Investigating Rickettsia Interspecies and Host-Specific Lipopolysaccharide Variation

研究立克次体种间和宿主特异性脂多糖变异

• 批准号: 10628037
• 负责人: Joseph J Gillespie
• 金额: $ 19.31万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10628037
• 关键词: Abbreviations; Acetates; Adopted; Alphaproteobacteria; Arthropods; Bacteria; Biochemical; Bioinformatics; Biological Assay; Biology; Carbohydrates; Cell Line; Cells; Chemicals; Cytosol; Darkness; Data; Deforestation; Development; Disease; Environment; Enzymes; Epitopes; Escherichia coli; Eukaryota; Eukaryotic Cell; Evolution; Fleas; Francisella tularensis; Future; Genbank; Gene Expression; Gene Modified; Genes; Genetic Variation; Genome; Goals; Health; Horizontal Gene Transfer; Human; Immune Evasion; Immune response; Immunologics; Infection; Inflammatory; Innate Immune System; Length; Ligation; Lipid A; Lipopolysaccharides; Measures; Membrane; Metabolic; Microbe; Modification; Mutagenesis; Operon; Parasites; Pathogenesis; Pathogenicity; Phenotype; Phosphorylcholine; Physiological; Polysaccharides; Proteins; Pseudogenes; Pseudomonas; Regulation; Reporting; Research; Rickettsia; Rickettsia Infections; Rickettsia typhi; Rocky Mountain Spotted Fever; Role; STEM research; Sampling; Structure; TLR4 gene; Testing; Tetracycline Resistance; Time; Trees; Typhus; Urbanization; Variant; Vertebrates; Virulence; Work; Yersinia pestis; analytical method; cell envelope; combat; comparative; design; efficacious treatment; endosymbiont; experimental study; human pathogen; in silico; insight; interest; novel; pathogen; phosphoethanolamine; prevent; rapid technique; receptor; resistant strain; spotted fever; targeted treatment; trait; vector; vector tick; vector transmission; vector-borne; vector-borne pathogen

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).

项目概要 立克次体是革兰氏阴性专性细胞内α变形菌和代谢寄生虫，具有广泛的 跨越立克次体树的真核宿主，媒介传播的病原体（即斑疹热群（SFG）和斑疹伤寒） 组（TG）疾病病原体）散布着许多内共生体和其他未知致病性的物种。 测序基因组的宝库可以进行强有力的比较，以阐明载体背后的机制 随着森林砍伐和城市化的加剧，这对人类健康至关重要。 美国各地立克次体疾病激增，四环素耐药菌株出现的可能性始终存在。 Gillespie 博士使用系统基因组学来识别谱系特定因素，随后对这些因素进行角色表征 与细菌 LPS 专家 Ernst 博士合作，研究了立克次体发病机制。 发现并非所有立克次体脂质 A 都是相同的 脂质 A，是脂多糖 (LPS) 的膜成分之一！ 已知最具促炎性的分子，在 SFG 立克次体中的一个可定义点处酰基链长度存在差异 进化 - 最致命的病原体之一（落基山斑疹热病原体立克次体）采用 2' 酰基 类似于在高效力大肠杆菌脂质 A 中发现的链，这意味着立克次体脂质 A 与宿主的相互作用存在差异。 我们还做出了另外两项发现，表明 LPS 在立克次体中是可变的。 首先是多糖合成操纵子（pso），它编码参与 LPS 合成的酶。 其次，编码两种酶的基因在立克次体基因组中存在高度差异。 磷酸乙醇胺（Ept 的 pEtN）和磷酸胆碱（LicD 的 ChoP）可能修饰 LPS 通过横向基因转移获得，并在一些不感染脊椎动物的非病原体中被假基因化！ 这些集体数据进一步证实了我们的假设，即脂多糖在不同的立克次体中是可变的； 由于节肢动物和脊椎动物细胞之间的生理和免疫学差异，我们认为立克次体 LPS 节肢动物和脊椎动物宿主环境之间的转换期间结构发生变化，类似于其他细菌（即 鼠疫耶尔森菌和土拉弗朗西斯菌），并且可能是沉默宿主先天免疫系统的机制。 在该提案中，设计了两个独立（但互补）的具体目标来首先检验这些假设。 (AIM 1)，我们将使用 FLATn，这是一种以最少的输入样品且无需化学物质即可产生脂质 A 结构的快速方法 提取，以确定十种不同的立克次体感染节肢动物和 接下来 (AIM 2)，这些物种的一个子集将用于表征 CaMo 结构和测量 ps、ept 和 licD 的基因表达；此外，我们将表征除了 LPS 和我的伤寒立克次体 pEtN 其他 LPS 修饰基因的立克次体基因组最近已扩展了可用的基因组。 GenBank 上新立克次体的组装，因此我们预计会在 LPS 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深入了解媒介传播动力学，识别物种特异性特征， 并为未来测试 LPS 免疫刺激潜力（脂质 A）和表位识别（CaMo）的分析奠定基础。