J Bacteriol. 2025 Jan 30. e0043924
Sergii Krysenko,
Carine Sao Emani,
Moritz Bäuerle,
Maria Oswald,
Andreas Kulik,
Christian Meyners,
Doris Hillemann,
Matthias Merker,
Gareth Prosser,
Inken Wohlers,
Felix Hausch,
Heike Brötz-Oesterhelt,
Agnieszka Mitulski,
Norbert Reiling,
Wolfgang Wohlleben.
Mycobacterium tuberculosis is well adapted to survive and persist in the infected host, escaping the host's immune response. Since polyamines such as spermine, which are synthesized by infected macrophages, are able to inhibit the growth of M. tuberculosis, the pathogen needs strategies to cope with these toxic metabolites. The actinomycete Streptomyces coelicolor, a close relative of 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 in vitro enzymatic assays we determined that GlnA3Mt (Rv1878) possesses genuine gamma-glutamylspermine synthetase catalytic activity. We further showed that purified His-Strep-GlnA3Mt, as well as native GlnA3Mt, prefer spermine as a substrate over 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. Gene expression analysis of spermine-treated M. tuberculosis revealed no difference in the level of glnA3Mt expression relative to untreated cells, whereas a gene encoding a previously characterized efflux pump (Mmr; rv3065) was significantly upregulated. This suggests that bacterial survival under elevated spermine concentrations can not only be achieved by detoxification of spermine itself but also by mechanisms resulting in decreased spermine levels in the bacteria.
IMPORTANCE: Upon Mycobacterium 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 glutamylspermine synthetase GlnA3 may be responsible for the resistance of M. tuberculosis against toxic spermine. Here we show that GlnA3Mt can indeed covalently modify spermine via glutamylation. However, GlnA3Mt is probably not the only resistance mechanism since a glnA3 null mutant of M. tuberculosis can survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. Thus a combination of GlnA3Mt and specific efflux pumps acting as putative spermine transporters may constitute an active spermine-detoxification system in M. tuberculosis.
Keywords: GS-like enzyme; GlnA3; Rv3065; glutamylation; infection; polyamine metabolism; tuberculosis