Table_4_Features and Colonization Strategies of Enterococcus faecalis in the Gut of Bombyx mori.xlsx

The complex gut microbiome is a malleable microbial community that can undergo remodeling in response to many factors, including the gut environment and microbial properties. Enterococcus has emerged as one of the predominant gut commensal bacterial and plays a fundamental role in the host physiology and health of the major economic agricultural insect, Bombyx mori. Although extensive research on gut structure and microbiome diversity has been carried out, how these microbial consortia are established in multifarious niches within the gut has not been well characterized to date. Here, an Enterococcus species that was stably associated with its host, the model organism B. mori, was identified in the larval gut. GFP–tagged E. faecalis LX10 was constructed as a model bacterium to track the colonization mechanism in the intestine of B. mori. The results revealed that the minimum and optimum colonization results were obtained by feeding at doses of 105 CFU/silkworm and 107 CFU/silkworm, respectively, as confirmed by bioassays and fluorescence-activated cell sorting analyses (FACS). Furthermore, a comprehensive genome-wide exploration of signal sequences provided insight into the relevant colonization properties of E. faecalis LX10. E. faecalis LX10 grew well under alkaline conditions and stably reduced the intestinal pH through lactic acid production. Additionally, the genomic features responsible for lactic acid fermentation were characterized. We further expressed and purified E. faecalis bacteriocin and found that it was particularly effective against other gut bacteria, including Enterococcus casselifavus, Enterococcus mundtii, Serratia marcescens, Bacillus amyloliquefaciens, and Escherichia coli. In addition, the successful colonization of E. faecalis LX10 led to drastically increased expression of all adhesion genes (znuA, lepB, hssA, adhE, EbpA, and Lap), defense genes (cspp, tagF, and esp), regulation gene (BfmRS), secretion gene (prkC) and immune evasion genes (patA and patB), while the expression of iron acquisition genes (ddpD and metN) was largely unchanged or decreased. This work establishes an unprecedented conceptual model for understanding B. mori–gut microbiota interactions in an ecological context. Moreover, these results shed light on the molecular mechanisms of gut microbiota proliferation and colonization in the intestinal tract of this insect.

复杂的肠道微生物组（gut microbiome）是一种可塑的微生物群落，可响应多种因素发生重塑，其中包括肠道环境与微生物自身特性。肠球菌属（Enterococcus）已成为主要的肠道共生菌之一，在重要经济农业昆虫家蚕（Bombyx mori）的宿主生理与健康中发挥着基础性作用。尽管学界已对肠道结构与微生物组多样性开展了大量研究，但目前对于这些微生物群落如何在肠道内多样微生态位中建立仍未得到充分阐释。本研究从家蚕幼虫肠道中分离得到一种与宿主、模式生物家蚕（B. mori）稳定共生的肠球菌属物种。我们构建了带有绿色荧光蛋白（GFP）标签的粪肠球菌（E. faecalis）LX10菌株，以此作为模式菌以追踪其在家蚕肠道内的定殖机制。生物测定与荧光激活细胞分选分析（fluorescence-activated cell sorting analyses，FACS）结果证实，以10⁵菌落形成单位（colony-forming unit，CFU）/蚕的剂量饲喂时可获得最低定殖效果，而10⁷ CFU/蚕的饲喂剂量则可获得最佳定殖效果。进一步通过全基因组信号序列的全面分析，我们深入解析了粪肠球菌LX10的相关定殖特性。粪肠球菌LX10在碱性条件下生长良好，并可通过产生乳酸稳定降低肠道pH值。此外，我们还对其参与乳酸发酵的基因组特征进行了系统表征。我们进一步表达并纯化了粪肠球菌细菌素，发现其对多种其他肠道细菌具有显著抑制活性，包括卡塞拉肠球菌（Enterococcus casselifavus）、蒙氏肠球菌（Enterococcus mundtii）、粘质沙雷氏菌（Serratia marcescens）、解淀粉芽孢杆菌（Bacillus amyloliquefaciens）以及大肠杆菌（Escherichia coli）。此外，粪肠球菌LX10的成功定殖可显著上调所有粘附相关基因（znuA、lepB、hssA、adhE、EbpA及Lap）、防御相关基因（cspp、tagF及esp）、调控基因（BfmRS）、分泌基因（prkC）以及免疫逃逸基因（patA与patB）的表达水平；而铁摄取相关基因（ddpD与metN）的表达则基本无变化或出现下调。本研究建立了一个前所未有的概念模型，可用于在生态背景下解析家蚕与肠道菌群的互作关系。此外，本研究结果还为阐明该昆虫肠道内菌群增殖与定殖的分子机制提供了新的见解。