Table_3_Prediction of Pathogenic Factors in Dysbiotic Gut Microbiomes of Colorectal Cancer Patients Using Reverse Microbiomics.docx

BackgroundGut microbiome plays a crucial role in the formation and progression of colorectal cancer (CRC). To better identify the underlying gene-level pathogenic mechanisms of microbiome-associated CRC, we applied our newly developed Reverse Microbiomics (RM) to predict potential pathogenic factors using the data of microbiomes in CRC patients.
ResultsOur literature search first identified 40 bacterial species enriched and 23 species depleted in the guts of CRC patients. These bacteria were systematically modeled and analyzed using the NCBI Taxonomy ontology. Ten species, including 6 enriched species (e.g., Bacteroides fragilis, Fusobacterium nucleatum and Streptococcus equinus) and 4 depleted species (e.g., Bacteroides uniformis and Streptococcus thermophilus) were chosen for follow-up comparative genomics analysis. Vaxign was used to comparatively analyze 47 genome sequences of these ten species. In total 18 autoantigens were predicted to contribute to CRC formation, six of which were reported with experimental evidence to be correlated with drug resistance and/or cell invasiveness of CRC. Interestingly, four human homology proteins (EDK89078.1, EDK87700.1, EDK89777.1, and EDK89145.1) are conserved among all enriched strains. Furthermore, we predicted 76 potential virulence factors without homology to human proteins, including two riboflavin synthase proteins, three ATP-binding cassettes (ABC) transporter protein family proteins, and 12 outer membrane proteins (OMPs). Riboflavin synthase is present in all the enriched strains but not in depleted species. The critical role of riboflavin synthase in CRC development was further identified from its hub role in our STRING-based protein−protein interaction (PPI) network analysis and from the finding of the riboflavin metabolism as the most significantly enriched pathway in our KEGG pathway analysis. A novel model of the CRC pathogenesis involving riboflavin synthase and other related proteins including TpiA and GrxC was further proposed.
ConclusionsThe RM strategy was used to predict 18 autoantigens and 76 potential virulence factors from CRC-associated microbiome data. In addition to many of these autoantigens and virulence factors experimentally verified as reported in the literature, our study predicted many new pathogenetic factors and developed a new model of CRC pathogenesis involving the riboflavin synthase from the enriched colorectal bacteria and other associated proteins.

背景
肠道微生物组在结直肠癌（colorectal cancer, CRC）的发生与进展中发挥关键作用。为更好地明确微生物组相关结直肠癌潜在的基因层面致病机制，本研究采用课题组新开发的反向微生物组学（Reverse Microbiomics, RM）方法，利用结直肠癌患者的微生物组数据预测潜在致病因子。

结果
本研究首先通过文献检索，确定了结直肠癌患者肠道内富集的40种细菌与耗竭的23种细菌。采用NCBI分类学本体（NCBI Taxonomy ontology）对上述细菌进行系统性建模与分析。选取其中10种细菌开展后续比较基因组学分析，包括6种富集菌（如脆弱拟杆菌（Bacteroides fragilis）、具核梭杆菌（Fusobacterium nucleatum）及马链球菌（Streptococcus equinus））与4种耗竭菌（如单形拟杆菌（Bacteroides uniformis）及嗜热链球菌（Streptococcus thermophilus））。采用Vaxign软件对这10种细菌的47个基因组序列进行比较分析。本研究共预测出18种潜在参与结直肠癌发生的自身抗原，其中6种已有实验证据表明其与结直肠癌的耐药性及/或细胞侵袭性相关。有趣的是，4种人类同源蛋白（EDK89078.1、EDK87700.1、EDK89777.1及EDK89145.1）在所有富集菌株中均保守存在。此外，本研究还预测出76种无人类同源性的潜在毒力因子，包括2种核黄素合酶蛋白、3种ATP结合盒（ATP-binding cassettes, ABC）转运蛋白家族蛋白及12种外膜蛋白（outer membrane proteins, OMPs）。核黄素合酶在所有富集菌株中均存在，但在耗竭菌株中未检测到。通过基于STRING数据库的蛋白质-蛋白质相互作用（protein−protein interaction, PPI）网络分析，以及KEGG通路富集分析发现核黄素代谢为最显著富集的通路，进一步证实了核黄素合酶在结直肠癌发生发展中的关键作用。本研究进一步提出了一种涉及核黄素合酶及TpiA、GrxC等相关蛋白的结直肠癌致病新模型。

结论
本研究采用反向微生物组学策略，从结直肠癌相关微生物组数据中预测出18种自身抗原与76种潜在毒力因子。除部分自身抗原与毒力因子已被文献报道的实验研究所证实外，本研究还预测出多种全新致病因子，并构建了一种由富集肠道细菌来源的核黄素合酶及其他相关蛋白参与的结直肠癌致病新模型。