Rotation 1: Characterising HCMV gene functions

BBSRC战略主题：恙虫病东方体（OT）是立克次氏体目（Rickettsiales）的一种革兰氏阴性、专性细胞内α-蛋白杆菌，包括其他人类和动物病原体以及非致病性无脊椎动物内共生体，如沃尔巴克氏体。OT是螨传播的，并导致丛林斑疹伤寒。我们的研究重点是OT细菌细胞生物学的基础知识及其与真核宿主细胞的相互作用。OT通过网格蛋白介导的内吞作用或巨胞饮作用进入细胞。然后细菌逃离核内体，劫持宿主微管向细胞核运输并直接在细胞质中复制，形成核周菌落。已知OT以非裂解性“出芽”机制离开宿主细胞，使人联想到病毒退出，释放被宿主细胞膜包围的单个细菌。然而，参与这种成熟的因素和退出机制仍然不清楚。在纤恙螨中，进食诱导OT从受感染的唾液腺细胞中出芽。此外，OT已被证明可分化为两种不同的状态：杆状且具有免疫活性的细胞内细菌（IB）和圆形且无免疫活性的细胞外细菌（EB）。这种成熟的复杂性是不共享的其他成员的立克次氏体科，这些国家的传染性和行为是至关重要的了解生理相关的感染周期。解决的主要问题，哪些因素影响成熟和宿主细胞退出OT以前是不可能的，由于缺乏一个强大的和信息出芽测定。在Salje实验室的轮换项目期间，我开发了一种基于qPCR和IFM的检测方法，可以对这一过程进行研究，包括明显阻止成熟和出芽的阳性对照。因此，这个博士项目的一个关键目标是研究如何触发宿主细胞退出。使用这种检测方法，我将首先测试一系列宿主细胞条件的影响，包括营养消耗和流入，ER应激，氧化应激和钙信号传导。我的初步数据表明，营养的可用性和感染的多样性可能是有希望的候选人进行进一步调查。 一个重要的问题是OT是否“决定”单独离开或以协调一致的方式离开，这意味着细菌之间的传感途径和可能的通信。OT在萌芽过程中的实时成像将提供更多关于OT行为的信息。此外，对成熟细菌中表达的蛋白质的研究不仅可以为我们提供OT成熟的有价值的标志物，而且还可以阐明细菌开启的信号传导或感知宿主细胞条件的机制。该项目还旨在使用实时成像确定OT保留质膜包膜的时间，以及它是否富含任何特定的宿主蛋白质，因为这可能对重新感染不同的宿主细胞类型有影响。最后，OT运输到质膜的机制仍然未知;潜在的候选者（在病毒退出中发挥作用）包括驱动蛋白和肌动蛋白运输。我建议使用出芽试验测试运输抑制剂对OT出口的影响;将对破坏OT出口的途径进行进一步调查。

BBSRC strategic theme: Bioscience for an integrated understanding of healthOrientia tsutsugamushi (OT) is a gram-negative, obligate intracellular alpha-proteobacterium of the Rickettsiales, which include other human and animal pathogens and non-pathogenic invertebrate endosymbionts such as Wolbachia. OT is mite-borne and causes scrub typhus. Our research focuses on the fundamentals of OT bacterial cell biology and its interaction with eukaryotic host cells. OT enters cells by clathrin-mediated endocytosis or macropinocytosis. Bacteria then escape endosomes, hijack host microtubules to traffic towards the nucleus and replicate directly in the cytoplasm, forming perinuclear colonies. It is known that OT leave host cells in a non-lytic 'budding' mechanism reminiscent of virus exit, releasing individual bacteria surrounded by host cell membrane. However, the factors involved in this maturation and the mechanisms of exit are still not understood. In Leptotrombidium mites, feeding induces OT to bud out of infected salivary gland cells. In addition, OT has been shown to differentiate into two distinct states; intracellular bacteria (IB), which are rod-shaped and translationally active, and extracellular bacteria (EB), which are round and translationally inactive. The complexity of this maturation is not shared by other members of the Rickettsiaceae family, and the infectivity and behaviour of these states is crucial to understanding the physiologically relevant infection cycle. Addressing the major question of which factors affect maturation and host cell exit in OT has previously been impossible due to the lack of a robust and informative budding assay. During my rotation project in the Salje lab, I developed a qPCR and IFM-based assay which allows this process to be investigated, including positive controls which visibly arrest maturation and budding. One key aim of this PhD project is therefore to investigate how host cell exit is triggered. Using this assay, I will initially test the effect of a range of host cell conditions, including nutrient depletion and influx, ER stress, oxidative stress and calcium signalling. My preliminary data has shown that nutrient availability and multiplicity of infection may be promising candidates for further investigation. One important question is whether OT 'decide' to leave individually or in a concerted manner, implying sensing pathways and possible communication between bacteria. Live imaging of OT during the budding process will provide more information on OT behaviour at this time. Additionally, investigation of the proteins expressed in maturing bacteria could not only give us a valuable marker for maturation of OT, but may also elucidate mechanisms which are switched on by bacteria for signalling or sensing host cell conditions. This project also aims to determine how long OT retains the plasma membrane envelope, using live imaging, and whether this is enriched in any particular host proteins, as this may have implications for reinfection into different host cell types. Finally, the mechanism by which OT traffics to the plasma membrane is still unknown; potential candidates (which play a role in virus exit) include kinesins and actin trafficking. I propose to test the effect of trafficking inhibitors on OT exit using the budding assay; further investigation will be carried out on pathways which abrogate OT exit when disrupted.