Department of Respiratory and Critical Care Medicine, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, China; School of Medicine, Tongji University, Shanghai, China.
Department of Respiratory and Critical Care Medicine, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, China; School of Medicine, Tongji University, Shanghai, China.
Int J Antimicrob Agents. 2024 Jan;63(1):107025. doi: 10.1016/j.ijantimicag.2023.107025. Epub 2023 Nov 4.
Nitrogen is indispensable for the synthesis of biomacromolecules. The correlation between nitrogen metabolism and Mycobacterium abscessus (M. abscessus) biofilm formation is unclear. This study constructed global nitrogen regulator gene GlnR (Mab_0744) knockout (ΔglnR) and complementation (ΔglnR::glnR) M. abscessus strains.
Global nitrogen regulator gene glnR (Mab_0744) knockout (ΔglnR) and complementation (ΔglnR::glnR) M. abscessus strains were constructed. Sauton's medium was used to culture M. abscessus pellicle biofilm. To test the antibiotic susceptibility of pellicle biofilm, clarithromycin, amikacin, cefoxitin or imipenem was added to the medium under biofilms after 14 days of incubation. RT-qPCR and ChIP-qPCR were performed to analyse the transcriptional regulatory function of GlnR.
GlnR knockout decreased the growth rate of planktonic cells, reduced biofilm mass and wrinkle formation, and diminished the resistance of biofilms to antibiotics. However, the susceptibility of planktonic cells to antibiotics was not changed by glnR knockout. The growth rate of planktonic ΔglnR cells was accelerated by adding nitrogen sources to the medium; the addition of glutamine or sodium glutamate rescued ΔglnR biofilm morphology and resistance to amikacin, cefoxitin, clarithromycin and imipenem. GlnR bound the promoter region and activated the transcription of eight nitrogen metabolic pathway genes (i.e. glnA, amt, ansP, nirB, nirD, glnD, glnK and narK3), which are closely related to glutamine/glutamate biosynthesis and, thus, regulate biofilm formation.
This study provides insights into the mechanisms of M. abscessus biofilm formation and its resistance to antibiotics.
氮是生物大分子合成所必需的。氮代谢与脓肿分枝杆菌（M. abscessus）生物膜形成的关系尚不清楚。本研究构建了全局氮调节基因 GlnR（Mab_0744）敲除（ΔglnR）和互补（ΔglnR::glnR）脓肿分枝杆菌菌株。
构建全局氮调节基因 glnR（Mab_0744）敲除（ΔglnR）和互补（ΔglnR::glnR）脓肿分枝杆菌菌株。使用 Sauton 培养基培养脓肿分枝杆菌菌膜生物膜。为了测试生物膜中抗生素的敏感性，在孵育 14 天后，在生物膜下的培养基中加入克拉霉素、阿米卡星、头孢西丁或亚胺培南。进行 RT-qPCR 和 ChIP-qPCR 以分析 GlnR 的转录调控功能。
GlnR 敲除降低了浮游细胞的生长速度，减少了生物膜的质量和皱缩形成，并降低了生物膜对抗生素的抗性。然而，glnR 敲除并未改变浮游细胞对抗生素的敏感性。向培养基中添加氮源可加速浮游 ΔglnR 细胞的生长；添加谷氨酰胺或谷氨酸可挽救 ΔglnR 生物膜形态并增强其对阿米卡星、头孢西丁、克拉霉素和亚胺培南的抗性。GlnR 结合启动子区域并激活与谷氨酰胺/谷氨酸生物合成密切相关的八个氮代谢途径基因（即 glnA、amt、ansP、nirB、nirD、glnD、glnK 和 narK3）的转录，从而调节生物膜形成。
本研究深入了解了脓肿分枝杆菌生物膜形成及其对抗生素耐药的机制。
