基于GlcNAc代谢的核心细菌合作维持胃菌群稳态的研究

Dysbiosis of microbiota causes a variety of diseases. Finding out factors responsible for maintenance of the stability of microbiota is crucial for understanding mechanisms of dysbiosis. Our previous studies on gastric microbiota identified a core bacteria co-occurrence network, which is essential for maintaining the normal structure and stability of gastric microbiota. Combined with findings from genomic analyses, we hypothesize that N-acetylglucosamine (GlcNAc), produced by Arthrobacter through catalysis of glycan, could act as “public goods” to enhance bacterial cooperation and growth in a way of cross-feeding. This study is to illuminate the metabolic pathway of GlcNAc in co-occurring core bacteria through characterizing involved enzymes and genomic analyses; To demonstrate cross-feeding between co-occurring core bacteria with GlcNAc through in vivo and in vitro studies, which facilitates cooperation between core bacteria; To analyze alterations of the stability and functions of artificial gastric microbiota transplanted to germ-free mice when metabolism of GlcNAc is disrupted. The study will finally demonstrate the effect of bacteria cooperation mediated by GlcNAc on the stability of gastric microbiota. It will be much of helps for understanding mechanisms of dysbiosis, discovering novel intervention targets and approaches for curing dysbiosis.

菌群紊乱是多种疾病的致病因素。明确维持菌群稳态的决定因素是阐明菌群紊乱发生机制的关键问题。前期研究中，我们通过胃菌群系列分析发现，正常胃菌群结构和稳态的维持依赖于特定核心细菌间的共存网络；体外实验表明细菌间合作是共存关系建立的基础；依据基因组学分析结果我们认为节杆菌分解多糖产生的GlcNAc可以作为共有养料，通过交叉喂饲作用促进共存细菌间的合作生长。本研究拟通过酶生化分析和基因组学分析明确共存核心细菌的GlcNAc代谢通路；细菌共培养和无菌小鼠核心细菌移植实验证实GlcNAc作为共有养料介导细菌交叉喂饲，进而揭示共存细菌间的合作机制；探讨GlcNAc代谢缺失情况下无菌小鼠细菌移植模型中人工胃菌群稳态与功能变化，最终阐明基于GlcNAc的细菌合作维持胃菌群稳态的作用与机制。本研究将为揭示胃菌群紊乱发生机制、明确菌群调整的靶点，探讨菌群紊乱治疗策略奠定基础。

为明确维持菌群稳态的决定因素从而阐明菌群紊乱发生机制，本研究探讨了正常胃菌群核心细菌间的共存网络对结构和稳态的维持作用，进一步分析了GlcNAc促进核心细菌间合作形成的作用与机制。1）基因组学分析和酶生化分析发现胃菌群核心细菌包括节杆菌、戈登氏菌、根瘤菌、假诺卡氏菌具备GlcNAc相关代谢的通路Ko00520，并且节杆菌还具备GlcNAc聚糖的分解代谢通路（ko00511）。代谢组学分析发现节杆菌、戈登氏菌、根瘤菌、假诺卡氏菌糖代谢以及氨基酸代谢过程活跃，分解多糖产生的GlcNAc重要中间代谢产物N-乙酰氨基葡萄糖1-磷酸在各细菌中含量升高。2）为了探讨交叉喂饲在核心细菌共存网络形成中的作用，细菌共培实验发现硫磺色节杆菌与戈登氏菌联合培养24小时后细菌数量明显高于硫磺色节杆菌或戈登氏菌单独培养；含40nM GlcNAc硫磺色节杆菌与戈登氏菌的联合培养细菌数量显著高于单独培养的硫磺色节杆菌、戈登氏菌，提示共存细菌间存在基于GlcNAc相互合作机制。3）为进一步分析GlcNAc介导的核心细菌间合作作用对胃菌群稳态稳定作用，通过无菌小鼠细菌定植实验探讨胃菌群改变情况。结果表明具备GlcNAc聚糖的分解代谢通路的野生节杆菌与节杆菌突变株对小鼠胃粘膜菌群结构的影响明显不同，两组小鼠胃菌群的结构呈现明显分离。组成分析发现突变节杆菌组胃菌群中的梭状芽孢杆菌、棒状杆菌、根瘤菌属、戈登氏菌、节杆菌等菌属的相对丰度比野生组显著下降。这表明节杆菌基于GlcNAc的胃菌群稳定作用。Network分析显示组胃菌群的网络互作明显简化，进一步证实野生节杆菌能够维持胃菌群稳态。代谢分析显示突变型节杆菌组糖代谢相关酶的相对频率均值明显降低；病理分析发现胃幽门腺组织异常增生，炎性因子IFN-γ、IL-17 mRNA水平表达增加。. 本研究结果阐明了GlcNAc通过促进细菌间合作从而维持胃菌群稳态的作用，细菌GlcNAc代谢异常会导致胃菌群失调，从而引起胃黏膜炎症和过度增殖。最新文献报道认为多糖有效预防动物模型中自发性胃癌发生，这提示GlcNAc代谢可以作为干预菌群失调的新靶点，为临床建立干预菌群失调新措施提供新的思路。