Downregulation of Rv0189c, encoding a dihydroxyacid dehydratase, affects growth of Mycobacterium tuberculosis in vitro and in mice

Abstract

Dihydroxyacid dehydratase (DHAD), a key enzyme involved in branched-chain amino acid (BCAA) biosynthesis, catalyses the synthesis of 2-ketoacids from dihydroxyacids. InMycobacterium tuberculosis, DHAD is encoded by geneRv0189c, and it shares 40 % amino acid sequence identity and conserved motifs with DHAD ofEscherichia coliencoded byilvD. In this study,Rv0189cwas overexpressed inE. coliand the resultant protein was characterized as a homodimer (∼155 kDa). Functional characterization ofRv0189cwas established by biochemical testing and by genetic complementation of an intron-disruptedilvD-auxotrophic mutant ofE. colito prototrophy. Growth ofM. tuberculosis,E. coliBL21(DE3) and recombinantE. coliBL21(DE3) ΔilvDcarryingRv0189cwas inhibited by transient nitric oxide (NO) exposure in minimal medium but growth was restored if the medium was supplemented with BCAA (isoleucine, leucine and valine). This suggested that inactivation ofRv0189cby NO probably inhibited bacterial growth. The role ofRv0189cinM. tuberculosiswas elucidated by antisense and sense RNA constructs. Growth ofM. tuberculosistransformed with a plasmid encoding antisense mRNA was markedly poor in the lungs of infected mice and in Middlebrook 7H9 broth compared to that of sense and vector-alone transformants, but growth was normal when the medium was supplemented with BCAA. Upregulation ofRv0189cwas observed during the early exponential phase of growth, under acid stress andex vivo, suggesting thatRv0189chas a role in the survival ofM. tuberculosisduring normal and stress conditions. It may be concluded that the DHAD encoded byRv0189cis essential for the survival ofM. tuberculosisand could be a potential drug/vaccine target, as it is absent in mammals.