GlnA3Mtis able to glutamylate spermine but it is not essential for the detoxification of spermine inMycobacterium tuberculosis

Abstract

ABSTRACTMycobacterium tuberculosisis 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 ofM. tuberculosis, the pathogen needs strategies to cope with toxic spermine. The actinomyceteStreptomyces coelicolor, closely related toM. tuberculosismakes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional inM. tuberculosis. In the current study we demonstrated thatM. tuberculosisgrowth was inhibited by the polyamine spermine. Using a glutamine synthetase-basedin vitroenzymatic activity assay we determined that GlnA3Mt(Rv1878) is a gamma-glutamylspermine synthetase. In anin vitrophosphate release assay we showed that purified His-Strep-GlnA3Mtas well as native GlnA3Mtprefer spermine as a substrate to putrescine, cadaverine, spermidine or other monoamines and amino acids, suggesting that GlnA3Mtmay play a specific role in the detoxification of the polyamine spermine. However, the deletion of theglnA3gene inM. tuberculosisdid not result in growth inhibition or enhanced sensitivity ofM. tuberculosisin the presence of high spermine concentrations. Subsequent RNAsequencing ofM. tuberculosisbacteria revealed that the gene cluster consisting of the efflux pump-encodingrv3065-rv3066-rv3067genes is upregulated upon spermine treatment, suggesting its involvement in bacterial survival under elevated spermine concentrations.IMPORTANCEAntibiotics for the treatment ofMycobacterium tuberculosisinfections attack classical bacterial targets, such as the cell envelope or the ribosome. UponM. tuberculosisinfection macrophages synthesize the polyamine spermine which - at elevated concentrations - is toxic forM. tuberculosis. Based on our investigations of spermine resistance in the closely related actinomyceteStreptomyces 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 aglnA3mutant ofM. tuberculosiscan survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. The functional role of GlnA3Mtas well as of the spermine transporter in the pathogenicity ofM. tuberculosisis of special interest for their validation as new targets of novel anti-tubercular drugs.