Disruption of MenT2 toxin impairs the growth of Mycobacterium tuberculosis in guinea pigs

Abstract

Toxin–antitoxin (TA) systems are abundantly present in the genomes of various bacterial pathogens. TA systems have been implicated in either plasmid maintenance or protection against phage infection, stress adaptation or disease pathogenesis. The genome of Mycobacterium tuberculosis encodes for more than 90 TA systems and 4 of these belong to the type IV subfamily (MenAT family). The toxins and antitoxins belonging to type IV TA systems share sequence homology with the AbiEii family of nucleotidyl transferases and the AbiEi family of putative transcriptional regulators, respectively. Here, we have performed experiments to understand the role of MenT2, a toxin from the type IV TA system, in mycobacterial physiology and disease pathogenesis. The ectopic expression of MenT2 using inducible vectors does not inhibit bacterial growth in liquid cultures. Bioinformatic and molecular modelling analysis suggested that the M. tuberculosis genome has an alternative start site upstream of the annotated menT2 gene. The overexpression of the reannotated MenT2 resulted in moderate growth inhibition of Mycobacterium smegmatis . We show that both menT2 and menA2 transcript levels are increased when M. tuberculosis is exposed to nitrosative stress, in vitro. When compared to the survival of the wild-type and the complemented strain, the ΔmenT2 mutant strain of M. tuberculosis was more resistant to being killed by nitrosative stress. However, the survival of both the ΔmenT2 mutant and the wild-type strain was similar in macrophages and when exposed to other stress conditions. Here, we show that MenT2 is required for the establishment of disease in guinea pigs. Gross pathology and histopathology analysis of lung tissues from guinea pigs infected with the ∆menT2 strain revealed significantly reduced tissue damage and inflammation. In summary, these results provide new insights into the role of MenT2 in mycobacterial pathogenesis.

Mycobacterium tuberculosis strain with deletions in menT3 and menT4 is attenuated and confers protection in mice and guinea pigs

“…Here, we report that relative to the parental strain, Δ menT4 Δ T3 strain was attenuated for growth at both acute and chronic stages of infection in guinea pigs. As observed in the case of ∆mazF3∆9∆6 or ∆vapC22 or Δ higB1 or Δ vapBC3 or Δ vapBC4 or Δ vapBC11 or Δ menT2 , the growth defect associated with Δ menT4 Δ T3 strain was more prominent in spleens and during the chronic stage of infection in guinea pigs 21 , 23 , 25 – 27 , 35 . In agreement with guinea pig data, we observed that the deletion of menT3 and menT4 also impaired the growth of M. tuberculosis in mice tissues.…”

“…However, deletions in either relE1 or relE2 or relE3 or vapC28 or vapC21 or darTG did not reduce M. tuberculosis growth in guinea pigs or mice 21 , 24 , 33 , 34 . In addition to type II TA systems, we have recently shown that MenT2 toxin belonging to the MenAT subfamily is also essential for M. tuberculosis pathogenesis in guinea pigs 35 . Previously, high throughput screening assays such as transposon site hybridization (TRASH) and designer array for defined mutant analysis (DeADMAn) have been performed to identify genes necessary for in vivo growth of M. tuberculosis 36 , 37 .…”

“…The cultures were harvested by centrifugation and washed twice with 1x PBS. Subsequently, apolar and polar lipids were extracted as previously described 35 . An equal amount of different lipid fractions was loaded on silica plates and resolved using one-dimensional TLC in the different solvent systems as described previously 35 .…”

“…Subsequently, apolar and polar lipids were extracted as previously described 35 . An equal amount of different lipid fractions was loaded on silica plates and resolved using one-dimensional TLC in the different solvent systems as described previously 35 . Different lipids were visualized by staining the TLC with 5% molybdophosphoric acid in ethanol and subsequent charring at 100 °C.…”

Nucleotidyltransferase toxin MenT targets and extends the aminoacyl acceptor ends of serine tRNAs in vivo to control Mycobacterium tuberculosis growth

“…tuberculosis encodes four MenAT TA systems, comprised of a nucleotidyltransferase (NTase) toxin and a cognate antitoxin belonging to one of three different families (Dy et al, 2014;Xu et al, 2023;Cai et al, 2020). Although, in vivo, menAT2 was recently shown to be required for M. tuberculosis pathogenesis in guinea pigs (Gosain et al, 2022), only menAT1 and menAT3 were shown to act as bona fide TA systems in their native host M. tuberculosis (Xu et al, 2023;Cai et al, 2020). The MenA3 antitoxin inhibits MenT3 through phosphorylation of a serine residue in the catalytic site (Yu et al, 2020), whilst MenA1 forms an asymmetric heterotrimeric complex with two MenT1 protomers, suggesting a different mode of inhibition (Xu et al, 2023).…”