Analysis of the role of VapBC-type toxin-antitoxin modules in growth, stress tolerance and drug tolerance in mycobacteria
Date
2011-07-20
Authors
Ahidjo, Bintou Ahmadou
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Abstract
Forty seven of the toxin-antitoxin modules in the genome of
Mycobacterium tuberculosis belong to the VapBC family in which the VapC toxin
is a member of the PIN-domain protein family associated with nuclease activity.
The role of VapBCs in the physiology of M. tuberculosis and the cellular
function(s) served by their expansion are unknown but is the subject of intense
investigation as a result of the evidence suggesting an association between TAmodule
function and stress adaptation as well as phenotypic drug tolerance in
certain organisms. In this study, the function of ten vapBC modules from M.
tuberculosis and the single vapBC from M. smegmatis was investigated. Of the
vapCs assessed, Rv0549c, Rv0595c, Rv2549c and Rv2829c were growth
inhibitory when conditionally expressed under the control of a tetracycline (Tet)-
regulated promoter in both M. smegmatis and M. tuberculosis, with Rv0549c
being less toxic than the others. The toxicity of Rv2549c in M. smegmatis
correlated with the level of protein expressed, suggesting that in order for toxicity
to be observed, the VapC level must exceed a certain threshold. Low levels of
protein expression were demonstrated for Rv2456 following induction, which may
account for the lack of toxicity observed for this, and the remaining ‘non-toxic’
VapCs. In addition, given that Rv3320c was toxic in both M. smegmatis and M.
tuberculosis only when expressed in the absence of the Tet repressor, protein
expression levels, rather than differences in (nuclease) activity appeared to be the
principal determinant of VapC toxicity in this assay system. VapC toxicity was
neutralized by co-expression of the cognate vapB antitoxin from both an operon
with the toxin, as well as from a different chromosomal locus. However, noncognate
antitoxins could not abrogate VapC toxicity thus demonstrating a
specificity of interaction between VapCs and their cognate VapBs. Deletion of
selected vapBC genes did not affect mycobacterial growth in vitro or
mycobacterial stress adaptation, but rendered it more susceptible to growth
inhibition following toxic VapC expression. However, toxicity of ‘non-toxic’
VapCs was not increased in deletion mutant strains, even when the mutation
eliminated the cognate VapB, presumably due to insufficient levels of VapC
expression in this genetic background. Interestingly, low levels of VapC appeared VapCs that are induced as a result of stochastic and/or environmentally-induced
vapBC expression might have a significant effect on the physiology of
mycobacteria. The above-mentioned findings suggest that the vapBC family may
provide an abundant source of nuclease activity in M. tuberculosis, which can
vary as a function of regulated expression of individual modules, and the
rates/mechanisms of antitoxin degradation. Such activity is likely to have a
profound impact on the physiology of M. tuberculosis.