The mycobacterial mutasome: composition and recruitment in live cells

Abstract

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded “mycobacterial mutasome” – minimally comprising DnaE2 polymerase and ImuA′ and ImuB accessory proteins – remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

Distinctive roles of translesion polymerases DinB1 and DnaE2 in diversification of the mycobacterial genome through substitution and frameshift mutagenesis

“…This shared role raises several questions about how DinB1 and DnaE2 cooperate or compete at the replication fork. Both DinB1 and DnaE2 interact with the β clamp, DinB1 directly and DnaE2 via the ImuB protein (Gessner et al, 2021; Warner et al, 2010). Whether these two proteins compete for the same binding site, are recruited based on the type of damage, or can occupy different sites on the β clamp remains to be determined.…”

“…This shared role raises several questions about how DinB1 and DnaE2 cooperate or compete at the replication fork. Both DinB1 and DnaE2 interact with the β clamp, DinB1 directly and DnaE2 via the ImuB protein (Gessner et al, 2021;Warner et al, 2010).…”

RecA-NT homology motif in ImuB is essential for mycobacterial ImuA’-ImuB protein interaction and mutasome function

“…Therefore, to confirm the inferred disruption of the ImuAʹ-ImuB complex, a biochemical approach was taken. We recently reported successful expression of ImuA' and ImuB proteins in complex 5 , noting that, in the absence of ImuB, ImuAʹ appears to be mostly insoluble. We took advantage of this observation by predicting that mutants affecting the interaction between the two proteins would be expected to yield lower amounts of soluble ImuAʹ rescued by co-expression with ImuB.…”

“…ImuB-β co-localisation in vivo 5 ; however, the precise molecular function of ImuB is not known. The function of ImuAʹ is even less clear, but homology to RecA makes it tempting to postulate a structural role for ImuAʹ similar to that of RecA in binding components of a mutagenic complex and regulating its activity 2,6,7 .…”

Anti-tuberculosis treatment strategies and drug development: challenges and priorities

“…Though not falling under the category of tolerance per se, other indirect mechanisms facilitate the emergence of resistance and could be targeted to prevent or limit it. For example, a preliminary study suggests that disruption of the ImuB – DnaN (β-clamp) interaction by griselimycin prevents DnaE2-mediated induced mutagenesis, suggesting that the mycobacterial mutasome might be a good target for novel ‘anti-evolution’ drugs aimed at protecting against emergent resistance 148 ; inhibition of bacterial factors that promote mutagenesis as an anti-evolution strategy 149 ; inhibition of intrabacterial metabolism by promiscuous cytochromes and other enzymes 150 , 151 that may constitute a source of induced resistance; and inhibition of nonspecific drug efflux pumps shown to extrude anti-TB drugs 152 – 154 .…”