Understanding the mechanisms utilised by Coxiella burnetii for intracellular replication within the host phagolysosome
AffiliationMicrobiology & Immunology
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2022-08-26. This item is currently available to University of Melbourne staff and students only, login required.
© 2020 Miku Kuba
The human pathogen Coxiella burnetii is the causative agent of Q fever, a febrile illness which may lead to chronic disease in a small percentage of infected individuals. C. burnetii is a unique Gram negative intracellular bacterium which replicates within a host cell lysosome derived vacuole, termed the Coxiella containing vacuole (CCV). Currently, the exact mechanisms which allow C. burnetii to replicate within this normally hostile compartment are unknown. In order to understand how C. burnetii survives within this intracellular niche, this research investigated carbon metabolism of both intracellular and axenically cultivated bacteria, using steady state metabolic profiling and 13C stable isotope labelling. Both C. burnetii populations were shown to assimilate exogenous [13C]glutamate and [13C]glucose, with concomitant labelling of intermediates in glycolysis and gluconeogenesis, and in the TCA cycle. Significantly, the two populations displayed metabolic pathway profiles reflective of the nutrient availabilities within their propagated environments. Disruption of the C. burnetii glucose transporter, CBU0265, by transposon mutagenesis led to a significant decrease in [13C]glucose utilisation but did not abolish glucose usage, suggesting that C. burnetii express additional hexose transporters that may be able to compensate for the loss of CBU0265. This was supported by intracellular infection of human cells and in vivo studies in the Galleria mellonella insect model showing loss of CBU0265 had no impact on intracellular replication or virulence. Using this mutagenesis and [13C]glucose labelling approach, this study identified a second glucose transporter, CBU0347, the disruption of which also showed significant decreases in 13C label incorporation. Despite maintaining a relatively small genome, C. burnetii have retained seemingly redundant strategies to obtain glucose. This suggests that glucose may be an important metabolite for C. burnetii. Together, these analyses indicate that C. burnetii may use multiple carbon sources in vivo and exhibits greater metabolic flexibility than expected. In addition, this thesis also investigated the novel and unique C. burnetii protein CBU2072, a small, 18.3 kDa protein, which has subsequently been named essential for intracellular replication A (EirA), as loss of this protein prevents replication of C. burnetii within the CCV. Intracellular replication of the EirA mutant can be restored during co infection of the same vacuole with C. burnetii wild type, which is analogous to the phenotype observed for mutants of the Dot/Icm type 4B secretion system in C. burnetii. EirA localises to the C. burnetii inner membrane, and absence of this protein leads to the loss of Dot/Icm effector translocation. These data together contribute important understanding of the unique mechanisms involved in C. burnetii pathogenesis within the host phagolysosome.
KeywordsBacterial Pathogenesis; Virulence Factor; Carbon Metabolism; Glucose Transport; Intracellular replication; Gram negative Bacteria; Host-Pathogen Interactions; Coxiella burnetii; Type IVB Secretion System; Dot/Icm Secretion System; Metabolomics
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