脓肿分枝杆菌MBT结构鉴定与比较演化分析

Structural identification and phylogenetic analysis of MBT from Mycobacteroides abscessus

摘要:

【背景】作为临床最常见的非结核条件致病分枝杆菌，脓肿分枝杆菌(Mycobacteroides abscessus)因其天然、多耐药等特性成为目前临床治疗的一大挑战。作为分枝杆菌限制性营养元素——铁摄取的关键系统，分枝杆菌素(mycobactin，MBT)、羧基分枝杆菌素(carboxymycobactin，cMBT)与病原分枝杆菌的毒力、耐药等密切相关。【目的】丰富分枝杆菌MBT、cMBT结构数据，探究MBT在致病分枝杆菌起源过程中的演化规律。【方法】在MALDI-TOF-MS与FT-MS/MS解析脓肿MBT、cMBT结构的基础上，进一步开展其活性分析与生物合成基因簇比较基因组分析。【结果】虽然脓肿分枝杆菌MBT、cMBT母核修饰模式与海洋分枝杆菌最相似，R1、R2、R3、R5等位置的修饰完全相同，而且脂肪酸链均位于R4位置；但脂肪酸链长度不同[C10-17 (MBT)、C4-8 (cMBT)]，为新结构。Fe-cMBT不仅以浓度依赖方式促进脓肿分枝杆菌生长，而且利用效率显著高于FeCl₃，相关结果表明MBT-cMBT是脓肿分枝杆菌高效获取铁元素的关键系统。与MBT结构结果一致，mbt-1基因簇共线性分析及mbt-1、mbt-2系统发育分析结果均表明脓肿分枝杆菌与海洋分枝杆菌(M.marinum)亲缘关系最近，而非结核分枝杆菌(M.tuberculosis)或耻垢分枝杆菌(M.smegmatis) (基于16S rRNA基因序列分析)。进一步分析发现，M.marinum、M.tuberculosis、M.bovis等病原分枝杆菌脂肪酸链长度变化范围仅4 C，而M.abscessus、M.fortuitum、M.avium和M.smegmatis等条件致病与非致病菌的脂肪酸链长度变化范围为7-11 C，暗示MBT同系物脂肪酸链长度变化范围与分枝杆菌不同生活方式、环境之间可能存在关联。【结论】作为获取铁元素的关键系统，具有独特结构的脓肿分枝杆菌MBT-cMBT在致病、耐药等方面的作用及起源、演化规律值得深入研究。

Abstract:

[Background] As one of the common non-tuberculous conditionally pathogenic mycobacteria, Mycobacteroides abscessus is a major clinical challenge because of its natural multi-drug resistance. Mycobactin (MBT) and carboxymycobactin (cMBT), the crucial systems for mycobacteria to acquire iron, one of the limiting nutrients, are closely associated with virulence and drug resistance. [Objective] To reveal the structure of MBT in M. abscessus and explore the evolution of MBT in pathogenic mycobacteria. [Methods] The structures of MBT and cMBT were analyzed by MALDI-TOF-MS and FT-MS/MS. Further, the biological activities of MBT and cMBT were determined. The MBT biosynthesis gene clusters were compared between M. abscessus and several representative mycobacteria. [Results] M. abscessus and M. marinum had similar modification patterns of MBT and cMBT core structure. In particular, the modifying groups at R1, R2, R3, and R5 were exactly the same, and the fatty acid chains were both located at R4. However, the MBT and cMBT of M. abscessus are a new structure because of the different lengths of the fatty acid chains (10-17 C for MBT and 4-8 C for cMBT). The growth of M. abscessus in iron-deprived media could be significantly recovered by supplying with Fe-cMBT in a dosage-dependent manner, and M. abscessus can much efficiently absorb Fe-cMBT than FeCl₃, which proved that MBT-cMBT system was vital for M. abscessus to acquirie iron from the environment. Synteny and phylogenetic analysis of the MBT biosynthetic gene cluster mbt-1 showed that M. abscessus was closely related to M. marinum rather than M. tuberculosis and M. smegmatis (referring to 16S rRNA gene phylogenetic tree). This result is consistent with that based on the structure of MBT. Further analysis revealed that the variation range of the fatty acid chain length of pathogenic mycobacteria such as M. marinum, M. tuberculosis, and M. bovis was only 4 C, while that of conditionally pathogenic and non-pathogenic bacteria such as M. abscessus, M. fortuitum, M. avium, and M. smegmatis was 7-11 C, which suggested that the range variation of fatty acid chain length of MBT might be associated with the lifestyles and habitats of mycobacteria. [Conclusion] As an important system for obtaining iron with unique structure, M. abscessus MBT deserved further study, especially its roles in pathogenesis and drug resistance, as well as its evolution in pathogenic mycobacteria.