Abstract
The zoonotic pathogen Coxiella burnetii, the causative agent of the human disease Q fever, is an ever-present danger to global public health. Investigating novel metabolic pathways necessary for C. burnetii to replicate within its unusual intracellular niche may identify new therapeutic targets. Recent studies employing stable isotope labelling established the ability of C. burnetii to synthesize lactate, despite the absence of an annotated synthetic pathway on its genome. A noncanonical lactate synthesis pathway could provide a novel anti-Coxiella target if it is essential for C. burnetii pathogenesis. In this study, two C. burnetii proteins, CBU1241 and CBU0823, were chosen for analysis based on their similarities to known lactate synthesizing enzymes. Recombinant GST-CBU1241, a putative malate dehydrogenase (MDH), did not produce measurable lactate in in vitro lactate dehydrogenase (LDH) activity assays and was confirmed to function as an MDH. Recombinant 6xHis-CBU0823, a putative NAD+-dependent malic enzyme, was shown to have both malic enzyme activity and MDH activity, however, did not produce measurable lactate in either LDH or malolactic enzyme activity assays in vitro. To examine potential lactate production by CBU0823 more directly, [13C]glucose labelling experiments compared label enrichment within metabolic pathways of a cbu0823 transposon mutant and the parent strain. No difference in lactate production was observed, but the loss of CBU0823 significantly reduced 13C-incorporation into glycolytic and TCA cycle intermediates. This disruption to central carbon metabolism did not have any apparent impact on intracellular replication within THP-1 cells. This research provides new information about the mechanism of lactate biosynthesis within C. burnetii, demonstrating that CBU1241 is not multifunctional, at least in vitro, and that CBU0823 also does not synthesize lactate. Although critical for normal central carbon metabolism of C. burnetii, loss of CBU0823 did not significantly impair replication of the bacterium inside cells.
Highlights
Coxiella burnetii is the Gram negative bacteria that causes the multifaceted human disease Q fever, an ever-present global public health threat [1]
Clustal Omega alignment of select malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) protein sequences revealed that CBU1241 and the putative MDH from Legionella pneumophila str
CBU1241 was more similar to the putative Thermus thermophilus MDH (56% identity) and the pig cytoplasmic MDH isozyme (46% identity) than to E. coli MDH (24% identity) (S1A Fig and S2A Table)
Summary
Coxiella burnetii is the Gram negative bacteria that causes the multifaceted human disease Q fever, an ever-present global public health threat [1]. Lactate production can be beneficial to bacteria by several means. It can be the end product of efficient metabolism pathways, play a role in redox and energy homeostasis, be metabolized an alternative carbon source, or modify host immune response [9,10,11,12,13,14,15,16,17,18]. Should unusual enzyme(s) responsible for lactate production in C. burnetii be determined and found to play an important role in the organism’s metabolism and pathogenicity, they may represent useful future antiCoxiella targets
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