Krysenko Sergii, Emani Carine Sao, Bäuerle Moritz, Oswald Maria, Kulik Andreas, Meyners Christian, Hillemann Doris, Merker Matthias, Prosser Gareth, Wohlers Inken, Hausch Felix, Brötz-Oesterhelt Heike, Mitulski Agnieszka, Reiling Norbert, Wohlleben Wolfgang
Interfaculty Institute of Microbiology and Infection Medicine Tübingen (IMIT), Department of Microbiology and Biotechnology, University of Tübingen, Tübingen, Baden-Württemberg, Germany.
Cluster of Excellence 'Controlling Microbes to Fight Infections', University of Tübingen, Tübingen, Baden-Württemberg, Germany.
J Bacteriol. 2025 Feb 20;207(2):e0043924. doi: 10.1128/jb.00439-24. Epub 2025 Jan 30.
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 , the pathogen needs strategies to cope with these toxic metabolites. The actinomycete , a close relative of makes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional in . In the current study, we demonstrated that growth was inhibited by the polyamine spermine. Using enzymatic assays we determined that GlnA3 (Rv1878) possesses genuine gamma-glutamylspermine synthetase catalytic activity. We further showed that purified His-Strep-GlnA3 as well as native GlnA3 prefer spermine as a substrate over putrescine, cadaverine, spermidine, or other monoamines and amino acids, suggesting that GlnA3 may play a specific role in the detoxification of the polyamine spermine. However, the deletion of the gene in did not result in growth inhibition or enhanced sensitivity of in the presence of high spermine concentrations. Gene expression analysis of spermine-treated revealed no difference in the level of expression relative to untreated cells, whereas a gene encoding a previously characterized efflux pump (Mmr; ) 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.
Upon infection macrophages synthesize the polyamine spermine, which at elevated concentrations is toxic for . Based on our investigations of spermine resistance in the closely related actinomycete , we hypothesized that the glutamylspermine synthetase GlnA3 may be responsible for the resistance of against toxic spermine. Here we show that GlnA3 can indeed covalently modify spermine via glutamylation. However, GlnA3 is probably not the only resistance mechanism since a null mutant of can survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. Thus a combination of GlnA3 and specific efflux pumps acting as putative spermine transporters may constitute an active spermine-detoxification system in .
能很好地适应在受感染宿主体内生存并持续存在，逃避宿主的免疫反应。由于诸如精胺之类的多胺由受感染的巨噬细胞合成，能够抑制[病原体名称]的生长，病原体需要应对这些有毒代谢产物的策略。放线菌[放线菌名称]是[病原体名称]的近亲，它利用γ-谷氨酰化途径在功能上中和精胺。因此，我们思考在[病原体名称]中是否有类似的途径起作用。在当前研究中，我们证明了[病原体名称]的生长受到多胺精胺的抑制。通过[酶名称]酶活性测定，我们确定GlnA3（Rv1878）具有真正的γ-谷氨酰精胺合成酶催化活性。我们进一步表明，纯化的His-Strep-GlnA3以及天然的GlnA3相比于腐胺、尸胺、亚精胺或其他单胺和氨基酸，更倾向于将精胺作为底物，这表明GlnA3可能在多胺精胺的解毒中发挥特定作用。然而，在[病原体名称]中缺失[基因名称]基因并没有导致生长抑制，也没有使[病原体名称]在高浓度精胺存在下的敏感性增强。对经精胺处理的[病原体名称]进行基因表达分析发现，与未处理细胞相比，[基因名称]的表达水平没有差异，而一个编码先前已鉴定的外排泵（Mmr；[外排泵名称]）的基因显著上调。这表明在精胺浓度升高的情况下细菌的存活不仅可以通过精胺本身的解毒来实现，还可以通过导致细菌内精胺水平降低的机制来实现。
在[病原体名称]感染后，巨噬细胞合成多胺精胺，高浓度时对[病原体名称]有毒性。基于我们对密切相关的放线菌[放线菌名称]中精胺抗性的研究，我们推测谷氨酰精胺合成酶GlnA3可能负责[病原体名称]对有毒精胺的抗性。在这里我们表明GlnA3确实可以通过谷氨酰化共价修饰精胺。然而，GlnA3可能不是唯一的抗性机制，因为[病原体名称]的[基因名称]缺失突变体在精胺胁迫下仍能存活。基因表达研究表明一个外排泵可能参与抗性。因此，GlnA3和作为假定精胺转运体的特定外排泵的组合可能构成[病原体名称]中一个活跃的精胺解毒系统。
