Multiple mutations often needed to make TB bacteria drug resistant

The anti-TB drug ethambutol blocks bacterial genes required for synthesis of the bug's protective cell wall. Several mutations in these bacterial genes (collectively called the embCAB operon) have been identified in drug-resistant strains of TB, and single mutations are widely thought to confer resistance in one fell swoop. But not all bugs carrying embCAB mutations become ethambutol-resistant and not all resistance strains contain these mutations, suggesting that the story is much more complicated.

David Alland, director of the Center for Emerging and Re-Emerging Pathogens at Rutgers New Jersey Medical School, and colleagues had previously shown expressing single embCAB mutations in drug sensitive bugs rendered them only slightly more drug resistant than normal and failed to explain full-blown resistance. The group now identifies new mutations that contribute to drug resistance, with the level of resistance depending on the unique combination of mutations in a given bacterial isolate.

One of the newly identified mutations -- in a bacterial gene called Rv3806c -- ramps up production of a substrate used by the embCAB-encoded enzymes to generate the bug's cell wall. This excess substrate then binds to the enzymes, potentially limiting the amount of drug that can bind. However, the Rv3806c mutation alone only modestly increased drug resistance. But when combined with other mutations, it generated high-level ethambutol resistance. Surprisingly, they also discovered "synonymous" DNA mutations (ones that don't change the amino acid sequence of the resulting protein) in a related protein called Rv3792 that also contributed to drug resistance.

Alland's group suggests that bugs with single mutations, for example those in Rv3806c and Rv3792, represent a pre-resistant state, in which the bug is poised for full-blown drug resistance upon the acquisition of a 'second hit' mutation. Identification of patients infected with 'pre-resistant' bugs may allow doctors to increase drug dosages or alter treatment strategies before full-scale drug resistance develops.

This work was supported in part by National Institute of Allergy and Infectious Diseases, US National Institutes of Health grants AI080653, AI065663 and AI037139 and by Pathogen Functional Genomics Resource Center contract N01-AI5447.