Abstract
M. tuberculosis N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmUMtb) is a bi-functional enzyme engaged in the synthesis of two metabolic intermediates N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and UDP-GlcNAc, catalyzed by the C- and N-terminal domains respectively. UDP-GlcNAc is a key metabolite essential for the synthesis of peptidoglycan, disaccharide linker, arabinogalactan and mycothiols. While glmU Mtb was predicted to be an essential gene, till date the role of GlmUMtb in modulating the in vitro growth of Mtb or its role in survival of pathogen ex vivo / in vivo have not been deciphered. Here we present the results of a comprehensive study dissecting the role of GlmUMtb in arbitrating the survival of the pathogen both in vitro and in vivo. We find that absence of GlmUMtb leads to extensive perturbation of bacterial morphology and substantial reduction in cell wall thickness under normoxic as well as hypoxic conditions. Complementation studies show that the acetyl- and uridyl- transferase activities of GlmUMtb are independently essential for bacterial survival in vitro, and GlmUMtb is also found to be essential for mycobacterial survival in THP-1 cells as well as in guinea pigs. Depletion of GlmUMtb from infected murine lungs, four weeks post infection, led to significant reduction in the bacillary load. The administration of Oxa33, a novel oxazolidine derivative that specifically inhibits GlmUMtb, to infected mice resulted in significant decrease in the bacillary load. Thus our study establishes GlmUMtb as a strong candidate for intervention measures against established tuberculosis infections.
Highlights
The cell wall, which contains a number of virulence determinants, is the first line of defence for survival of the pathogen in the hostile host environment [1]
As the tetracycline-inducible system is an effective means to regulate gene expression [36], we introduced the integration-proficient pST-KirT-glmU construct into Mtb H37Rv (Fig 1B)
A comparative analysis of growth by spotting of serially diluted cultures of Rv and RvΔglmU grown in the presence versus absence of ATc showed that GlmUMtb depletion by addition of ATc led to complete inhibition of growth, with no growth detected after 6 days (Fig 2B)
Summary
The cell wall, which contains a number of virulence determinants, is the first line of defence for survival of the pathogen in the hostile host environment [1]. GlmUMtb is a bi-functional enzyme, with acetyltransferase and uridyltransferase activities catalyzed by the C- and N- terminal domains respectively (Fig 1A) [6,7]. The carboxy-terminal domain of GlmUMtb transfers the acetyl moiety from acetyl CoA onto glucosamine-1-phosphate to generate N-acetylglucosamine-1-phosphate (GlcNAc-1-P). The N-terminal uridyltransferase domain of GlmUMtb catalyzes the transfer of UMP (from UTP) to GlcNAc1-P to form UDP-GlcNAc (Fig 1A) [6]. The UDP-GlcNAc produced is among the central metabolites that is required for the synthesis of peptidoglycan, lipid A of LAM, arabinogalactan, Rha-GlcNAc linkers, mycothiol (required for maintaining redox homeostasis) [8,9,10,11,12,13,14]. No studies have addressed the question of whether both the activities of GlmUMtb are independently essential for the growth or survival of the bacterium
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