Lashermes A, Mathieu E, Marinelli L, Léjard V, Dervyn E, Martin-Gallausiaux C, Jules M, Lapaque N, Doré J, Multon M-C, Greifenberg D M, Plomer M, Righetto Z, Perez Iii M, Blottière H M
Université Paris-Saclay, 27057INRAE, AgroParisTech, Micalis Institute, 78350, Jouy-en-Josas, France.
Université Paris-Saclay, 27057INRAE, MetaGenoPolis, 78350, Jouy-en-Josas, France.
Benef Microbes. 2025 Jan 3;16(3):347-362. doi: 10.1163/18762891-bja00050.
Probiotics are widely used for their health promoting effects, though a lot remain to be discovered, particularly on their mechanisms of action at the molecular level. The functional genomic approach is an appropriate method to decipher how probiotics may influence human cell fate and therefore contribute to their health benefit. In the present work, we focused on Shouchella clausii (formerly named Bacillus then Alkalihalobacillus clausii), a spore-forming bacterium that is commercially available as a probiotic for the prevention and the treatment of intestinal dysbiosis and related gastrointestinal disorders, such as diarrhoea. Several studies have demonstrated that S. clausii treatment modulated inflammatory and immune responses, as well as gut barrier functions. A functional genomic strategy was implemented to decipher the mechanisms by which S. clausii exerts its probiotic effects on human intestinal epithelial cells. To do so, a large genomic DNA fragment library was constructed for each of the four strains: O/C, N/R, SIN and T. A high throughput in vitro screening in human epithelial cells was then conducted, using the reporter gene strategy, targeting the nuclear factor kappa B (NF-κB) pathway and interleukin-10 (IL-10) gene expression. After an exhaustive in vitro screening of approximately a thousand clones per library, several clones modulating the NF-κB pathway in the HT-29 reporter cell line were identified. Among clone lysates, 1.1% (O/C), 1.4% (N/R), 2.0% (SIN), and 1.2% (T) were identified as biologically active on immune reporter systems (NF-κB and IL-10 expression). After transposon mutagenesis and a new set of screening and sequencing, 23 coding sequences (CDS) were identified, including one encoding for the glutamine synthetase, associated with NF-κB modulation, and six CDS for IL-10 modulation. The functional genomic strategy that was applied to S. clausii was an original approach to identify gene candidates that may explain the mechanisms of action of probiotics. However, further work is needed to validate the identified leads.
益生菌因其促进健康的作用而被广泛使用，尽管仍有许多有待发现，特别是在分子水平上的作用机制。功能基因组学方法是一种合适的方法，用于解读益生菌如何影响人类细胞命运，从而有助于其对健康的益处。在本研究中，我们聚焦于克劳氏肖凯拉菌（原名为芽孢杆菌，后更名为克劳氏嗜碱芽孢杆菌），这是一种形成芽孢的细菌，作为益生菌可用于预防和治疗肠道菌群失调及相关胃肠道疾病，如腹泻。多项研究表明，克劳氏肖凯拉菌治疗可调节炎症和免疫反应以及肠道屏障功能。我们实施了一种功能基因组学策略，以解读克劳氏肖凯拉菌对人肠道上皮细胞发挥益生菌作用的机制。为此，为四个菌株（O/C、N/R、SIN和T）分别构建了一个大型基因组DNA片段文库。然后使用报告基因策略，针对核因子κB（NF-κB）途径和白细胞介素-10（IL-10）基因表达，在人上皮细胞中进行高通量体外筛选。在对每个文库约一千个克隆进行详尽的体外筛选后，在HT-29报告细胞系中鉴定出了几个调节NF-κB途径的克隆。在克隆裂解物中，1.1%（O/C）、1.4%（N/R）、2.0%（SIN）和1.2%（T）被确定对免疫报告系统（NF-κB和IL-10表达）具有生物活性。经过转座子诱变以及新一轮的筛选和测序，鉴定出了23个编码序列（CDS），其中包括一个编码谷氨酰胺合成酶的序列，与NF-κB调节相关，还有六个CDS与IL-10调节相关。应用于克劳氏肖凯拉菌的功能基因组学策略是一种识别可能解释益生菌作用机制的基因候选物的原创方法。然而，需要进一步的工作来验证所确定的线索。
