Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
PLoS Negl Trop Dis. 2018 Apr 2;12(4):e0006351. doi: 10.1371/journal.pntd.0006351. eCollection 2018 Apr.
Chagas disease also known as American trypanosomiasis is caused by the protozoan Trypanosoma cruzi. Over the last 30 years, Chagas disease has expanded from a neglected parasitic infection of the rural population to an urbanized chronic disease, becoming a potentially emergent global health problem. T. cruzi strains were assigned to seven genetic groups (TcI-TcVI and TcBat), named discrete typing units (DTUs), which represent a set of isolates that differ in virulence, pathogenicity and immunological features. Indeed, diverse clinical manifestations (from asymptomatic to highly severe disease) have been attempted to be related to T.cruzi genetic variability. Due to that, several DTU typing methods have been introduced. Each method has its own advantages and drawbacks such as high complexity and analysis time and all of them are based on genetic signatures. Recently, a novel method discriminated bacterial strains using a peptide identification-free, genome sequence-independent shotgun proteomics workflow. Here, we aimed to develop a Trypanosoma cruzi Strain Typing Assay using MS/MS peptide spectral libraries, named Tc-STAMS2.
METHODS/PRINCIPAL FINDINGS: The Tc-STAMS2 method uses shotgun proteomics combined with spectral library search to assign and discriminate T. cruzi strains independently on the genome knowledge. The method is based on the construction of a library of MS/MS peptide spectra built using genotyped T. cruzi reference strains. For identification, the MS/MS peptide spectra of unknown T. cruzi cells are identified using the spectral matching algorithm SpectraST. The Tc-STAMS2 method allowed correct identification of all DTUs with high confidence. The method was robust towards different sample preparations, length of chromatographic gradients and fragmentation techniques. Moreover, a pilot inter-laboratory study showed the applicability to different MS platforms.
This is the first study that develops a MS-based platform for T. cruzi strain typing. Indeed, the Tc-STAMS2 method allows T. cruzi strain typing using MS/MS spectra as discriminatory features and allows the differentiation of TcI-TcVI DTUs. Similar to genomic-based strategies, the Tc-STAMS2 method allows identification of strains within DTUs. Its robustness towards different experimental and biological variables makes it a valuable complementary strategy to the current T. cruzi genotyping assays. Moreover, this method can be used to identify DTU-specific features correlated with the strain phenotype.
恰加斯病，又称美洲锥虫病，是由原生动物克氏锥虫引起的。在过去的 30 年中，恰加斯病已从农村地区被忽视的寄生虫感染扩展为城市化的慢性疾病，成为潜在的全球突发卫生问题。克氏锥虫株被分为七个遗传群（TcI-TcVI 和 TcBat），称为离散型分类单位（DTUs），它们代表一组在毒力、致病性和免疫特征上存在差异的分离株。事实上，不同的临床表现（从无症状到高度严重的疾病）已尝试与克氏锥虫的遗传变异性相关联。由于这个原因，已经引入了几种 DTU 分型方法。每种方法都有其自身的优点和缺点，例如高复杂性和分析时间，并且它们都是基于遗传特征的。最近，一种新的方法使用一种无需肽鉴定、独立于基因组序列的鸟枪法蛋白质组学工作流程来区分细菌菌株。在这里，我们旨在使用 MS/MS 肽谱文库开发一种克氏锥虫株分型测定法，称为 Tc-STAMS2。
方法/主要发现：Tc-STAMS2 方法使用鸟枪法蛋白质组学结合谱文库搜索，独立于基因组知识对克氏锥虫株进行分配和区分。该方法基于使用基因分型的克氏锥虫参考株构建 MS/MS 肽谱文库。对于鉴定，未知克氏锥虫细胞的 MS/MS 肽谱使用谱匹配算法 SpectraST 进行鉴定。Tc-STAMS2 方法能够以高置信度正确识别所有 DTUs。该方法对不同的样品制备、色谱梯度长度和碎裂技术具有稳健性。此外，一项试点实验室间研究表明了其在不同 MS 平台上的适用性。
这是第一项开发基于 MS 的克氏锥虫株分型平台的研究。事实上，Tc-STAMS2 方法允许使用 MS/MS 谱作为鉴别特征对克氏锥虫株进行分型，并允许区分 TcI-TcVI DTUs。与基于基因组的策略类似，Tc-STAMS2 方法允许鉴定 DTU 内的菌株。其对不同实验和生物学变量的稳健性使其成为当前克氏锥虫基因分型测定法的一种有价值的补充策略。此外，该方法可用于鉴定与菌株表型相关的 DTU 特异性特征。
