潜能随着磷基取代基的增加而增加，并且不同NeisSeries菌株LOS内LA结构的变化是天然免疫系统对NeisSeries感染应答程度的主要决定因素。根据TLR4在LOS识别中的关键作用，很明显，LOS对TLR4的不适当信号可能在NeisSeries感染过程中产生重要后果，导致过度反应，如脑膜炎双球菌败血症和淋球菌性盆腔炎。因此，在本文提出的研究中，我们试图通过脑膜炎球菌89I株的基因缺失和可变表达突变来定义决定炎症信号的LA结构元件，以产生具有单一不变LA部分的LOS，该LA部分在磷酸化和/或酰化状态下不同。其具体目的是：(1)在LA结构变异的背景下，确定调节TLR4依赖的先天免疫反应诱导NeisSeries LOS的分子机制。我们将构建遗传变量表达LA突变体，并通过确定突变体LOS通过TLR4传递信号、成熟树突状细胞和极化T细胞、诱导microRNA、与TLR4-MD-2复合体结合以及在电子计算机模拟中诱导与TLR4-MD-2分子相互作用的差异来预测LA的生物活性。(2)确定LA结构和功能的预测模型与致病株和非致病株表达的LA的生物活性和相对丰度之间的相关性。我们将把致病和非致病菌株的LA分子的表达与它们诱导炎性细胞因子的潜力联系起来，LA分子是TLR4诱导的分子决定因素。我们的研究有望建立炎症信号的LA结构决定因素，为开发治疗奈瑟氏菌感染的抗生素确定新的靶点，使新的方法能够识别超强毒力和高度炎症的菌株，并帮助LOS疫苗的设计。 公共卫生相关性： 由于抗生素耐药性的增加以及B群脑膜炎奈瑟菌和淋病奈瑟菌疫苗的缺乏，控制脑膜炎奈瑟菌和淋病奈瑟菌的感染仍然存在问题。在美国，由B群细菌引起的感染在军队中仍然是一个问题，C群疾病仍然是社区获得性脑膜炎的原因之一。美国报告的淋病发病率从1975年到1997年下降了74%，1998年有所上升，到目前为止基本上保持不变。美国的发病率仍然是所有工业化国家中最高的，估计每年有70万新病例。重要的是，流行病学研究提供了强有力的证据表明，淋球菌感染促进了艾滋病毒-1感染的传播。鉴于美国脑膜炎双球菌脑膜炎和淋病发病率的统计数据，相应数量的退伍军人可能会感染这些感染。因此，我们提出的了解人类对感染的免疫反应的研究与退伍军人管理局的健康和患者护理任务相关。

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. PUBLIC HEALTH RELEVANCE: Control of infections due to Neisseria meningitidis and N. gonorrhoeae remains problematic due to increased antibiotic resistance and the lack of vaccines for serogroup B N. meningitidis and N. gonorrhoeae. In the U.S., infection caused by serogroup B organisms remains a problem in the military, and group C disease remains a cause of community-acquired meningitis. Following a 74% decline in the reported rate of gonorrhea in the U.S. from 1975 to 1997, the rate increased in 1998 and has remained essentially unchanged to date. The U.S. rate remains the highest of any industrialized nation, with an estimated 700,000 new cases per year. Importantly, epidemiologic studies provide strong evidence that gonococcal infection facilitates the transmission of HIV-1 infection. Given the statistics on the incidence of meningococcal meningitis and gonorrhea in the U.S., a corresponding number of veterans will potentially contract these infections. Thus, our proposed research to understand human immune responses to infection is relevant to the health and patient care missions of the VA.