Open AccessCharacterisation of putative lactate synthetic pathways of Coxiella burnetii
Author(s) -
Janine Hofmann,
Mebratu A. Bitew,
Miku Kuba,
David P. De Souza,
Hayley J. Newton,
Fiona M. Sansom
Publication year - 2021
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0255925
Subject(s) - coxiella burnetii , biology , biochemistry , malate dehydrogenase , lactate dehydrogenase , malic enzyme , lactate dehydrogenase a , citric acid cycle , glycolysis , metabolic pathway , enzyme , adenylate kinase , dehydrogenase , microbiology and biotechnology
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, [ 13 C]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 13 C-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.