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. ### Competing Interest Statement The authors have declared no competing interest.