Modulation of Human Cells by Virulent Francisella tularensis

摘要：图拉氏方济氏菌(FT)是图拉热病的病原体，可通过多种途径感染人类，包括通过媒介传播。然而，吸入细菌和由此导致的肺炎图拉热症是最危险的疾病形式。这是由于未经治疗的患者的潜伏期短(3-5天)、非特异性症状以及高死亡率(大于80%)。此外，英国《金融时报》已经被美国和前苏联武器化，使其成为一个可行的生物武器候选者。尽管世界各地对FT的研究已有80多年的历史，但人们对这种细菌与宿主的动态相互作用知之甚少，尤其是在气溶胶感染之后。 我的实验室已经证实，与小鼠细胞类似，人类树突状细胞和巨噬细胞对FT感染非常敏感，但无法产生促炎细胞因子或经历成熟。此外，毒力FT能积极干扰人DC和巨噬细胞对二次刺激的反应能力。了解FT主动抑制DC和巨噬细胞功能的机制是我实验室的核心指导。我们正在以两种不同的方式处理这一指令。 具体目标1：我们正在分析弗朗西塞氏菌脂类在介导抗炎反应中所起的作用。细菌表面的结构是宿主细胞遇到的第一组分。因此，在FT感染的背景下，这些结构可能有助于早期、快速地抑制人类树突状细胞。细菌脂类就是这样一种结构。我们最近鉴定了FT中存在的一种抑制炎症的活性脂质物种。我们已经测试了这种脂质的合成版本，并证实它也会损害炎症反应。我们已经确定了完整的细菌和纯化的脂类都利用的受体之一，以及促进免疫抑制反应所需的宿主信号分子。重要的是，我们还证明了FT脂类在体外可以限制其他感染源驱动的致病性炎症反应，包括寨卡病毒。我们已经筛选了FT脂类(包括合成脂质体)通过多种途径递送后在体内的非靶向作用，并准备在体内检测它们对病毒介导的炎症的疗效。我们已经提交了一项将FT脂质用作新型抗炎疗法的专利申请。我们目前正在确定FT中存在的其他有助于抑制炎症的脂类，以及它们干扰人类细胞功能的其他机制。 具体目标2：我们正在探索与FT外表面相关的碳水化合物在指导人类细胞免疫抑制程序中的作用。FT的主要外表面碳水化合物结构是O-抗原(O-Ag)胶囊。通常情况下，胶囊被认为只是掩盖了细菌表面存在的蛋白质，这些蛋白质可能会刺激炎症反应。然而，我们的数据表明，FT胶囊直接抑制了人类细胞的促炎反应。利用胶囊合成中具有特定缺陷的突变体和纯化的FT胶囊，我们目前正在鉴定胶囊调控的特定受体和宿主信号通路，以启动人类细胞的抗炎计划。 具体目标3：我们已经制定了一项计划，与拥有执行这些分析的独特协议、技术和能力的ProImmune公司一起筛选原始人类T细胞池。我们正在通过筛选在土拉氏原虫强毒力感染后存活的动物的免疫反应，来确定这些检测中的免疫优势抗原。

Summary: Francisella tularensis (FT), the causative agent for tularemia, can infect humans by a number of routes, including vector-borne transmission. However, it is inhalation of the bacterium, and the resulting pneumonic tularemia, that represents the most dangerous form of disease. This is due to the short incubation time (3-5 days), non-specific symptoms, and a high mortality rate (greater than 80%) in untreated individuals. Furthermore, FT has been weaponized by both the United States and the former Soviet Union making it a viable candidate for use as a biological weapon. Despite over 80 years of research on FT around the world, very little is understood about the dynamic interaction of this bacterium with the host, especially following aerosol infection. My laboratory has established that, similarly to murine cells, human dendritic cells and macrophages are acutely susceptible to infection with FT, but fail to produce pro-inflammatory cytokines or undergo maturation. Further, virulent FT actively interferes with the ability of human DC and macrophages to respond to secondary stimuli. Understanding the mechanism by which FT actively suppresses DC and macrophage function is a central directive of my laboratory. We are tackling this directive in two different ways. Specific Aim 1: We are analyzing the role Francisella lipids play in mediating anti-inflammatory responses. Structures present on the surface of bacteria are the first components encountered by the host cell. Thus, it is possible that, in the context of FT infections, these structures contribute to the early, rapid suppression of human dendritic cells. Bacterial lipids represent one such structure. We have recently identified one of the active lipid species present in FT that inhibit inflammation. We have tested a synthetic version of this lipid and confirmed that it also impairs inflammatory responses. We have identified one of the receptors utilized by both the intact bacterium and purified lipid as well as a host signaling molecule required to promote immunosuppressive responses. Importantly, we have also demonstrated that FT lipids can limit pathogenic inflammatory responses driven by other infectious agents in vitro, including Zika virus. We have screened FT lipids (including synthetic liposomes) for off target effects in vivo following delivery via multiple routes and are preparing to examine their efficacy against viral mediated inflammation in vivo. We have filed a patent application for use of FT lipids as novel anti-inflammatory therapeutics. We are currently identifying the other lipids present in FT that contribute to suppression of inflammation and additional mechanisms by which they interfere with functions in human cell. Specific Aim 2: We are exploring the role of carbohydrates associated with the outer surface of FT in directing immunosuppressive programs in human cells. The major outer surface carbohydrate structure of FT is the O-Antigen (O-Ag) capsule. Typically capsules are thought to simply cover up proteins present on the bacterial surface that could stimulate an inflammatory response. However, our data demonstrates that FT capsule directly inhibits pro-inflammatory responses in human cells. Utilizing mutants with specific defects in capsule synthesis along with purified FT capsule, we are currently identifying the specific receptors and host signaling pathways modulated by capsule to initiate an anti-inflammatory program in human cells. Specific Aim 3: We have established a plan to screen pools of nave human T cells with the company ProImmune who possesses unique protocols, techniques and capabilities to perform these assays. We are in the process of identifying immunodominate antigens to be targeted in these assays by screening the immune response among animals which have survived virulent F. tularensis infection.