GlnA3Mt is able to glutamylate spermine but it is not essential for the detoxification of spermine in Mycobacterium tuberculosis

Mycobacterium tuberculosis is well adapted to survive and persist in the infected host, escaping the host immune response. Since polyamines, which are synthesized by infected macrophages are able to inhibit the growth of M. tuberculosis, the pathogen needs strategies to cope with toxic spermine. The actinomycete Streptomyces coelicolor, closely related to M. tuberculosis makes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional in M. tuberculosis. In the current study we demonstrated that M. tuberculosis growth was inhibited by the polyamine spermine. Using a glutamine synthetase-based in vitro enzymatic activity assay we determined that GlnA3Mt (Rv1878) is a gamma-glutamylspermine synthetase. In an in vitro phosphate release assay we showed that purified His-Strep-GlnA3Mt as well as native GlnA3Mt prefer spermine as a substrate to putrescine, cadaverine, spermidine or other monoamines and amino acids, suggesting that GlnA3Mt may play a specific role in the detoxification of the polyamine spermine. However, the deletion of the glnA3 gene in M. tuberculosis did not result in growth inhibition or enhanced sensitivity of M. tuberculosis in the presence of high spermine concentrations. Subsequent RNAsequencing of M. tuberculosis bacteria revealed that the gene cluster consisting of the efflux pump-encoding rv3065-rv3066-rv3067 genes is upregulated upon spermine treatment, suggesting its involvement in bacterial survival under elevated spermine concentrations. IMPORTANCEAntibiotics for the treatment of Mycobacterium tuberculosis infections attack classical bacterial targets, such as the cell envelope or the ribosome. Upon M. tuberculosis infection macrophages synthesize the polyamine spermine which - at elevated concentrations - is toxic for M. tuberculosis. Based on our investigations of spermine resistance in the closely related actinomycete Streptomyces coelicolor, we hypothesized that the glutamyl-sperminesynthetase GlnA3 may be responsible for resistance against toxic spermine. Here we show that the mycobacterial glutamyl-sperminesynthetase indeed can inactivate spermine by glutamylation. However, GlnA3 is probably not the only resistance mechanism since a glnA3 mutant of M. tuberculosis can survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. The functional role of GlnA3Mt as well as of the spermine transporter in the pathogenicity of M. tuberculosis is of special interest for their validation as new targets of novel anti-tubercular drugs.