Garg Rajni, Tripathi Deeksha, Kant Sashi, Chandra Harish, Bhatnagar Rakesh, Banerjee Nirupama
School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.
International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Infect Immun. 2015 Jan;83(1):120-9. doi: 10.1128/IAI.02274-14. Epub 2014 Oct 13.
The virulence of Mycobacterium tuberculosis is intimately related to its distinctive cell wall. The biological significance of poly-α-L-glutamine (PLG), a component in the cell wall of virulent mycobacteria, has not been explored adequately. The focus of this study is to investigate the role of a locus, Rv0574c, coding for a polyglutamate synthase-like protein, in the synthesis of poly-α-L-glutamine in the context of mycobacterial virulence. Evaluation of Rv0574c gene expression in M. tuberculosis demonstrated its growth-phase-linked induction with concomitant accumulation of poly-α-L-glutamine in the cell wall. Rv0574c was activated under conditions prevalent in the tubercular granuloma, e.g., hypoxia, nitric oxide, and CO2. For functional characterization, we produced a deletion mutant of the Rv0574c gene by allelic exchange. The mutant produced smaller amounts of poly-α-L-glutamine in the cell wall than did the wild-type bacterium. Additionally, the increased sensitivity of the mutant to antitubercular drugs, SDS, lysozyme, and mechanical stress was accompanied by a drastic reduction in the ability to form biofilm. Growth of the ΔRv0574c strain was normal under in vitro conditions but was retarded in THP-1 macrophages and in the lungs and spleen of BALB/c mice. This was in agreement with histopathology of the lungs showing slow growth and less severe pathology than that of the wild-type strain. In summary, this study demonstrates that the protein encoded by the Rv0574c locus, by virtue of modulating PLG content in the cell wall, helps in maintaining cellular integrity in a hostile host environment. Also, its involvement in protecting the pathogen from host-generated lethal factors contributes to the infectious biology of M. tuberculosis.
结核分枝杆菌的毒力与其独特的细胞壁密切相关。毒力型分枝杆菌细胞壁成分聚-α-L-谷氨酰胺（PLG）的生物学意义尚未得到充分研究。本研究的重点是调查编码聚谷氨酸合酶样蛋白的Rv0574c位点在分枝杆菌毒力背景下聚-α-L-谷氨酰胺合成中的作用。对结核分枝杆菌中Rv0574c基因表达的评估表明，其表达与生长阶段相关，同时细胞壁中聚-α-L-谷氨酰胺积累。Rv0574c在结核肉芽肿中普遍存在的条件下被激活，例如缺氧、一氧化氮和二氧化碳。为了进行功能表征，我们通过等位基因交换产生了Rv0574c基因的缺失突变体。该突变体细胞壁中产生的聚-α-L-谷氨酰胺比野生型细菌少。此外，突变体对抗结核药物、SDS、溶菌酶和机械应激的敏感性增加，同时形成生物膜的能力急剧下降。ΔRv0574c菌株在体外条件下生长正常，但在THP-1巨噬细胞以及BALB/c小鼠的肺和脾中生长受到抑制。这与肺组织病理学一致，显示其生长缓慢且病理变化比野生型菌株轻。总之，本研究表明，Rv0574c位点编码的蛋白质通过调节细胞壁中的PLG含量，有助于在恶劣的宿主环境中维持细胞完整性。此外，它参与保护病原体免受宿主产生的致死因子的影响，这有助于结核分枝杆菌的感染生物学。
