Coxiella burnetii control of the host transcription factors TFEB and TFE3
AffiliationMicrobiology & Immunology
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2022-11-05. This item is currently available to University of Melbourne staff and students only, login required.
© 2020 Bhavna Padmanabhan
Coxiella burnetii, the etiological agent of the zoonotic disease Q fever, is an obligate Gram-negative intracellular bacterial pathogen that replicates inside the lysosome-derived CCV (Coxiella-containing vacuole) within mammalian hosts. The CCV maintains the degradative and acidic nature of the host lysosome despite C. burnetii directing the massive expansion of this compartment to accommodate the replicating pathogen. To establish this unique replicative niche, C. burnetii requires the Dot/Icm type IV secretion system (T4SS). This T4SS translocates approximately 150 effectors into the host cell to modulate various cellular processes. To date, the functional role of very few of these effectors have been defined. Given the CCV’s origins it is not surprising that C. burnetii infection increases host autophagy and lysosome biogenesis. To investigate this at the protein level, we employed an elegant SILAC based proteome analysis of human cells infected with C. burnetii. This validated that many proteins involved in these processes are increased in abundance during infection. This prompted us to examine the role of the human transcription factor EB (TFEB) and its close homologue TFE3 during C. burnetii infection. TFEB is a master transcription regulator directly controlling the expression of a network of genes responsible for autophagy and lysosome biogenesis. 3 day’s post-infection with C. burnetii, TFEB/TFE3 is activated as demonstrated by TFEB/TFE3 trafficking from the cytoplasm into the nucleus. The nuclear translocation of TFEB/TFE3 appears to be controlled by C. burnetii as blocking bacterial translation with chloramphenicol leads to TFEB/TFE3 movement back into the cytoplasm. siRNA silencing of tfeb and tfe3 additionally demonstrated their contribution towards the intracellular success of C. burnetii. Interestingly, these host factors did not contribute to the replication of C. burnetii but in the absence of TFEB and TFE3 the CCV did not undergo its typical massive expansion. This research was able to demonstrate that C. burnetii induced activation of TFEB/TFE3 was dependent on the Dot/Icm T4SS thus we hypothesized that an effector(s) of this system may manipulate TFEB/TFE3. An unbiased visual screen was conducted to identify effectors that influence this process and two putative C. burnetii effector proteins, namely CBU1701 and CBU2016, were identified. We demonstrated that ectopic expression of these proteins leads to nuclear localisation of TFEB. Subsequent characterisation of the impact of CBU1701 and CBU2016, demonstrated that they influence the host proteome in similar ways but with surprisingly little impact on TFEB- regulated proteins. These results indicated that nuclear localisation of TFEB in response to CBU1701 and CBU2016 may be uncoupled from activation of this host transcription factor. In addition to characterising the impact of these effector proteins on host cellular function, we set out to understand the role they play in intracellular replication and virulence of C. burnetii. Genetic manipulation of this pathogen is in its infancy and remains technically challenging however this study reports the successful production of multiple mutant strains. Initial characterisation experiments demonstrate that CBU1701 and CBU2016 likely make important contributions to the establishment of the C. burnetii intracellular niche. Overall, the research carried out in this thesis has worked towards elucidating the contribution of TFEB and TFE3 to C. burnetii infection and developing an understanding of the molecular players in this process. Significant tool development and foundational findings reported here will pave the way to a deeper understanding of the interplay between intracellular bacterial pathogens and the host response to infection. Additionally, using C. burnetii effector proteins as a novel biological toolbox may uncover important insights that will impact our understanding of a range of human molecular pathways that impact human health.
KeywordsCoxiella burnetii; CCV; Coxiella containing vacuole; TFE3; Lysosome; biogenesis; intracellular; replication; siRNA; effectors; CBU1701; CBU2016; Dot/Icm; host; transcription factor; proteome; mutant; TFEB
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