Phylogenomics of 8,839 Clostridioides difficile genomes reveals recombination-driven evolution and diversification of toxin A and B.
Phylogenomics of 8,839 Clostridioides difficile genomes reveals recombination-driven evolution and diversification of toxin A and B.
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DOI：
10.1371/journal.ppat.1009181
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发表时间：
2020-12
期刊：
影响因子：
6.7
通讯作者：
Doxey AC
中科院分区：
文献类型：
--
作者：
Mansfield MJ;Tremblay BJ;Zeng J;Wei X;Hodgins H;Worley J;Bry L;Dong M;Doxey AC
艰难梭状芽胞杆菌是全球抗生素相关性胃肠道感染的主要原因。艰难梭菌致病基因(PaLoc)编码一种或两种同源毒素，即毒素A(TcdA)和毒素B(TcdB)，是艰难梭菌致病所必需的。然而，毒素序列的变异给诊断分析、治疗和疫苗的发展带来了重大挑战。在这里，我们对8,839株艰难梭菌及其毒素进行了全面的系统基因组分析，其中包括我们从NCBI Short Read档案中收集的6,492株基因组。共有5,175个TcdA和8,022个TcdB基因聚为7个(A1-A7)和12个(B1-B12)不同的亚型，这构成了艰难梭菌毒素亚型的新方法的基础。我们开发了一种单倍型着色算法来可视化所有毒素序列的氨基酸变异，这表明TcdB通过在其整个序列中进行广泛的同源重组而多样化，并通过不同的重组事件形成新的亚型。相反，TcdA主要在其C末端重复区域的重复数上有所不同，这表明重组介导的TcdB的多样化在艰难梭菌的进化中提供了一种选择性优势。然后，通过对波士顿布里格姆妇女医院的351株艰难梭菌临床分离株的分类，验证了毒素亚型的应用，展示了其临床实用价值。亚型将TcdB分为两个功能和抗原组，由基因内重组产生，包括两个不同的细胞环绕表型，是否识别卷曲的蛋白作为受体，以及是否能有效地被FDA批准的唯一治疗性抗体Bzlooxumab中和。我们的分析还确定了TcdB结构中八个普遍保守的表面斑块，代表了开发广谱治疗的理想靶点。最后，我们建立了一个开放的在线数据库(DiffBase)，作为艰难梭菌毒素收集和分类的中央枢纽，这将帮助临床医生决定针对特定毒素变体的治疗策略，并允许研究人员监测艰难梭菌正在进行的进化和多样化。艰难梭状芽胞杆菌是全球抗生素相关性胃肠道感染的主要原因。两种毒素(TcdA和TcdB)是导致致病性差异的两种毒素，但这些毒素中的基因变异使广谱诊断、治疗和疫苗的发展复杂化。在这里，我们提供了艰难梭菌现有毒素序列的全球分类和分析，并引入了一个新的开放在线数据库(Diffbase.uwater lo.ca)，以满足临床和研究界尚未满足的需求。我们的分析将TcdA和TcdB基因分成7个和12个不同的组，这为基于序列的艰难梭菌毒素亚型提供了一种新的方法。我们的分析表明，重组尤其驱动了TcdB的广泛多样化，导致TcdB亚型具有不同的抗原性、功能性和表型特性。作为我们方法的验证，我们能够快速分型来自布里格姆妇女医院的351株临床菌株的新数据集，预测它们的表型和临床特征。最后，基于序列分析，我们确定了TcdB中的保守区，它们代表了艰难梭菌普遍治疗和诊断发展的理想靶点。
Clostridioides difficile is the major worldwide cause of antibiotic-associated gastrointestinal infection. A pathogenicity locus (PaLoc) encoding one or two homologous toxins, toxin A (TcdA) and toxin B (TcdB), is essential for C. difficile pathogenicity. However, toxin sequence variation poses major challenges for the development of diagnostic assays, therapeutics, and vaccines. Here, we present a comprehensive phylogenomic analysis of 8,839 C. difficile strains and their toxins including 6,492 genomes that we assembled from the NCBI short read archive. A total of 5,175 tcdA and 8,022 tcdB genes clustered into 7 (A1-A7) and 12 (B1-B12) distinct subtypes, which form the basis of a new method for toxin-based subtyping of C. difficile. We developed a haplotype coloring algorithm to visualize amino acid variation across all toxin sequences, which revealed that TcdB has diversified through extensive homologous recombination throughout its entire sequence, and formed new subtypes through distinct recombination events. In contrast, TcdA varies mainly in the number of repeats in its C-terminal repetitive region, suggesting that recombination-mediated diversification of TcdB provides a selective advantage in C. difficile evolution. The application of toxin subtyping is then validated by classifying 351 C. difficile clinical isolates from Brigham and Women’s Hospital in Boston, demonstrating its clinical utility. Subtyping partitions TcdB into binary functional and antigenic groups generated by intragenic recombinations, including two distinct cell-rounding phenotypes, whether recognizing frizzled proteins as receptors, and whether it can be efficiently neutralized by monoclonal antibody bezlotoxumab, the only FDA-approved therapeutic antibody. Our analysis also identifies eight universally conserved surface patches across the TcdB structure, representing ideal targets for developing broad-spectrum therapeutics. Finally, we established an open online database (DiffBase) as a central hub for collection and classification of C. difficile toxins, which will help clinicians decide on therapeutic strategies targeting specific toxin variants, and allow researchers to monitor the ongoing evolution and diversification of C. difficile. Clostridioides difficile is a major worldwide cause of antibiotic-associated gastrointestinal infection. Two toxins (TcdA and TcdB) are responsible for C. diffile pathogenicity, but genetic variants within these toxins complicate the development of broad-spectrum diagnostics, therapeutics and vaccines. Here we provide a global classification and analysis of available C. difficile toxin sequences and introduce a new open online database (diffbase.uwaterloo.ca) to serve the unmet needs of the clinical and research community. Our analysis partitions TcdA and TcdB genes into 7 and 12 distinct groups which provides a new method for sequence-based C. difficile toxin subtyping. Our analysis revealed that recombination has driven extensive diversification of TcdB in particular, resulting in TcdB subtypes with distinct antigenic, functional, and phenotypic properties. As validation of our method, we were able to rapidly subtype a new dataset of 351 clinical strains from Brigham and Women’s Hospital, predicting their phenotypic and clinical features. Lastly, based on sequence analysis, we identified conserved regions in TcdB that represent ideal targets for the development of universal C. difficile therapeutics and diagnostics.