Ehrlichia genes required for tick colonization and virulence

蜱定植和毒力所需的埃里希体基因

• 批准号: 9412419
• 负责人: Ulrike Gertrud Munderloh
• 金额: $ 19.19万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-15 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9412419
• 关键词: Agar; Anaplasma; Anaplasmataceae; Anaplasmosis; Animal Model; Antibiotic Therapy; Antibiotics; Antibodies; Asia; Bacteria; Bacterial Proteins; Base Sequence; Bioinformatics; Biological Assay; Black-legged Tick; Cell Culture Techniques; Cells; Cessation of life; Clinical; Complement; DNA cassette; Diagnostic; Disease; Ehrlichia; Ehrlichia chaffeensis; Ehrlichiosis; Environment; Essential Genes; Europe; Family; Fever; Future; Gene Proteins; Genes; Genetic; Genome; Goals; Growth; Heartland virus; Human; In Vitro; Individual; Infectious Agent; Inflammatory; Knowledge; Laboratory mice; Lead; Libraries; Link; Mammals; Maps; Mediating; Minnesota; Molecular; Mus; Mutagenesis; Mutate; Nature; North America; Onset of illness; PAWR protein; Pathogenicity; Patients; Prevention; Reaction; Recovery; Resources; Rocky Mountain Spotted Fever; Scrub Typhus; Sequence Analysis; Shapes; Signs and Symptoms; Site; Syndrome; Thrombocytopenia; Ticks; Time; Undifferentiated; Virulence; Virulence Factors; Virus Diseases; Wisconsin; Zoonoses; base; cell type; differential expression; gene function; human disease; human model; monocyte; mouse model; mutant; novel; pathogen; prevent; screening; trait; transmission process; vaccine development; vector

Tick-borne bacteria in the family Anaplasmataceae cause emerging zoonoses across the globe, such as human anaplasmosis and ehrlichioses, where competent vector ticks occur. Due to the non-specific signs and symptoms they cause, and absence of diagnostic antibodies at the time of illness onset, they may be confused with better recognized febrile illnesses such as rickettsioses or viral diseases. Severe human ehrlichiosis is characterized by a pro- inflammatory syndrome, but the bacterial gene products responsible for inducing adverse host reactions are unknown. Determination of gene function in the Anaplasmataceae is complicated by their obligately intracellular nature, yet, this would allow identification of targets for prevention and treatment of disease. Previous animal models of ehrlichiosis do not reproduce features of human disease, which makes it difficult to investigate virulence factors. Our group has isolated a novel human ehrlichiosis agent (Ehrlichia muris-like agent, EMLA) in vitro from both a human patient and a blacklegged tick. EMLA causes disease in laboratory mice that recapitulates human monocytic ehrlichiosis, making it now possible to investigate the factors that cause clinical ehrlichiosis in a mouse model. To elucidate the molecular basis of pathogenicity and tick transmission in ehrlichiae, we propose to generate a library of mutant EMLA using random mutagenesis and characterization by Illumina sequencing-based insertion site determination. We will use a transposon containing a cassette that can subsequently be replaced with a functional gene copy for complementation. We will implement our plan through completion of the following specific aims: AIM 1: Produce a library of Ehrlichia muris-like agent (EMLA) mutants with a replaceable selection cassette in tick and human cell cultures. AIM 2a: Map insertion sites of transposons by Illumina-based sequence analysis of mutant pools followed by assignment to individual mutant lines using PCR. 2b. Identification of mutants defective for invasion and successful colonization of human and tick cells using cell invasion/colonization screens, bioinformatics analysis, and genetic complementation. Ehrlichia species differentially express genes depending on the host cell in which they reside. We will raise mutants in human and tick cells to recover bacteria with disrupted genes that are essential for colonization of only one host cell type, as well as those that govern pathogenicity but are not essential. We realize that genes required for colonization of and transmission by ticks are important for ability of ehrlichiae to infect mammals. With the availability of the human, the EMLA, and the Ixodes scapularis genomes, the conditions are now right to identify ehrlichia genes that mediate interactions with its host and vector in vitro, setting the stage for more comprehensive studies in a subsequent project.

无形体科中的蜱传细菌在全球范围内引起新出现的人畜共患病，例如人类 无形体病和埃利希体病，其中发生有能力的媒介蜱。由于非特异性体征和症状，他们 原因，以及疾病发作时缺乏诊断抗体，它们可能会与更容易识别的抗体相混淆 发热性疾病，例如立克次体病或病毒性疾病。严重的人类埃利希体病的特点是亲 炎症综合征，但引起宿主不良反应的细菌基因产物尚不清楚。 无形体科中基因功能的测定由于其绝对细胞内性质而变得复杂，然而，这 将有助于确定预防和治疗疾病的目标。先前的埃立克体病动物模型 不能再现人类疾病的特征，这使得研究毒力因子变得困难。我们组有 在体外从人类患者和 黑腿蜱。 EMLA 在实验室小鼠中引起疾病​​，重现人类单核细胞埃利希体病， 现在可以在小鼠模型中研究导致临床埃利希体病的因素。到 为了阐明埃里希亚科致病性和蜱传播的分子基础，我们建议建立一个库 使用随机诱变和基于 Illumina 测序的插入位点表征的突变 EMLA 决心。我们将使用包含盒的转座子，随后可以用功能基因替换该盒 复制以补充。我们将通过完成以下具体目标来实施我们的计划： 目标 1：在蜱和蜱中产生埃利希体样因子 (EMLA) 突变体文库，并带有可更换的选择盒 人类细胞培养物。 目标 2a：通过基于 Illumina 的突变体池序列分析来绘制转座子插入位点图谱，然后 使用 PCR 分配单个突变株系。 2b.鉴定入侵缺陷和成功的突变体 使用细胞侵袭/定植筛选、生物信息学分析和遗传对人类和蜱细胞进行定植 互补。 埃里希体的基因表达差异取决于它们所处的宿主细胞。我们将培育突变体 人类和蜱细胞恢复具有破坏基因的细菌，这些基因对于仅一种宿主细胞的定植至关重要 类型，以及那些控制致病性但不是必需的。我们意识到定植所需的基因 蜱传播对于埃利希亚科病毒感染哺乳动物的能力很重要。随着人类的可用性， EMLA 和肩胛硬蜱基因组，现在有条件鉴定介导埃利希体基因 与其宿主和载体在体外相互作用，为后续项目中更全面的研究奠定了基础。

项目成果

Proposal to reclassify Ehrlichia muris as Ehrlichia muris subsp. muris subsp. nov. and description of Ehrlichia muris subsp. eauclairensis subsp. nov., a newly recognized tick-borne pathogen of humans.

建议将鼠埃利希体重新分类为鼠埃利希体亚种。

• 发表时间: 2017-07
• 影响因子: 2.8
• 作者: Pritt, Bobbi S;Allerdice, Michelle E J;Sloan, Lynne M;Paddock, Christopher D;Munderloh, Ulrike G;Rikihisa, Yasuko;Tajima, Tomoko;Paskewitz, Susan M;Neitzel, David F;Hoang Johnson, Diep K;Schiffman, Elizabeth;Davis, Jeffrey P;Goldsmith, Cynthia
• 通讯作者: Goldsmith, Cynthia