Proteogenomic Investigation of Strain Variation in Clinical <i>Mycobacterium tuberculosis</i> Isolates

Mycobacterium tuberculosis consists of a large number of different strains that display unique virulence characteristics. Whole-genome sequencing has revealed substantial genetic diversity among clinical M. tuberculosis isolates, and elucidating the phenotypic variation encoded by this genetic diversity will be of the utmost importance to fully understand M. tuberculosis biology and pathogenicity. In this study, we integrated whole-genome sequencing and mass spectrometry (GeLC–MS/MS) to reveal strain-specific characteristics in the proteomes of two clinical M. tuberculosis Latin American-Mediterranean isolates. Using this approach, we identified 59 peptides containing single amino acid variants, which covered ∼9% of all coding nonsynonymous single nucleotide variants detected by whole-genome sequencing. Furthermore, we identified 29 distinct peptides that mapped to a hypothetical protein not present in the M. tuberculosis H37Rv reference proteome. Here, we provide evidence for the expression of this protein in the clinical M. tuberculosis SAWC3651 isolate. The strain-specific databases enabled confirmation of genomic differences (i.e., large genomic regions of difference and nonsynonymous single nucleotide variants) in these two clinical M. tuberculosis isolates and allowed strain differentiation at the proteome level. Our results contribute to the growing field of clinical microbial proteogenomics and can improve our understanding of phenotypic variation in clinical M. tuberculosis isolates.结核分枝杆菌（Mycobacterium tuberculosis）包含大量具备独特毒力特征的不同菌株。全基因组测序已揭示临床结核分枝杆菌分离株间存在显著的遗传多样性，阐明该遗传多样性所编码的表型变异，对于全面解析结核分枝杆菌的生物学特性与致病性至关重要。本研究整合全基因组测序与质谱（GeLC–MS/MS）技术，对两株临床拉美-地中海型结核分枝杆菌分离株的蛋白质组进行菌株特异性特征分析。通过该研究策略，我们共鉴定出59个携带单氨基酸变异的肽段，覆盖全基因组测序检测到的全部编码区错义单核苷酸变异的约9%。此外，我们还鉴定出29条独特肽段，这些肽段对应于结核分枝杆菌H37Rv参考蛋白质组中未收录的假设蛋白。本研究证实了该蛋白在临床结核分枝杆菌SAWC3651分离株中的表达。基于菌株特异性数据库，我们得以确认这两株临床结核分枝杆菌分离株的基因组差异（即大片段差异基因组区域与错义单核苷酸变异），并实现了蛋白质组层面的菌株分型。本研究结果为日益发展的临床微生物蛋白质基因组学领域提供了新的支撑，同时有助于深化我们对临床结核分枝杆菌分离株表型变异的认知。