The zoonotic pathogen Coxiella burnetii poses a serious threat to global public health. C. burnetii replicates intracellularly within a unique vacuole derived from the phagolysosome, known as the Coxiella-containing vacuole (CCV). To cause disease, C. burnetii must not only survive the bactericidal environment within this vacuole, but also obtain energy and nutrients to replicate. Investigating the metabolic pathways required by C. burnetii to survive inside host cells may identify novel therapeutic targets.
Recent stable isotope labelling studies revealed that C. burnetii is capable of synthesising lactate, despite the apparent absence of a genetic pathway for lactate production. In this study we are investigating two potential lactate-producing pathways. Malolactic enzymes, found in lactic acid bacteria, convert malate to lactate. Our bioinformatic analysis revealed that Cbu823, currently annotated as a NAD-dependent malic enzyme, possesses 43% identity with the malolactic enzyme of the lactic acid bacterium Oenococcus oeni. As C. burnetii already possesses a putative malate dehydrogenase, MDH (Cbu1241), Cbu0823 may function as a malolactic enzyme. Alternatively, Cbu1241 may perform a dual function and possess MDH and LDH activity.
We have successfully expressed and purified recombinant Cbu0823 and Cbu1241 as 6xHis N-terminal fusion and GST N-terminal fusion proteins respectively. In vitro enzyme assays demonstrated that Cbu1241 possesses MDH activity but lacks LDH activity, at least in vitro, and that Cbu0823 has malic enzyme activity. Current work is examining the malolactic enzyme activity of Cbu0823. Future work will examine the role of this pathway in C. burnetii intracellular replication.