The basis of strain-specific differences in virulence in the human pathogen Orientia tsutsugamushi

项目总结 媒介传播的专性胞内细菌是世界范围内发病率和死亡率的重要原因。一 这种病原体，东方体，导致被忽视的热带疾病丛林斑疹伤寒，这是 估计每年至少导致100万人感染，如果不这样做，就会导致高死亡率 及时接受治疗，并使用适当的抗生素。？ 菌株多样性，不同的菌株在患者和动物模型中表现出不同水平的毒力。一个 钩端螺旋体致病的主要因素是炎症的过度激活。 免疫反应。然而，描述恙虫病原虫如何诱导这种反应的详细机制 仍然不为人所知。恙虫病原虫在遗传上是难以处理的，因此基于功能基因组学 识别毒力因素的方法是不可能的。在这里，我们建议利用特定于菌株的 自然变异，以揭示对毒力至关重要的关键途径。在我们的初步工作中，我们量化了 卡普和UT176两株临床分离株的毒力测定结果表明，两株临床分离株具有较强的致病力。 在小鼠模型中，结果明显不同。我们还使用了双重RNAseq来鉴定这两种细菌 以及在人类培养细胞中生长时这两个菌株之间差异表达的宿主基因 (HUVEC)，鉴定出细菌表面蛋白TSA22、TSA47、TSA56和Ankyrins Ank6、Ank16、Ank17、 Ank20)和宿主(SOCS2、IL33、其他趋化因子和细胞因子)因子与毒力密切相关。 我们最近还生成了6株钩端螺旋体的完整基因组序列，带来了 全部测序的菌株总数最多为8个。这些菌株都将用于本研究。我们的 初步工作和以前的报告引导我们提出了这样的假设，即在 恙虫病原虫毒力相关基因表达的差异对其毒力的影响 基因，导致病原体清除或炎症诱导的致病机制。这一假设 将在以下目标中加以解决。在目标1中，我们将增加我们初步关联的能力 通过量化共8株钩端螺旋体的毒力和基因表达谱进行分析， 并将使用这些信息来识别与毒力密切相关的细菌和宿主因素。在AIM 2，我们将使用体外免疫报告试验来探索特定细菌基因在毒力中的作用， 重点关注我们初步研究和目标1中确定的因素。这项研究的结果将指导未来 致力于开发针对这种病原体的新诊断工具和治疗方法。

PROJECT SUMMARY Vector-borne obligate intracellular bacteria are an important cause of morbidity and mortality worldwide. One such pathogen, Orientia tsutsugamushi, causes the neglected tropical disease scrub typhus, which is estimated to cause infection in at least 1 million people each year, and is associated with high mortality if not treated promptly and with the appropriate antibiotics. The species O. tsutsugamushi is characterised by high strain diversity, where different strains exhibit different levels of virulence in patients and animal models. A major factor in the pathogenicity induced by O. tsutsugamushi is an over-activation of the inflammatory immune response. However, the detailed mechanisms describing how O. tsutsugamushi induces this response remain unknown. O. tsutsugamushi is genetically intractable and therefore functional genomics based approaches to identify virulence factors are not possible. Here we propose to take advantage of strain-specific natural variation to uncover key pathways important for virulence. In our preliminary work we have quantified the virulence of two clinical isolate strains of O. tsutsugamushi, Karp and UT176, and shown that they have significantly different outcomes in a mouse model. We have also used dual RNAseq to identify both bacterial and host genes that are differentially expressed between these two strains when grown in human cultured cells (HUVEC), and identified bacterial (surface proteins TSA22, TSA47, TSA56 and Ankyrins Ank6, Ank16, Ank17, Ank20) and host (SOCS2, IL33, other chemokines and cytokines) factors that strongly correlate with virulence. We have also recently generated complete genome sequences for 6 strains of O. tsutsugamushi, bringing the total number of completely sequenced strains up to 8. These strains will all be used in this study. Our preliminary work and previous reports led us to the hypothesis that strain specific differences in the virulence of O. tsutsugamushi are driven by differences in expression of bacterial virulence-related genes, resulting in either pathogen clearance or inflammatory induced pathogenesis. This hypothesis will be addressed in the following aims. In Aim 1, we will increase the power of our preliminary correlative analysis by quantifying the virulence and gene expression profiles of a total of 8 strains of O. tsutsugamushi, and will use this information to identify bacterial and host factors that robustly associate with virulence. In Aim 2, we will use an in vitro immune reporter assay to probe the role of specific bacterial genes in virulence, focusing on the factors identified in our preliminary studies and in Aim 1. Results of this study will guide future work on developing novel diagnostic tools and therapies against this pathogen.