Ehrlichia genes required for tick colonization and virulence

壁虱传播的无浆体科细菌可引起全球新出现的人畜共患病，如人类 无浆体病和埃立克体病，在这些地方会出现有能力的媒介蜱虫。由于他们的非特定体征和症状 病因，以及发病时缺乏诊断抗体，可能会与更好的识别混淆 发热性疾病，如立克次体病或病毒性疾病。严重的人类埃立克体病的特征是 炎症综合征，但导致宿主不良反应的细菌基因产物尚不清楚。 无浆体科基因功能的测定由于其固有的细胞内性质而变得复杂，然而，这 将能够确定预防和治疗疾病的目标。以前的埃立克体病动物模型 不复制人类疾病的特征，这使得研究毒力因素变得困难。我们的团队已经 从一名病人和一名病人体内分离出一种新的人类埃立克体病病原体(类埃立克体样制剂，EMLA)。 黑腿扁虱。EMLA在实验室小鼠中引起疾病，这种疾病重现人类单核细胞埃立克体病， 这使得现在有可能在小鼠模型上调查导致临床埃立克体病的因素。至 为了阐明埃立克体致病力和壁虱传播的分子基础，我们建议建立一个 随机诱变突变型EMLA及其插入位点的光测序鉴定 决心。我们将使用包含盒式磁带的转座子，该盒式磁带随后可以被功能基因取代。 复制以供补充。我们将通过完成以下具体目标来实施我们的计划： 目的1：建立鼠类埃利希菌(EMLA)突变体文库，该文库带有可替换的TICK和 人类细胞培养。 目的2a：通过基于Illumina的突变库序列分析来定位转座子的插入位点，然后 利用聚合酶链式反应对单个突变系进行分配。2B。入侵缺陷突变体的鉴定及成功 利用细胞侵袭/定植筛选、生物信息学分析和遗传学技术对人和扁虱细胞进行定植 互补性。 埃利希菌物种根据寄主细胞的不同而表达不同的基因。我们将在中国培育变种人 人类和扁虱细胞恢复携带破坏基因的细菌，这些基因对只有一个宿主细胞的定植至关重要 类型，以及控制致病性但不是必需的那些。我们意识到人类定居所需的基因 而硬蜱传播对埃立克次体感染哺乳动物的能力具有重要作用。随着人类的出现， EMLA和肩部硬蜱的基因组，现在条件是正确的，以确定介导埃立克病毒的基因 在体外与宿主和载体相互作用，为后续项目中更全面的研究奠定了基础。

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.