Characterisation of putative lactate synthetic pathways of Coxiella burnetii.

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. Rec...

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Autores principales:	Janine Hofmann, Mebratu A Bitew, Miku Kuba, David P De Souza, Hayley J Newton, Fiona M Sansom
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/59adb9cd552d42529f646706aa8879d9
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spelling	oai:doaj.org-article:59adb9cd552d42529f646706aa8879d92021-12-02T20:18:09ZCharacterisation of putative lactate synthetic pathways of Coxiella burnetii.1932-620310.1371/journal.pone.0255925https://doaj.org/article/59adb9cd552d42529f646706aa8879d92021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0255925https://doaj.org/toc/1932-6203The 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.Janine HofmannMebratu A BitewMiku KubaDavid P De SouzaHayley J NewtonFiona M SansomPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 8, p e0255925 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Janine Hofmann Mebratu A Bitew Miku Kuba David P De Souza Hayley J Newton Fiona M Sansom Characterisation of putative lactate synthetic pathways of Coxiella burnetii.
description	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.
format	article
author	Janine Hofmann Mebratu A Bitew Miku Kuba David P De Souza Hayley J Newton Fiona M Sansom
author_facet	Janine Hofmann Mebratu A Bitew Miku Kuba David P De Souza Hayley J Newton Fiona M Sansom
author_sort	Janine Hofmann
title	Characterisation of putative lactate synthetic pathways of Coxiella burnetii.
title_short	Characterisation of putative lactate synthetic pathways of Coxiella burnetii.
title_full	Characterisation of putative lactate synthetic pathways of Coxiella burnetii.
title_fullStr	Characterisation of putative lactate synthetic pathways of Coxiella burnetii.
title_full_unstemmed	Characterisation of putative lactate synthetic pathways of Coxiella burnetii.
title_sort	characterisation of putative lactate synthetic pathways of coxiella burnetii.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/59adb9cd552d42529f646706aa8879d9
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Characterisation of putative lactate synthetic pathways of Coxiella burnetii.

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