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Mycofactocin is critical for Mycobacterium tuberculosis adaptation into microaerophilic conditions

ABSTRACT: Mycofactocin is a new class of peptide-derived redox cofactor whose structural elucidation and functional characterization have recently received enormous focus. Among the six-gene cluster, mftD mediates a probable penultimate step in mycofactocin biosynthesis. MftD is a putative lactate dehydrogenase predicted critical for Mycobacterium tuberculosis survival under oxygen-limited conditions. In this study, mftD transcripts levels were found significantly increased in M. tuberculosis cells adapted to 0.01% oxygen. mftD deletion mutant of M. tuberculosis exhibited survival deficit in in vitro hypoxia and wayne models. However, mftD functions was found dispensable for M. tuberculosis L-Lactate metabolism. Rather surprisingly, the growth fitness of mftD mutant was increased in glucose under aerobic conditions. While the cause of this in vitro phenotype remains unestablished, the levels of NAD(P)H and glucose-6-phosphate dehydrogenase activity was found decreased in ΔmftD when compared to its parental strain. Increased growth fitness did not have any major impact on bacterial cell shape and size except the formation of extracellular fibril-like structures in subpopulations of ΔmftD. Cell-surface proteins analysis showed that genetic deficiency of mycofactocin likely to predispose accumulation of cofactor-free protein aggregates resembling destabilization of the flavoproteome upon riboflavin deprivation. Nevertheless, like in vitro findings, disruption of mftD increased the fitness of M. tuberculosis in C57BL/6J mice at early phase and resulted in growth stasis in a Nos2-/- hypoxia mouse model. Collectively these results establish the relevance of MftD in M. tuberculosis growth, redox and cofactor homeostasis, and pathogenesis.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mycobacterium Smegmatis

SUBMITTER: Christian Frese

LAB HEAD: Christian Frese

PROVIDER: PXD023045 | Pride | 2021-07-05

REPOSITORIES: Pride

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Project description:Fang2010 - Genome-scale metabolic network of Mycobacterium tuberculosis (iNJ661m) This model is described in the article: Development and analysis of an in vivo-compatible metabolic network of Mycobacterium tuberculosis. Fang X, Wallqvist A, Reifman J. BMC Syst Biol 2010; 4: 160 Abstract: BACKGROUND: During infection, Mycobacterium tuberculosis confronts a generally hostile and nutrient-poor in vivo host environment. Existing models and analyses of M. tuberculosis metabolic networks are able to reproduce experimentally measured cellular growth rates and identify genes required for growth in a range of different in vitro media. However, these models, under in vitro conditions, do not provide an adequate description of the metabolic processes required by the pathogen to infect and persist in a host. RESULTS: To better account for the metabolic activity of M. tuberculosis in the host environment, we developed a set of procedures to systematically modify an existing in vitro metabolic network by enhancing the agreement between calculated and in vivo-measured gene essentiality data. After our modifications, the new in vivo network contained 663 genes, 838 metabolites, and 1,049 reactions and had a significantly increased sensitivity (0.81) in predicted gene essentiality than the in vitro network (0.31). We verified the modifications generated from the purely computational analysis through a review of the literature and found, for example, that, as the analysis suggested, lipids are used as the main source for carbon metabolism and oxygen must be available for the pathogen under in vivo conditions. Moreover, we used the developed in vivo network to predict the effects of double-gene deletions on M. tuberculosis growth in the host environment, explore metabolic adaptations to life in an acidic environment, highlight the importance of different enzymes in the tricarboxylic acid-cycle under different limiting nutrient conditions, investigate the effects of inhibiting multiple reactions, and look at the importance of both aerobic and anaerobic cellular respiration during infection. CONCLUSIONS: The network modifications we implemented suggest a distinctive set of metabolic conditions and requirements faced by M. tuberculosis during host infection compared with in vitro growth. Likewise, the double-gene deletion calculations highlight the importance of specific metabolic pathways used by the pathogen in the host environment. The newly constructed network provides a quantitative model to study the metabolism and associated drug targets of M. tuberculosis under in vivo conditions. This model is hosted on BioModels Database and identified by: MODEL1507180018. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Previous findings have demonstrated that the NADH/NAD+ ratio has a strong impact on the glycolytic flux in C. glutamicum under anaerobic conditions in the absence of external electron acceptors. During an attempt to rewire anaerobic metabolism to achieve high yield formation of ethanol, we inactivated the malate dehydrogenase and lactate dehydrogenase in a C. glutamicum strain expressing pyruvate decarboxylase and alcohol dehydrogenase, to eliminate formation of the by-products succinate and lactate, respectively. This modification increased the yield of ethanol but had a negative effect on glycolysis, which we found to correlate with an elevated NADH/NAD+ ratio. The pyruvate dehydrogenase (PDH) of C. glutamicum is active under anaerobic conditions, and can potentially exacerbate the negative effect on glycolysis, due to NADH formation. To reduce PDH activity under anaerobic conditions, we decided to replace the gene encoding the E3 subunit of PDH with its Escherichia coli counterpart, as E. coli PDH has been reported to be functional under aerobic conditions only. The resultant strain JS133 produced far less acetate with a further increased ethanol production, however, the glycolytic flux was still low. After observing differences in glycolytic flux for JS133 on glucose and fructose, we speculated that the pentose phosphate pathway (PPP) might be involved in the reduced flux on glucose. To prove this, we deleted the zwf gene, encoding glucose-6-phosphate dehydrogenase, which is the entry point into PPP, and immediately observed a stimulating effect on glycolysis. Subsequent characterization revealed a direct correlation between the intracellular NADH/NAD+ and NADPH/NADP+ ratios under anoxic conditions. Based on these findings we managed to re-channel the metabolism of C. glutamicum successfully towards either to ethanol or D-lactate with 92% and 98% of the theoretical yield respectively, which is the highest yields for D-lactate production thus far reported in the literature.

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Mycofactocin is critical for Mycobacterium tuberculosis adaptation into microaerophilic conditions

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