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; 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 立克人士是革兰氏阴性的细胞内字母内细菌和代谢寄生虫，范围很广 真核宿主。穿过立克树，媒介传播病原体（即斑点发烧组（SFG）和斑疹伤寒 组（TG）疾病剂）散布在许多内共生植物和其他未知的致病性。 测序基因组的宝库可以进行鲁棒的比较，以阐明矢量后面的机制 传播和发病机理。这对于人类健康至关重要，因为森林砍伐和城市化正在加剧 在美国的立克疾病中，会出现抗四环素抗菌菌株的尖峰。 吉莱斯皮博士使用系统基因学来识别谱系的特定因素，这些因素随后以角色为特征 在立克的发病机理中。与细菌LPS专家Ernst合作，导致了最近的 发现并非所有立克脂质A都是平等的！脂质A，LPS的膜成分 已知最促炎的分子在SFG Rickettsiae中定义的点上的酰基链长度分歧 进化 - 最致命的病原体之一（落基山斑点发烧特工R. Rickettsi）适应2’酰基 像高度潜在的大肠杆菌脂质A中发现的链！这意味着立克西亚脂质A与宿主的相互作用可变 MD-2/TLR4受体。我们还做出了另外两个发现LPS在Rickettsia中变化的发现 物种。首先，多糖合成歌剧（PSO），它编码参与LPS合成的酶 碳水化合物部分（Camo）在立克基因组中高度分歧。第二，编码两个酶的基因 潜在用磷酸乙醇胺（EPT的PETN）和磷酸胆碱（LICD的CHOP）修改LPS 通过侧向基因转移获得，并在某些不感染脊椎动物的非path仪中进行伪造！ 这些集体数据加强了我们的假设，即LPS在潜水员身分中有变化。此外，给定 节肢动物和脊椎动物细胞之间的生理和免疫学差异，我们阳性立克LPS 类似于其他细菌（即 耶尔森氏菌和弗朗西斯氏菌，可能是使宿主先天免疫系统沉默的机制。 在此提案中，两个独立（但完整的）特定目标旨在检验这些假设。第一的 （AIM 1），我们将使用Flatn，一种快速方法来产生脂质A结构，其输入样品最少，没有化学 提取，以确定十个潜水物立克的脂质A酰基链长度的变异性感染了节肢动物和 脊椎动物细胞。接下来（AIM 2），这些物种的一个子集将用于表征迷彩结构和量表 PS，EPT和LICD的基因表达；此外，我们将描述Rickettsia Typhi Petn在LPS和MINE中添加 Rickettsia基因组用于其他LPS修饰基因。最近有可用的基因组扩展 GenBank上新的立克西亚的集会，因此我们预计会出现有关LPS生物学的重要发现。 我们对Rickettsia LPS的描述将对矢量传输动态产生见解，确定规范特异性特征， 并为未来测定的阶段测试LPS免疫刺激潜力（脂质A）和表位识别（CAMO）。