Dynamics of Anaplasma phagocytophilum infection through tick development

 描述（由申请人提供）：人粒细胞无形体病（HGA）是一种由专性细胞内细菌嗜吞噬细胞无形体（Ap）引起的新兴疾病，新发人类病例数量每年都在增加。在北美，Ap通过蜱类传播给人类和动物，蜱类包括肩突硬蜱和太平洋硬蜱，其中I。肩胛肌是造成绝大多数人类病例的原因。令人印象深刻的进步已经取得了理解流行病学，临床，生理和分子方面的Ap与哺乳动物的相互作用，而在细胞水平上了解这种细菌和它的媒介蜱之间的亲密关系，取得了很小的进展。一个重要的限制是很难在蜱内定位Ap。我们已经采用了新技术，使我们能够通过在整个蜱虫的固定切片中标记独特的细菌RNA序列来精确定位蜱虫内的单个细菌，从而可以在完整的蜱虫解剖结构的背景下可视化AP感染的组织和细胞，并将它们与其他细菌（包括病原体和内共生体）区分开来。此外，我们已经产生了一个库的单插入AP突变体中，有许多命中的基因只在蜱细胞感染过程中转录，这些克隆的突变体将允许我们，在未来的研究中，以确定与蜱定植过程的每个阶段，从收购到唾液腺感染的P基因。现在是认真分析亚太地区殖民化的时候了。 主向量I.肩胛肌这项研究的主要目标有两个。首先，我们将使用共聚焦显微镜观察并定量分析蜱组织中的Ap。感染的幼虫是肩胛骨幼虫和成虫，因为这些是传播所涉及的生命阶段。为此，我们将使用AP特异性荧光RNA探针检查充血幼虫、未进食蜱虫和充血蜱虫、未进食和部分进食的成年蜱虫的组织学切片。在蜱虫的不同生命阶段，将对每种感染器官类型的Ap感染的可测量特征，如平均桑椹胚（含细菌的细胞内包涵体）直径和占据的细胞质比例进行定量。定量PCR将用于估计Ap负荷。其次，我们将研究蜱体内细胞之间细菌运输的机制。将检查通过血淋巴血浆传播、通过活感染血细胞转运和直接细胞间接触。将对转化为表达绿色荧光蛋白的AP进行实时成像，以确定其是否定位于吞噬性血细胞的破坏性溶酶体区室或复制以在细胞质中产生桑椹胚。将在体外对血细胞和血淋巴血浆进行进一步的活力试验，以确定任一组分是否能够单独感染培养细胞。为了测试直接接触感染，将从I.肩胛肌培养物，并在限制细胞外活力的含抗生素的培养基存在下与培养的细胞接触。

 DESCRIPTION (provided by applicant): Human granulocytic anaplasmosis (HGA) is an emerging disease caused by the obligate intracellular bacterium Anaplasma phagocytophilum (Ap), and numbers of new human cases are increasing each year. In North America, Ap is transmitted to humans and animals by the ticks Ixodes scapularis and Ixodes pacificus, with I. scapularis responsible for the vast majority of human cases. Impressive strides have been made in understanding epidemiological, clinical, physiological, and molecular aspects of Ap's interaction with mammals, whereas little progress has been made in understanding the intimate relationship between this bacterium and its vector tick at the cellular level. One significant limitation has been the difficulty in localizing Ap within the tick. We have adapted new techniques that allow us to precisely locate individual bacteria within a tick by labeling unique bacterial RNA sequences in fixed sections of whole ticks, making it possible to visualize Ap-infected tissues and cells in the context of intact tick anatomy, and to distinguish them from other bacteria, including pathogens and endosymbionts. In addition, we have generated a library of single insertion Ap mutants in which there are numerous hits to genes only transcribed during infection of tick cells; these cloned mutants will allow us, in future studies, to identify p genes associated with each stage of the tick colonization process, from acquisition to salivary gland infection. The time is now right to proceed with a careful analysis of Ap colonization of its principal vector, I. scapularis. The main goals of this study are twofold. First, we will analyze te spread of Ap through tick tissues, using confocal microscopy to visualize and quantify Ap in I. scapularis nymphs and adults infected as larvae as these are the life stages involved in transmission. To do so, we will examine histological sections of engorged larvae, unfed nymphs, and engorged nymphs, unfed and partially fed adult ticks using an Ap-specific fluorescent RNA probe. Measurable characteristics of Ap infection such as mean morula (an intracellular inclusion containing bacteria) diameter and proportion of occupied cellular cytoplasm will be quantified for each infected organ type at the various life stages of the tick. Quantitative PCR will be used to estimate Ap load. Second, we will investigate the mechanisms of bacterial trafficking between cells within the body of the tick. Dissemination via hemolymph plasma, transport by viably infected hemocytes, and direct cell-to-cell contact will be examined. Ap transformed to express green-fluorescent protein will be imaged live to determine if it localizes to the destructive lysosomal compartments of phagocytic hemocytes or replicates to generate morulae in the cytoplasm. Further viability tests will be performed in vitro with hemocytes and hemolymph plasma to determine if either component, separately, is capable of infecting cultured cells. To test direct contact infection, Ap-infected organs will be dissected from I. scapularis tiks and placed in contact with cultured cells in the presence of antibiotic-containing media restricting extracellular viability.