Ehrlichia genes required for tick colonization and virulence

无形体科蜱传细菌在地球仪引起人畜共患病，如人类 无形体病和埃立克体病，其中存在有能力的媒介蜱。由于非特异性体征和症状， 由于疾病的原因，以及在疾病发作时缺乏诊断抗体，它们可能会与更好地识别混淆。 发热性疾病，如立克次体病或病毒性疾病。严重的人类埃里希体病的特征是亲- 炎症综合征，但细菌基因产物负责诱导不良宿主反应是未知的。 无形体科中基因功能的确定由于其专性细胞内性质而复杂化，然而， 可以确定预防和治疗疾病的目标。先前的埃立克体病动物模型 不能复制人类疾病的特征，这使得研究毒力因子变得困难。我们集团 在体外从人患者和人中分离出一种新的人埃里希体病因子（小鼠埃里希体样因子，EMLA）， 黑腿扁虱EMLA在实验室小鼠中引起的疾病重现了人类单核细胞埃里希体病， 使得现在可以在小鼠模型中研究引起临床埃里希体病的因素。到 为了阐明埃立克体的致病性和蜱传播的分子基础，我们建议建立一个 使用随机诱变和通过基于Illumina测序的插入位点表征的突变体EMLA 保持战略定力我们将使用一个转座子，它包含一个随后可以被功能基因替换的盒 copy for complementation.我们将通过完成以下具体目标来实施我们的计划： 目的1：在蜱中产生具有可替换选择盒的鼠埃立克体样因子（EMLA）突变体文库， 人类细胞培养物。 AIM 2a：通过基于Illumina的突变体库序列分析， 使用PCR分配到单个突变株系。2b.鉴定入侵缺陷突变体和成功入侵突变体 使用细胞侵袭/定殖筛选、生物信息学分析和遗传学方法， 互补 埃里希体属物种差异表达基因取决于它们所驻留的宿主细胞。我们将培养变种人， 人和蜱细胞来回收具有对于仅一种宿主细胞的定殖所必需的破坏基因的细菌 类型，以及那些管理致病性，但不是必需的。我们意识到， 和蜱传播对于埃立克体感染哺乳动物的能力是重要的。随着人类的可用性， EMLA和肩突硬蜱基因组，现在的条件是正确的，以确定埃立克体基因，介导 在体外研究其与宿主和载体的相互作用，为后续项目中更全面的研究奠定基础。

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.