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: 10527408
• 负责人: Joseph J Gillespie
• 金额: $ 23.18万
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10527408
• 关键词: Abbreviations; Acetates; Adopted; Alphaproteobacteria; Arthropods; Bacteria; Biochemical; Bioinformatics; Biological Assay; Biology; Carbohydrates; Cell Line; Cells; Chemicals; Cytosol; Data; Deforestation; Development; Disease; Environment; Enzymes; Epitopes; Escherichia coli; Ethanolamines; 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; Innate Immune System; Lead; Length; Lipid A; Lipopolysaccharides; Measures; Membrane; Metabolic; Microbe; Modification; Mutagenesis; Operon; Parasites; Pathogenesis; Pathogenicity; Phenotype; Phosphorylcholine; Physiological; Play; 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).

项目摘要 立克次体是革兰氏阴性专性胞内α-变形菌和代谢性寄生虫， 真核生物宿主。在立克次体树中，媒介传播的病原体（即，斑疹热组（SFG）和斑疹伤寒 组（TG）病原体）散布有许多内共生体和其他致病性未知的物种。 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，因为森林砍伐和城市化加剧了 在美国，立克次体病的发病率激增，四环素耐药菌株的出现机会一直存在。 吉莱斯皮博士使用基因组学来识别谱系特异性因子，随后对其作用进行表征 立克次氏体发病机制与细菌脂多糖专家恩斯特博士合作， 发现并非所有立克次体脂质A都是平等的！脂质A，LPS的膜成分， 已知的最促炎分子，在SFG立克次体中在可定义的点上在酰基链长度上发散 进化-最致命的病原体之一（落基山斑疹热病原体R。立克次氏体）采用2'酰基 类似于在高效力的E. coli lipid A！这表明立克次体脂质A与宿主相互作用 MD-2/TLR 4受体。我们还取得了另外两项发现，表明LPS在立克次体中是可变的 物种首先，多糖合成操纵子（pso），其编码参与LPS合成的酶。 碳水化合物部分（CaMo）在立克次体基因组中高度不同。第二，编码两种酶的基因 用磷酸乙醇胺（pEtN，Ept）和磷酸胆碱（ChoP，LicD）潜在修饰LPS已经 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！ 这些集体数据加强了我们的假设，即LPS在不同的立克次体中是可变的;此外，考虑到 节肢动物和脊椎动物细胞之间的生理和免疫差异，我们认为立克次体LPS 在节肢动物和脊椎动物宿主环境之间的转换过程中，结构发生变化，类似于其他细菌（即， 鼠疫耶尔森氏菌和土拉弗朗西斯氏菌），并且可能是沉默宿主先天免疫系统的机制。 在这个建议中，两个独立的（但互补的）具体目标是为了测试这些假设。第一 (AIM 1），我们将使用FLATn，一种快速方法，以最少的输入样品和无化学品产生脂质A结构 提取，以确定脂质A酰基链长度的变化，为10个不同的立克次体感染节肢动物和 脊椎动物细胞下一步（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，我们还将描述伤寒立克次体pEtN加LPS和mine的特征， 其他LPS修饰基因的立克次体基因组。最近可用的基因组已经扩展， 基因库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LPS的描述将有助于了解媒介传播动力学，识别物种特异性特征， 并为将来测试LPS免疫刺激潜力（脂质A）和表位识别（CaMo）的测定奠定了基础。