Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland.
Department of Biochemistry, Faculty of Sciences, University of Geneva, Geneva, Switzerland.
Cell Microbiol. 2020 May;22(5):e13163. doi: 10.1111/cmi.13163. Epub 2020 Feb 11.
Mycobacterium marinum is a model organism for pathogenic Mycobacterium species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. These pathogens enter phagocytes and replicate within the Mycobacterium-containing vacuole, possibly followed by vacuole exit and growth in the host cell cytosol. Mycobacteria release siderophores called mycobactins to scavenge iron, an essential yet poorly soluble and available micronutrient. To investigate the role of M. marinum mycobactins, we purified by organic solvent extraction and identified by mass spectrometry the lipid-bound mycobactin (MBT) and the water-soluble variant carboxymycobactin (cMBT). Moreover, we generated by specialised phage transduction a defined M. marinum ΔmbtB deletion mutant predicted to be defective for mycobactin production. The M. marinum ΔmbtB mutant strain showed a severe growth defect in broth and phagocytes, which was partially complemented by supplying the mbtB gene on a plasmid. Furthermore, purified Fe-MBT or Fe-cMBT improved the growth of wild type as well as ΔmbtB mutant bacteria on minimal plates, but only Fe-cMBT promoted the growth of wild-type M. marinum during phagocyte infection. Finally, the intracellular growth of M. marinum ΔmbtB in Acanthamoeba castellanii amoebae was restored by coinfection with wild-type bacteria. Our study identifies and characterises the M. marinum MBT and cMBT siderophores and reveals the requirement of mycobactins for extra- and intracellular growth of the pathogen.
海分枝杆菌是致病性分枝杆菌物种（包括结核分枝杆菌，即结核病的病原体）的模式生物。这些病原体进入吞噬细胞并在含有分枝杆菌的空泡内复制，随后可能有空泡外溢并在宿主细胞胞质中生长。分枝杆菌释放称为分枝菌酸的铁载体来掠夺铁，铁是一种必需的但溶解度低且不易获得的微量营养素。为了研究海分枝杆菌分枝菌酸的作用，我们通过有机溶剂萃取进行纯化，并通过质谱法鉴定了脂结合分枝菌酸（MBT）和水溶性变体羧基分枝菌酸（cMBT）。此外，我们通过专门的噬菌体转导生成了一个定义明确的海分枝杆菌ΔmbtB 缺失突变体，该突变体预计在分枝菌酸产生方面存在缺陷。海分枝杆菌ΔmbtB 突变株在肉汤和吞噬细胞中的生长严重受损，通过质粒提供 mbtB 基因可部分补偿。此外，纯化的 Fe-MBT 或 Fe-cMBT 可改善野生型和ΔmbtB 突变菌在最小平板上的生长，但只有 Fe-cMBT 可促进野生型海分枝杆菌在吞噬细胞感染期间的生长。最后，通过与野生型细菌共感染，可恢复海分枝杆菌ΔmbtB 在粘质沙雷氏菌变形虫中的细胞内生长。我们的研究鉴定并表征了海分枝杆菌 MBT 和 cMBT 铁载体，并揭示了分枝菌酸对病原体的细胞内外生长的需求。
