Functional analysis and enzyme characterization of mannose-1-phosphate guanylyl transferase (ManB) from Mycobacterium tuberculosis

Mycobacterium tuberculosis cell wall consist variety of mannose containing glycoconjugates including lipomannan (LM) and lipoarabinomannan (LAM). These lipoglycans are involved in cell wall integrity and play role in virulence of M. tuberculosis by modulating host immune response. GDP-mannose, required for the synthesis of lipoglycans, is catalyzed by enzyme Mannose-1-phosphate guanylyl transferase (ManB). The enzyme with similar function has been studied in variety of species of prokaryotes and eukaryotes. However, biological role of ManB and its enzymatic activity remains uncharacterized in M. tuberculosis. In present study, we elucidated the role of enzyme by constructing manB knockdown strain of M. tuberculosis H37Ra. The manB knockdown decreased the cell growth and also effected the morphology of M. tuberculosis by altering the permeability of cell membrane. These findings provide the understanding on ManB function and suggesting that ManB could be the potential target for novel antituberculosis drug. Furthermore, we also characterized ManB enzyme by establishing 96 well plate colorimetric assay and determined the kinetic properties including initial velocity, optimum temperature, optimum pH and other kinetic parameters. Our established assay will be helpful for further high throughput screening of potential inhibitors against ManB. (C) 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Automated summary

In this study, we elucidated the role of the enzyme ManB in Mycobacterium tuberculosis by constructing a knockdown strain and characterized its enzymatic properties. The knockdown of ManB affected cell growth and morphology, indicating its importance in the bacterium. We also established a colorimetric assay for ManB, which can be utilized for high throughput screening of potential inhibitors. These findings contribute to the understanding of ManB function and its potential as a target for novel antituberculosis drugs.