NagA deletion of Mycobacterium tuberculosis leads to a defective cell wall integrity and invokes unique host signatures rendering the pathogen attenuated in guinea pigs.
Mtb N-acetylglucosamine-6-phosphate deacetylase (NagA, Rv3332) belonging to the amido-hydrolase superfamily catalyzes a reaction which generates vital amino-sugars required as precursors for Mtb cell wall biosynthesis or glycolysis. In this study, a nagA gene deletion mutant of Mtb was generated, which showed an altered cellular morphology and cell wall structure. In vitro experiments revealed the importance of NagA under lysosomal, hypoxic and antibiotic stress and in biofilm formation. Mtb?nagA mutant showed reduced bacillary count inside macrophages in comparison to the parental strain. Deletion of nagA rendered the pathogen attenuated in guinea pigs exhibiting significantly lower bacillary counts as well as much reduced pathological damage in infected tissues in comparison to the wild type strain. Moreover, guinea pigs infected with Mtb?nagA mutant showed cent percent survival rate. Further, RNA sequencing analysis of the lung tissues from infected guinea pigs revealed that Mtb?nagA infection led to significantly lesser changes in gene expression in the host compared to the uninfected control while inducing a stronger immune response.  In contrast, the virulent Mtb strain drastically altered the host transcriptome favouring its survival. To conclude, our study demonstrated the importance of NagA in M. tuberculosis pathogenicity establishing it as a potential anti-tubercular target. Further, we consider Mtb?nagA to have potential to be explored as a TB vaccine candidate. Overall design: To investigate the role of NagA in the growth and pathogenesis of M.tuberculosis, we knocked out nagA by homologous recombination technique. We infected guinea pigs with wild type (Rv), nagA mutant (NK) and complemented strain of Mtb, aerogenically and then performed host RNA-Sequencing analysis. Type of sample: RNA isolated from infected/uninfected lung tissue of guinea pigs Experimental conditions: 10 weeks post infection Sample groups: RNA samples from guinea pigs infected with M.tuberculosis wild type strain (Rv), Mtb?nagA mutant strain (NK) or uninfected animals (UN) Replicates: Three replicates for each group Controls/References: RNA samples isolated from Mtb wild type strain infected (Rv) and uninfected guinea pigs (UN)