AimsTo construct a conditional N-acetylglucosamine-1-phosphate transferase (WecA) knockdown strain of Mycobacterium smegmatis and to investigate the biological effect of WecA on mycobacterial growth, morphology and susceptibilities against anti-tuberculosis drugs. Methods and ResultsMycobacterium smegmatis wecA knockdown strain was constructed by using a tetracycline-inducible expression vector pMind and the expression of WecA was regulated by antisense RNA. The results of growth curves and the colony formation unit curves showed that the growth rate of WecA down-regulation strain was decreased and the amount of live bacterial cells dropped. In addition, the wecA knockdown strain exhibited dramatically morphological alterations through scanning electron microscopy observation. The susceptibility of WecA low-expression strain to anti-tuberculosis drugs was detected by using a rapid resazurin microtitre assay as well as a traditional agar dilution method. Notably, the wecA knockdown strain was more sensitive to rifampin, compared with the wecA normal-expression strain. In addition, the sensitivity of wild type Myco.smegmatis mc(2)155 strain against rifampin was also enhanced in the presence of a low concentration of tunicamycin, a natural WecA inhibitor. ConclusionsDown-regulation of WecA enhanced the sensitivity of Myco.smegmatis against rifampin. Significance and Impact of the StudyThese results provided a possibility of combined application of rifampin together with tunicamycin or other WecA inhibitors, which could be a new approach for the treatment of tuberculosis.