Bacillus cereus Group-Type Strain-Specific Diagnostic Peptides

The Bacillus cereus group consists of eight very closely related species and comprises both harmless and human pathogenic species such as Bacillus anthracis, Bacillus cereus, and Bacillus cytotoxicus. Numerous efforts have been undertaken to allow presumptive differentiation of B. cereus group species from one another. However, methods to rapidly and accurately distinguish these species are currently lacking. We confirmed that classical matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) biotyping cannot achieve reliable identification of each type strain. We therefore assigned type strain-specific diagnostic peptides to the B. cereus group based on comparisons of their proteomic profiles. The number of diagnostic peptides varied remarkably in a type strain-dependent manner. The accuracy of the reference database was crucial to validate candidate diagnostic peptides and led to a noteworthy reduction of verified diagnostic peptides. Diagnostic peptides ranged from one for B. weihenstephanensis to 62 for B. pseudomycoides and were associated with proteins involved in diverse biological processes, e.g. amino acid biosynthesis, cell envelope, cellular processes, energy metabolism, and transport processes. However, 45.6% of all diagnostic peptides comprised currently unclassified proteins or proteins of unknown function. In addition, a phylogenetic tree based on clustering of theoretical precursor masses deduced from in silico-generated tryptic peptides was reconstructed.蜡样芽孢杆菌群（Bacillus cereus group）包含8个亲缘关系极近的物种，涵盖无害及致人致病的菌株，例如炭疽芽孢杆菌（Bacillus anthracis）、蜡样芽孢杆菌（Bacillus cereus）以及产毒芽孢杆菌（Bacillus cytotoxicus）。已有诸多研究致力于实现蜡样芽孢杆菌群内各物种的推定区分，然而目前仍缺乏能够快速且精准区分这些物种的方法。本研究证实，经典基质辅助激光解吸电离飞行时间生物分型技术（matrix-assisted laser desorption/ionization-time-of-flight, MALDI-TOF）无法实现各模式菌株的可靠鉴定。为此，本研究基于各菌株的蛋白质组谱比对，为蜡样芽孢杆菌群鉴定得到模式菌株特异性诊断肽段。诊断肽段的数量依模式菌株的不同呈现显著差异。参考数据库的准确性是验证候选诊断肽段的关键因素，该因素使得经验证的诊断肽段数量出现显著缩减。诊断肽段的数量范围从魏文斯泰凡芽孢杆菌（Bacillus weihenstephanensis）的1条，至假拟分枝芽孢杆菌（Bacillus pseudomycoides）的62条不等，且这些肽段与参与多种生物学过程的蛋白质存在关联，例如氨基酸生物合成、细胞包膜、细胞生理过程、能量代谢以及物质转运过程。然而，所有诊断肽段中有45.6%对应的蛋白质为当前未分类蛋白或功能未知蛋白。此外，本研究基于对计算机模拟生成的胰蛋白酶酶解肽段所推导的理论前体质量进行聚类，重建了系统发育树。