Investigating Rickettsia Interspecies and Host-Specific Lipopolysaccharide Variation

项目总结 立克次体是革兰氏阴性的专性胞内甲蛋白细菌和代谢寄生虫，种类繁多 真核宿主。在整个立克次体树上，病媒传播的病原体(即斑点热群和斑疹伤寒 组(TG)病原体)中夹杂着许多致病性未知的内共生菌和其他物种。 测序基因组的宝库允许进行强有力的比较，以阐明载体背后的机制 传播和发病机制。这对人类健康至关重要，因为日益严重的森林砍伐和城市化正在加剧 全美立克次体疾病激增，四环素耐药菌株随时可能出现。 吉莱斯皮博士使用系统基因组学来确定特定于谱系的因素，这些因素随后被描述为角色 在立克次体发病机制中。与细菌内毒素专家恩斯特博士的合作导致了最近的 发现并不是所有的类脂立克次体都是一样的！脂蛋白A，脂多糖的膜成分，是 已知的最具致炎作用的分子，在SFG立克次体的某一可定义点的酰链长度上存在分歧 最致命的病原体之一的进化−(落基山斑点热致病原体立克次体)采用2‘-酰基 就像在高效的大肠杆菌脂质A中发现的那样的链！这意味着立克次体类脂A与宿主的相互作用是不同的。 MD-2/TLR4受体。我们还发现另外两个内毒素在立克次体中是可变的 物种。首先，多糖合成操纵子(PSO)，它编码参与合成内毒素的酶 碳水化合物部分(CAMO)在立克次体基因组中具有高度的差异性。第二，编码两种酶的基因 用磷乙醇胺(EPT修饰PETN)和磷脂酰胆碱(LICD修饰CHOP)修饰脂多糖的潜力已经被 通过横向基因转移获得，并在一些不会感染脊椎动物的非病原体中假基因！ 这些集体数据强化了我们的假设，即脂多糖在不同的立克次体中是可变的；此外，鉴于 节肢动物和脊椎动物细胞的生理和免疫学差异，我们推测立克次体内毒素 在节肢动物和脊椎动物寄主环境之间转换过程中的结构变化，类似于其他细菌(即 鼠疫耶尔森氏菌和图拉氏方济氏菌)，并可能是一种机制，以沉默宿主的天然免疫系统。 在这项提案中，设计了两个独立的(但互补的)具体目标来检验这些假设。第一 (目标1)，我们将使用Flatn，一种快速制备类脂A结构的方法，只需最少的输入样本和不含化学物质 提取，测定10种不同立克次体感染节肢动物和 脊椎动物细胞。下一步(目标2)，这些物种的子集将被用来描述伪装结构和测量 PS、EPT和licD的基因表达；此外，我们还将研究伤寒立克次体对内毒素和我的影响 立克次体基因组寻找更多的内毒素修饰基因。最近，现有的基因组得到了扩展。 在GenBank上为新立克次体组装，因此我们期待着在内毒素生物学方面取得重要发现。 我们对立克次体脂多糖的描述将有助于深入了解媒介传播动态，识别物种特有的特征， 并为未来测试脂多糖免疫刺激潜力(类脂A)和表位识别(CAMO)奠定了基础。

PROJECT SUMMARY Rickettsiae are Gram-negative obligate intracellular Alphaproteobacteria and metabolic parasites with a wide range of eukaryotic hosts. Across the Rickettsia tree, vector-borne pathogens (i.e., Spotted Fever Group (SFG) and Typhus Group (TG) disease agents) are interspersed with many endosymbionts and other species of unknown pathogenicity. A treasure trove of sequenced genomes allows for robust comparisons to illuminate mechanisms behind vector transmission and pathogenesis. This is crucial for human health, as rising deforestation and urbanization are fueling spikes in rickettsial diseases across the US, with the ever-present chance tetracycline-resistant strains will emerge. Dr. Gillespie uses phylogenomics to identify lineage specific factors that are subsequently characterized for roles in rickettsial pathogenesis. Teaming with Dr. Ernst, an expert on bacterial LPS, has resulted in the very recent discovery that not all Rickettsia lipid A is created equal! Lipid A, the membrane component of LPS that is among the most proinflammatory molecules known, diverges in acyl chain length at a definable point in SFG Rickettsiae evolution − one of the deadliest pathogens (the Rocky Mountain Spotted Fever agent R. rickettsi) adopts a 2’ acyl chain like that found in the highly potent E. coli lipid A! This implies Rickettsiae lipid A interacts variably with the host MD-2/TLR4 receptor. We have also made two other discoveries indicating LPS is variable across Rickettsia species. First, the polysaccharide synthesis operon (pso), which encodes enzymes involved in synthesis of LPS carbohydrate moiety (CaMo), is highly divergent across Rickettsia genomes. Second, genes encoding two enzymes that potential modify LPS with phosphoethanolamine (pEtN by Ept) and phosphorylcholine (ChoP by LicD) have been acquired by lateral gene transfer and are pseudogenized in some non-pathogens that don’t infect vertebrates! These collective data strengthen our hypothesis that LPS is variable across diverse Rickettsiae; further, given the physiological and immunological differences between arthropod and vertebrate cells, we posit that Rickettsia LPS structure changes during shifts between arthropod and vertebrate host environments, similar to other bacteria (i.e. Yersinia pestis and Francisella tularensis), and may be a mechanism for silencing the host innate immune system. In this proposal, two independent (yet complementary) Specific Aims are designed to test these hypotheses. First (AIM 1), we will use FLATn, a rapid method to yield lipid A structures with minimal input sample and no chemical extraction, to determine lipid A acyl chain length variability for ten diverse rickettsiae infecting both arthropod and vertebrate cells. Next (AIM 2), a subset of these species will be used to characterize CaMo structures and gauge gene expression of ps, ept, and licD; furthermore, we will characterize Rickettsia typhi pEtN addition to LPS and mine Rickettsia genomes for additional LPS modification genes. There has been a recent expansion of available genome assemblies for new Rickettsiae on GenBank, so we anticipate making important discoveries regarding LPS biology. Our description of Rickettsia LPS will yield insight on vector transmission dynamics, identify species-specific traits, and set the stage for future assays testing LPS immunostimulatory potential (lipid A) and epitope recognition (CaMo).