Data Sheet 10_Wilson’s disease-associated gut dysbiosis: novel insights into microbial functional alterations, virulence changes, and resistance markers.pdf

BackgroundAlthough the gut microbiota is associated with a variety of metabolic, inflammatory, and neurological disorders through microbial dysbiosis, current studies on the gut microbiota in Wilson’s disease (WD) remain limited. Critical gaps exist in understanding the roles of key functional microbial factors in WD pathogenesis, which hinders the acquisition of mechanistic insights into this disease.
ObjectiveThis study aims to characterize alterations in the gut microbiome associated with WD, with a particular emphasis on virulence factors (VFs) and antibiotic resistance genes (ARGs), as well as functional mobile genetic elements (MGEs), in order to elucidate their potential roles in disease progression and clinical manifestations.
MethodsWe analyzed fecal samples from 37 patients with WD and 33 healthy controls (HCs) using metagenomic sequencing, with a specific focus on examining virulence gene profiles and antibiotic resistance patterns and MGE composition in relation to liver function markers.
ResultsBeta diversity analysis revealed significant differences in the gut microbial community structure between patients with WD and HCs, and a distinct set of microbial taxa was identified that showed significant associations with clinical indicators. A gut microbial co-occurrence network identified key species playing central roles in the microbial community structure, including Prevotella stercorea, Firmicutes bacterium CAG 110, Bacteroides salyersiae, Lactococcus petauri, Streptococcus cristatus, Actinomyces sp. HMSC035G02, and Streptococcus viridans. Widespread functional dysbiosis was detected across multiple biological levels in patients with WD, with significant correlations identified between these microbial alterations and clinical indicators. Significant disruptions were identified in key metabolic pathways, including the Pentose Phosphate Pathway, Pyruvate Metabolism, and Starch and Sucrose Metabolism, which were associated with the dysregulation of carbohydrate-active enzymes (CAZymes). These alterations showed significant correlations with clinical markers of liver dysfunction (e.g., procollagen III N-terminal peptide PIIINP, aspartate transaminase/alanine transaminase AST/ALT). A total of 54 virulence factor (VF) genes exhibited differential abundance in WD, with 36 genes depleted and 18 enriched. Notably, these included colibactin genes (clbB, clbH) from Escherichia coli and type IV secretion system genes (aec19, pilB). These VFs were significantly associated with indicators of liver function (e.g., bilirubin levels) and coagulation abnormalities. Among the detected antibiotic resistance genes (ARGs), 21 exhibited disease-specific patterns in WD, notably tetQ (encoding tetracycline resistance), ErmB (conferring macrolide resistance), and cfxA6 (mediating cephamycin resistance). Furthermore, ARG profiles were associated with Bifidobacterium enrichment and showed significant correlations with lipid metabolism markers [e.g., triglycerides (TG), high-density lipoprotein cholesterol (HDL-C)]. Critically, we identified significant enrichment of 60 functional mobile genetic elements (MGEs) in WD, spanning categories involved in DNA replication/repair, phage activity, and conjugative transfer, indicating heightened genomic plasticity and horizontal gene transfer potential. Strikingly, correlation network analysis revealed strong and specific co-occurrence between key ARGs (e.g., ErmX) and defined suites of MGEs, suggesting MGE-facilitated dissemination of resistance determinants.
ConclusionWilson’s disease (WD) patients exhibit significant alterations in gut microbial community structure and functional dysbiosis, wherein the enrichment of virulence genes (such as colibactin genes clbB/clbH) and the specific antibiotic resistance genes (such as tetQ and ErmB), and the activation of mobile genetic elements are closely associated with clinical indicators including liver function impairment, coagulation abnormalities, and lipid metabolism disorders.

背景 尽管肠道菌群通过微生物群失调与多种代谢、炎症及神经系统疾病相关，但目前针对威尔逊病（Wilson’s disease, WD）的肠道菌群研究仍较为有限。在理解关键功能微生物因子在威尔逊病发病机制中的作用方面仍存在重大空白，这阻碍了对该疾病的机制解析。
研究目的 本研究旨在表征与威尔逊病相关的肠道菌群组改变，重点关注毒力因子（virulence factors, VFs）、抗生素抗性基因（antibiotic resistance genes, ARGs）以及功能型可移动遗传元件（mobile genetic elements, MGEs），以阐明它们在疾病进展与临床表现中的潜在作用。
研究方法 本研究采用宏基因组测序（metagenomic sequencing）技术，对37例威尔逊病患者与33例健康对照（healthy controls, HCs）的粪便样本进行分析，重点考察与肝功能指标相关的毒力基因谱、抗生素抗性模式以及可移动遗传元件组成。
研究结果 β多样性分析显示，威尔逊病患者与健康对照的肠道微生物群落结构存在显著差异，并鉴定出一组与临床指标显著相关的独特微生物类群。肠道微生物共现网络明确了在群落结构中发挥核心作用的关键物种，包括粪普雷沃氏菌（Prevotella stercorea）、厚壁菌门细菌CAG 110、萨氏拟杆菌（Bacteroides salyersiae）、彼特里乳球菌（Lactococcus petauri）、克里斯滕链球菌（Streptococcus cristatus）、放线菌属HMSC035G02菌株以及草绿色链球菌（Streptococcus viridans）。威尔逊病患者在多个生物学层面均存在广泛的功能失调，且这些微生物改变与临床指标存在显著相关性。研究发现包括磷酸戊糖途径、丙酮酸代谢、淀粉与蔗糖代谢在内的关键代谢通路发生显著紊乱，该现象与碳水化合物活性酶（carbohydrate-active enzymes, CAZymes）的调控失衡相关。上述改变与肝功能异常的临床标志物（如Ⅲ型前胶原N端肽PIIINP、天冬氨酸转氨酶/丙氨酸转氨酶AST/ALT）显著相关。本研究共鉴定出54个丰度存在差异的毒力因子（VF）基因，其中36个基因丰度降低，18个基因丰度升高。值得注意的是，这些基因包括来自大肠杆菌的大肠杆菌素基因（clbB、clbH）以及Ⅳ型分泌系统基因（aec19、pilB）。这些毒力因子与肝功能指标（如胆红素水平）及凝血异常显著相关。在检测到的抗生素抗性基因（ARGs）中，有21个在威尔逊病中呈现疾病特异性模式，包括四环素抗性基因tetQ、介导大环内酯类抗性的ErmB以及介导头孢霉素抗性的cfxA6。此外，抗生素抗性基因谱与双歧杆菌属富集相关，并与脂质代谢标志物[如甘油三酯（TG）、高密度脂蛋白胆固醇（HDL-C）]存在显著相关性。至关重要的是，本研究发现威尔逊病患者体内60个功能型可移动遗传元件（MGEs）显著富集，涵盖DNA复制/修复、噬菌体活性以及接合转移相关类别，提示基因组可塑性与水平基因转移潜能增强。令人瞩目的是，相关性网络分析揭示了关键抗生素抗性基因（如ErmX）与特定类别的可移动遗传元件之间存在强特异性共现关系，表明可移动遗传元件可促进抗性决定因子的传播。
结论 威尔逊病患者的肠道微生物群落结构与功能均存在显著改变，其中毒力基因（如大肠杆菌素基因clbB/clbH）、特异性抗生素抗性基因（如tetQ与ErmB）的富集，以及可移动遗传元件的激活，与肝功能损伤、凝血异常及脂质代谢紊乱等临床指标密切相关。