Rotation 1: Characterising HCMV gene functions

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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.