Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice

Abstract

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthesis pathway in bacteria. Bioinformatics analysis revealed that the Mycobacterium tuberculosis genome contains four genes (ilvB1, ilvB2, ilvG and ilvX) coding for the large catalytic subunit of AHAS, whereas only one gene (ilvN or ilvH) coding for the smaller regulatory subunit of this enzyme was found. In order to understand the physiological role of AHAS in survival of the organism in vitro and in vivo, we inactivated the ilvB1 gene of M. tuberculosis. The mutant strain was found to be auxotrophic for all of the three branched-chain amino acids (isoleucine, leucine and valine), when grown with either C 6 or C 2 carbon sources, suggesting that the ilvB1 gene product is the major AHAS in M. tuberculosis. Depletion of these branched chain amino acids in the medium led to loss of viability of the DilvB1 strain in vitro, resulting in a 4-log reduction in colony-forming units after 10 days. Survival kinetics of the mutant strain cultured in macrophages maintained with sub-optimal concentrations of the branched-chain amino acids did not show any loss of viability, indicating either that the intracellular environment was rich in these amino acids or that the other AHAS catalytic subunits were functional under these conditions. Furthermore, the growth kinetics of the DilvB1 strain in mice indicated that although this mutant strain showed defective growth in vivo, it could persist in the infected mice for a long time, and therefore could be a potential vaccine candidate.

Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

“…This confirmed that the selected clones were mc 2 ⌬ilvH mutants. The inactivation of the large subunit of acetohydroxyacid synthase, ilvB1 has been shown to be auxotrophic (54). Our results suggest that the small subunit of acetohydroxyacid synthase ilvH is also essential in the absence of BCAA complementation.…”

“…4B). AHAS is composed of the IlvB (large) and IlvH (small) subunits encoded by the ilvB and ilvH genes, respectively (54,55). The genomic organization of ilvH and its flanking regions is illustrated in Fig.…”

Two FgLEU2 Genes with Different Roles in Leucine Biosynthesis and Infection-Related Morphogenesis in Fusarium graminearum

“…Therefore, it is extremely urgent to develop novel, less-toxic and effective fungicides for the management of FHB. Enzymes involved in certain amino acids biosynthesis (e.g., BCAAs) have been regarded as attractive fungicide targets due to their absence in mammals and the important biological functions of the genes encoding for these enzymes in yeasts and filamentous fungi [13–21]. …”

MRA_1571 is required for isoleucine biosynthesis and improves Mycobacterium tuberculosis H37Ra survival under stress

“…Also, knockout strains of acetohydroxyacid synthase ( ilvB1 ) were attenuated for growth and virulence in mice15. L-threonine acts as a precursor for L-isoleucine and flux from threonine to isoleucine is under allosteric control of threonine dehydratase and acetohydroxy acid synthase (AHAS).…”