Mycobacterium tuberculosis ketol-acid reductoisomerase down-regulation affects its ability to persist, and its survival in macrophages and in mice

Abstract

Branched-chain amino acids (BCAAs) leucine, isoleucine and valine biosynthetic pathways have been reported from plants, fungi and bacteria including Mycobacterium tuberculosis (Mtb) but are absent in animals. This makes interventions with BCAAs biosynthesis an attractive proposition for antimycobacterial drug discovery. In the present study, Mycobacterium tuberculosis H37Ra (Mtb-Ra) ketol-acid reductoisomerase encoding ORF MRA_3031 was studied to establish its role in Mtb-Ra growth and survival. Recombinant knockdown (KD) and complemented (KDC) strains along with wild-type (WT) Mtb-Ra were studied under in-vitro and ex-vivo conditions. KD was defective for survival inside macrophages and showed time dependent decrease in its colony forming unit (CFU) counts, while, WT and KDC showed time dependent increase in CFUs, after macrophage infection. Also, KD showed reduced ability to form persister cells, had altered membrane permeability against ethidium bromide and nile red dyes, and had reduced biofilm maturation, compared to WT and KDC. The in-vivo studies showed that KD infected mice had lower CFU counts in lungs, compared to WT. In summary Mtb shows survival deficit in macrophages and in mice after ketol-acid reductoisomerase down-regulation.