Recruitment and function of ORP1L on the Coxiella burnetii parasitophorous vacuole

dc.contributor.advisorGilk, Stacey D.
dc.contributor.authorJustis, Anna Victoria
dc.contributor.otherSpinola, Stanley M.
dc.contributor.otherNelson, David
dc.contributor.otherArrizabalaga, Gustavo A.
dc.contributor.otherHarrington, Maureen A.
dc.date.accessioned2018-03-13T15:35:57Z
dc.date.available2018-03-13T15:35:57Z
dc.date.issued2017-12-07
dc.degree.date2018en_US
dc.degree.disciplineDepartment of Microbiology and Immunology
dc.degree.grantorIndiana Universityen_US
dc.degree.levelPh.D.en_US
dc.descriptionIndiana University-Purdue University Indianapolis (IUPUI)en_US
dc.description.abstractCoxiella burnetii, the zoonotic agent of human Q fever and chronic endocarditis, is an obligate intracellular bacterial pathogen. The Coxiella intracellular niche, a large, lysosome-like parasitophorous vacuole (PV), is essential for bacterial survival and replication. There is growing evidence that host cell cholesterol trafficking plays a critical role in PV development and maintenance, prompting an examination of the role of cholesterol-binding host protein ORP1L (Oxysterol binding protein-Related Protein 1, Long) during infection. ORP1L is a multi-functional cholesterol-binding protein involved in late endosome/lysosome (LEL) trafficking, formation of membrane contact sites between LEL and the endoplasmic reticulum (ER), and cholesterol transfer from LEL to the ER. ORP1L localizes to the PV at novel membrane contact sites between the ER and the PV membrane. Ectopically expressed ORP1L in Coxiella-infected cells localizes to the PV membrane early during infection, before significant PV expansion and independent of other PV-localized proteins. Further, the N-terminal ORP1L Ankyrin repeats are both necessary and sufficient for PV localization, suggesting that protein-protein interactions, and not protein-lipid interactions, are primarily involved in PV association. Coxiella employs a Type IVB Secretion System (T4BSS) to translocate effector proteins into the host cytoplasm and manipulate various cellular functions. ORP1L is not found on the PV of a Coxiella mutant lacking a functional T4BSS, indicating a secreted bacterial protein is likely responsible for ORP1L recruitment. We identified a Coxiella mutant with a transposon insertion in CBU_0352 that exhibits a 50% decrease in ORP1L recruitment, suggesting that Coxiella CBU_0352 interacts directly or indirectly with ORP1L. Finally, we found that ORP1L depletion using siRNA alters PV dynamics, resulting in smaller yet more fusogenic Coxiella PVs. Together, these data suggest that ORP1L is specifically recruited to the PV, where it plays a novel role in Coxiella PV development and interactions between the PV and the host cell.en_US
dc.identifier.doi10.7912/C2XH1C
dc.identifier.urihttps://hdl.handle.net/1805/15448
dc.identifier.urihttp://dx.doi.org/10.7912/C2/1752
dc.language.isoen_USen_US
dc.subjectCoxiella burnetiien_US
dc.subjectORP1Len_US
dc.subjectCholesterolen_US
dc.subjectEndoplasmic reticulumen_US
dc.subjectMembrane contact sitesen_US
dc.subjectVacuoleen_US
dc.titleRecruitment and function of ORP1L on the Coxiella burnetii parasitophorous vacuoleen_US
dc.typeThesis
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