Rotation 1: Characterising HCMV gene functions

BBSRC战略主题：全面了解健康的生物科学东方体(OT)是立克次体的革兰氏阴性、专性细胞内α-蛋白细菌，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菌，如沃尔巴克氏菌。OT是由螨类传播的，会引起丛林斑疹伤寒。我们的研究重点是OT细菌细胞生物学的基本原理及其与真核宿主细胞的相互作用。OT通过网状蛋白介导的内吞作用或巨噬细胞吞噬作用进入细胞。然后，细菌逃离内小体，劫持宿主微管，向细胞核输送，并直接在细胞质中复制，形成核周克隆。众所周知，OT使宿主细胞处于一种非裂解的“萌芽”机制中，这使人想起病毒退出，释放被宿主细胞膜包围的单个细菌。然而，这种成熟所涉及的因素和退出机制仍不清楚。在纤毛螨中，取食诱导OT从受感染的唾液腺细胞中萌发出来。此外，OT还被证明分为两种不同的状态：胞内细菌(IB)和胞外细菌(EB)，前者是杆状的，翻译上活跃，后者是圆形的，翻译上不活跃。这种成熟的复杂性与立克次体家族的其他成员不同，这些状态的传染性和行为对于理解生理上相关的感染周期至关重要。解决哪些因素影响OT的成熟和宿主细胞退出的主要问题以前是不可能的，因为缺乏可靠和信息丰富的发芽试验。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发芽的阳性对照。因此，这个博士项目的一个关键目标是研究宿主细胞退出是如何被触发的。使用这项测试，我将初步测试一系列宿主细胞条件的影响，包括营养耗竭和内流、内质网应激、氧化应激和钙信号。我的初步数据显示，营养的可获得性和感染的多样性可能是进一步研究的有希望的候选者。一个重要的问题是，OT是“决定”单独离开还是以一致的方式离开，这意味着细菌之间可能存在感知通路和可能的交流。在萌芽过程中对OT的实时成像将在此时提供关于OT行为的更多信息。此外，研究成熟细菌中表达的蛋白质不仅可以为OT的成熟提供一个有价值的标记，还可以阐明细菌启动哪些机制来信号或感知宿主细胞的条件。该项目还旨在利用实时成像确定OT保留质膜被膜多长时间，以及这是否富含任何特定的宿主蛋白，因为这可能意味着重新感染不同类型的宿主细胞。最后，OT转运到质膜的机制尚不清楚；潜在的候选者(在病毒出口中起作用)包括动蛋白和肌动蛋白转运。我建议使用萌芽试验来检验贩运抑制剂对OT退出的影响；将对被干扰时取消OT退出的途径进行进一步的研究。

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.