New therapeutic strategies for <i>Mycobacterium abscessus</i> pulmonary diseases – untapping the mycolic acid pathway

Alcaraz, Matthéo; Edwards, Thomas E.; Kremer, Laurent

doi:10.1080/14787210.2023.2224563

Cited by 4 publications

(2 citation statements)

References 152 publications

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“…We note that, even at the highest concentrations of ATc tested (500 and 1,000 ng/mL), both S Δ mmpL3 ::c and R Δ mmpL3 ::c grew very slowly in liquid media, presumably indicating that the promoter is leaky, as judged by the low levels of MmpL3 produced under these repressive conditions, which translates into a partial inhibition of TDM production and AG mycolylation. Nevertheless, despite the presence of residual levels of MmpL3 upon ATc-induced repression, these results highlight the vulnerability of MmpL3 and the ensuing implications for future chemotherapeutic developments for M. abscessus infections, as exemplified by the wide list of chemotypes inhibiting this transporter in M. abscessus ( 16 , 32 – 36 ). Recent sequencing of a saturated transposon library in M. abscessus provided a comprehensive prediction of essential genes in this mycobacterial species ( 40 ).…”

Section: Discussionmentioning

confidence: 84%

“…MmpL3 conditional mutants using the Tet system were successfully used to demonstrate mmpL3 gene essentiality in M. tuberculosis (Δ Rv0206c-mmpL3 Mtb using the TetR/Pip-OFF system) ( 30 ) as well as in M. smegmatis (Δ MSMEG0250-mmpL3 Msm using the acetamide-inducible system) ( 31 ), implying that MmpL3 represents an attractive pharmacological target in these species. Subsequent work from various groups identified a wide panel of chemical entities inhibiting MmpL3 in M. abscessus ( 16 , 32 – 36 ), although the essentiality of mmpL3 in M. abscessus remains to be established. To do so, the mCherry coding sequence was replaced by an influenza hemagglutinin (HA)-tagged mmpL3 version into the dual-reporter plasmid, generating the pMV306-TetOFF- mmpL3-HA ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Silencing essential gene expression in Mycobacterium abscessus during infection

Boudehen,

Tasrini,

Aguilera-Correa

et al. 2023

Microbiol Spectr

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Mycobacterium abscessus is a multi-drug-resistant non-tuberculous mycobacterial species causing tuberculosis-like lung infections. The functional characterization of the extensive repertoire of genes encoding virulence factors and drug targets has been largely restricted by the lack of powerful genetic tools. In this study, we evaluated the performances of a Tet-OFF system, previously optimized for M. tuberculosis and M. smegmatis . Fluorescence reporter M. abscessus strains were developed where mCherry was under the control of the Tet-OFF regulatory system on an integrative plasmid. The addition of anhydrotetracycline (ATc) correlated with a decrease in fluorescence intensity not only in solid and broth medium but also in colony biofilms, for both smooth and rough variants of M. abscessus . Next, unmarked mmpL3 conditional knockdown mutants were engineered in both smooth and rough M. abscessus . Biochemical studies indicated that the addition of ATc was associated with a dose-dependent decrease in (i) the production of the MmpL3 protein, (ii) the biosynthesis of trehalose dimycolate and mycolylated arabinogalactan, (iii) bacterial viability on agar and liquid medium as well as in biofilms. Importantly, intravenous injection of ATc in zebrafish embryos infected with the rough mmpL3 conditional mutant impeded bacterial growth and correlated with a significant gain in embryo survival. These results suggest that mmpL3 is essential for M. abscessus growth in vitro and in the infected host, thus validating MmpL3 as an attractive drug target. Together, this study demonstrates the potential of ATc-dependent repression to modulate the expression of M. abscessus genes in the infected host. IMPORTANCE Mycobacterium abscessus represents the most common rapidly growing mycobacterial pathogen in cystic fibrosis and is extremely difficult to eradicate. Essential genes are required for growth, often participate in pathogenesis, and encode valid drug targets for further chemotherapeutic developments. However, assessing the function of essential genes in M. abscessus remains challenging due to the limited spectrum of efficient genetic tools. Herein, we generated a Tet-OFF-based system allowing to knock down the expression of mmpL3 , encoding the mycolic acid transporter in mycobacteria. Using this conditional mutant, we confirm the essentiality of mmpL3 in planktonic cultures, in biofilms, and during infection in zebrafish embryos. Thus, in this study, we developed a robust and reliable method to silence the expression of any M. abscessus gene during host infection.

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