Singh Nirbhay, Chauhan Anu, Kumar Ram, Singh Sudheer Kumar
Molecular Microbiology and Immunology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector-10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India.
Molecular Microbiology and Immunology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector-10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India.
Microbes Infect. 2022 Nov-Dec;24(8):105000. doi: 10.1016/j.micinf.2022.105000. Epub 2022 Oct 27.
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.
已报道植物、真菌和包括结核分枝杆菌（Mtb）在内的细菌中存在支链氨基酸（BCAAs）亮氨酸、异亮氨酸和缬氨酸的生物合成途径，但动物体内不存在这些途径。这使得针对BCAAs生物合成进行干预成为抗分枝杆菌药物研发的一个有吸引力的方向。在本研究中，对结核分枝杆菌H37Ra（Mtb-Ra）中编码酮醇酸还原异构酶的开放阅读框MRA_3031进行了研究，以确定其在Mtb-Ra生长和存活中的作用。在体外和体内条件下研究了重组敲低（KD）菌株、互补（KDC）菌株以及野生型（WT）Mtb-Ra。巨噬细胞感染后，KD在巨噬细胞内存活存在缺陷，其菌落形成单位（CFU）计数呈时间依赖性下降，而WT和KDC的CFU则呈时间依赖性增加。此外，与WT和KDC相比，KD形成持留菌的能力降低，对溴化乙锭和尼罗红染料的膜通透性改变，生物膜成熟度降低。体内研究表明，与WT相比，KD感染的小鼠肺部CFU计数较低。总之，酮醇酸还原异构酶下调后，Mtb在巨噬细胞和小鼠体内的存活能力下降。
