A new dehydratase conferring innate resistance to thiacetazone and intra‐amoebal survival of <scp><i>M</i></scp><i>ycobacterium smegmatis</i>

Carrère-Kremer, Séverine; Blaise, Mickaël; Singh, Vipul K.; Alibaud, Laeticia; Tuaillon, Édouard; Halloum, Iman; Weerd, Robert van de; Guérardel, Yann; Drancourt, Michel; Takiff, Howard; Geurtsen, Jeroen; Kremer, Laurent

doi:10.1111/mmi.12992

Cited by 21 publications

(17 citation statements)

References 57 publications

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“…Then, 50 absorbance units were collected by centrifugation and washed three times with PBS. The extraction of the cell envelope lipids was performed in three steps to harvest the apolar, polar, and mycolic acid lipid fractions as previously described (47,48). Both apolar and polar lipid fractions were analyzed with two-dimensional TLC for PDIM and the LOS lipid contents system A and system E, respectively, as earlier described by Besra (43).…”

Section: Methodsmentioning

confidence: 99%

iniBAC induction Is Vitamin B12- and MutAB-dependent in Mycobacterium marinum

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Sparrius

Jim

et al. 2016

Journal of Biological Chemistry

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Tuberculosis can be treated with a 6-month regimen of antibiotics. Although the targets of most of the first-line antibiotics have been identified, less research has focused on the intrabacterial stress responses that follow upon treatment with antibiotics. Studying the roles of these stress genes may lead to the identification of crucial stress-coping mechanisms that can provide additional drug targets to increase treatment efficacy. A threegene operon with unknown function that is strongly up-regulated upon treatment with isoniazid and ethambutol is the ini-BAC operon. We have reproduced these findings and show that iniBAC genes are also induced in infected host cells, although with higher variability. Next, we set out to elucidate the genetic network that results in iniBAC induction in Mycobacterium marinum. By transposon mutagenesis, we identified that the operon is highly induced by mutations in genes encoding enzymes of the vitamin B12 biosynthesis pathway and the vitamin B12-dependent methylmalonyl-CoA-mutase MutAB. Lipid analysis showed that a mutA::tn mutant has decreased phthiocerol dimycocerosates levels, suggesting a link between iniBAC induction and the production of methyl-branched lipids. Moreover, a similar screen in Mycobacterium bovis BCG identified that phthiocerol dimycocerosate biosynthesis mutants cause the up-regulation of iniBAC genes. Based on these data, we propose that iniBAC is induced in response to mutations that cause defects in the biosynthesis of methyl-branched lipids. The resulting metabolic stress caused by these mutations or caused by ethambutol or isoniazid treatment may be relieved by iniBAC to increase the chance of bacterial survival.

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Section: Methodsmentioning

confidence: 99%

iniBAC induction Is Vitamin B12- and MutAB-dependent in Mycobacterium marinum

Boot

Sparrius

Jim

et al. 2016

Journal of Biological Chemistry

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“…We have previously shown that Mycobacterium smegmatis possesses a gene, MSMEG_6754, responsible for its remarkable resistance to TAC and involved in mycolic acid metabolism and that disruption of MSMEG_6754 in M. smegmatis was associated with impaired growth in Acanthamoeba castellanii (23). Interestingly, MSMEG_6754 encodes a putative dehydratase and is structurally related to the HadABC complex (23). Blast analyses revealed the presence of genes homologous to MSMEG_6754 in several NTMs, all of which possess intact hadABC genes (23).…”

Section: Significancementioning

confidence: 99%

“…Moreover, deletion of hadC in M. tuberculosis correlates with a loss of cording and attenuation of virulence in infected mice (22), thus emphasizing the intimate interplay between TAC resistance, mycolic acid biosynthesis, cording, and virulence. We have previously shown that Mycobacterium smegmatis possesses a gene, MSMEG_6754, responsible for its remarkable resistance to TAC and involved in mycolic acid metabolism and that disruption of MSMEG_6754 in M. smegmatis was associated with impaired growth in Acanthamoeba castellanii (23). Interestingly, MSMEG_6754 encodes a putative dehydratase and is structurally related to the HadABC complex (23).…”

Section: Significancementioning

confidence: 99%

“…Interestingly, MSMEG_6754 encodes a putative dehydratase and is structurally related to the HadABC complex (23). Blast analyses revealed the presence of genes homologous to MSMEG_6754 in several NTMs, all of which possess intact hadABC genes (23). The presence of such a homolog in Mabs, designated MAB_4780, prompted us to perform the biochemical/ structural characterization of MAB_4780 to investigate its biological function with respect to mycolic acid biosynthesis.…”

Section: Significancementioning

confidence: 99%

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Deletion of a dehydratase important for intracellular growth and cording renders rough Mycobacterium abscessus avirulent

Halloum

Carrère-Kremer

Blaise

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Mycobacterium abscessus (Mabs) is a rapidly growing Mycobacterium and an emerging pathogen in humans. Transitioning from a smooth (S) high-glycopeptidolipid (GPL) producer to a rough (R) low-GPL producer is associated with increased virulence in zebrafish, which involves the formation of massive serpentine cords, abscesses, and rapid larval death. Generating a cord-deficient Mabs mutant would allow us to address the contribution of cording in the physiopathological signs of the R variant. Herein, a deletion mutant of MAB_4780, encoding a dehydratase, distinct from the β-hydroxyacyl-ACP dehydratase HadABC complex, was constructed in the R morphotype. This mutant exhibited an alteration of the mycolic acid composition and a pronounced defect in cording. This correlated with an extremely attenuated phenotype not only in wild-type but also in immunocompromised zebrafish embryos lacking either macrophages or neutrophils. The abolition of granuloma formation in embryos infected with the dehydratase mutant was associated with a failure to replicate in macrophages, presumably due to limited inhibition of the phagolysosomal fusion. Overall, these results indicate that MAB_4780 is required for Mabs to successfully establish acute and lethal infections. Therefore, targeting MAB_4780 may represent an attractive antivirulence strategy to control Mabs infections, refractory to most standard chemotherapeutic interventions. The combination of a dehydratase assay with a highresolution crystal structure of MAB_4780 opens the way to identify such specific inhibitors.M. abscessus | zebrafish | cording | dehydratase | virulence

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“…Upon activation, TAC has recently been shown to bind to the HadA component of the HadABC dehydratase complex, leading to inhibition of mycolic acid biosynthesis (15). Interestingly, we and others have reported that NTM such as Mycobacterium avium (16), Mycobacterium smegmatis (17), and M. abscessus (18) are naturally resistant to TAC. However, a TAC analogue, SRI-224, was previously tested against M. avium and found to be more effective than TAC in vitro as well as in mice (16) and subsequently shown to be very active against M. tuberculosis (19).…”

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confidence: 99%

Resistance to Thiacetazone Derivatives Active against Mycobacterium abscessus Involves Mutations in the MmpL5 Transcriptional Repressor MAB_4384

Halloum

Viljoen

Khanna

et al. 2017

Antimicrob Agents Chemother

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Available chemotherapeutic options are very limited against Mycobacterium abscessus, which imparts a particular challenge in the treatment of cystic fibrosis (CF) patients infected with this rapidly growing mycobacterium. New drugs are urgently needed against this emerging pathogen, but the discovery of active chemotypes has not been performed intensively. Interestingly, however, the repurposing of thiacetazone (TAC), a drug once used to treat tuberculosis, has increased following the deciphering of its mechanism of action and the detection of significantly more potent analogues. We therefore report studies performed on a library of 38 TAC-related derivatives previously evaluated for their antitubercular activity. Several compounds, including D6, D15, and D17, were found to exhibit potent activity in vitro against M. abscessus, Mycobacterium massiliense, and Mycobacterium bolletii clinical isolates from CF and non-CF patients. Similar to TAC in Mycobacterium tuberculosis, the three analogues act as prodrugs in M. abscessus, requiring bioactivation by the EthA enzyme, MAB_0985. Importantly, mutations in the transcriptional TetR repressor MAB_4384, with concomitant upregulation of the divergently oriented adjacent genes encoding an MmpS5/MmpL5 efflux pump system, accounted for high cross-resistance levels among all three compounds. Overall, this study uncovered a new mechanism of drug resistance in M. abscessus and demonstrated that simple structural optimization of the TAC scaffold can lead to the development of new drug candidates against M. abscessus infections.

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A new dehydratase conferring innate resistance to thiacetazone and intra‐amoebal survival of Mycobacterium smegmatis

Cited by 21 publications

References 57 publications

iniBAC induction Is Vitamin B12- and MutAB-dependent in Mycobacterium marinum

iniBAC induction Is Vitamin B12- and MutAB-dependent in Mycobacterium marinum

Deletion of a dehydratase important for intracellular growth and cording renders rough Mycobacterium abscessus avirulent

Resistance to Thiacetazone Derivatives Active against Mycobacterium abscessus Involves Mutations in the MmpL5 Transcriptional Repressor MAB_4384

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