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1. Characterisation of putative lactate synthetic pathways of Coxiella burnetii.

Author

Janine Hofmann, Mebratu A Bitew, Miku Kuba, David P De Souza, Hayley J Newton, and Fiona M Sansom

Subjects
Medicine, Science
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.

Published
2021
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2. Characterisation of putative lactate synthetic pathways of Coxiella burnetii

Author

Hayley J. Newton, Miku Kuba, Fiona M. Sansom, Janine Hofmann, David P De Souza, and Mebratu A Bitew

Subjects
Metabolic Processes, THP-1 Cells, Enzyme Metabolism, Pathology and Laboratory Medicine, Biochemistry, chemistry.chemical_compound, Medicine and Health Sciences, Enzyme Chemistry, Multidisciplinary, biology, Chemistry, Biochemical Cofactors, Malolactic enzyme activity, Ketones, Bacterial Pathogens, Enzymes, Bioassays and Physiological Analysis, Coxiella burnetii, Medical Microbiology, Host-Pathogen Interactions, Physical Sciences, Medicine, Metabolic Labeling, Pathogens, Q Fever, Research Article, Pyruvate, Science, Citric Acid Cycle, Malic enzyme, Research and Analysis Methods, Microbiology, Malate dehydrogenase, Lactate dehydrogenase, Humans, Malic enzyme activity, Lactic Acid, Molecular Biology Techniques, Microbial Pathogens, Molecular Biology, Enzyme Assays, Chemical Compounds, Biology and Life Sciences, Proteins, bacterial infections and mycoses, biology.organism_classification, Citric acid cycle, Metabolic pathway, Metabolism, Cell Labeling, Vacuoles, Enzymology, bacteria, Biochemical Analysis, Acids
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.

Published
2021

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