Metabolic Regulation of Longevity and Immune Response in Caenorhabditis elegans by Ingestion of Lacticaseibacillus rhamnosus IDCC 3201 Using Multi-Omics Analysis

Abstract

Probiotics, specifically Lacticaseibacillus rhamnosus, have garnered attention for their potential health benefits. This study focuses on evaluating the probiotic properties of candidate probiotics L. rhamnosus IDCC 3201 (3201) using the Caenorhabditis elegans surrogate animal model, a well-established in vivo system for studying host-bacteria interactions. The adhesive ability to the host's gastrointestinal tract is a crucial criterion for selecting potential probiotic bacteria. Our findings demonstrated that 3201 exhibits significantly higher adhesive capabilities compared with Escherichia coli OP50 (OP50), a standard laboratory food source for C. elegans and is comparable with the widely recognized probiotic L. rhamnosus GG (LGG). In lifespan assay, 3201 significantly increased the longevity of C. elegans compared with OP50. In addition, preconditioning with 3201 enhanced C. elegans immune response against four different foodborne pathogenic bacteria. To uncover the molecular basis of these effects, transcriptome analysis elucidated that 3201 modulates specific gene expression related to the innate immune response in C. elegans. C-type lectin-related genes and lysozyme-related genes, crucial components of the immune system, showed significant upregulation after feeding 3201 compared with OP50. These results suggested that preconditioning with 3201 may enhance the immune response against pathogens. Metabolome analysis revealed increased levels of fumaric acid and succinic acid, metabolites of the citric acid cycle, in C. elegans fed with 3201 compared with OP50. Furthermore, there was an increase in the levels of lactic acid, a well-known antimicrobial compound. This rise in lactic acid levels may have contributed to the robust defense mechanisms against pathogens. In conclusion, this study demonstrated the probiotic properties of the candidate probiotic L. rhamnosus IDCC 3201 by using multi-omics analysis.

Keywords: C. elegans; Lacticaseibacillus rhamnosus; immune response; multi-omics analysis.

Fig. 1. Evaluation of the adhesive ability of Lacticaseibacillus rhamnosus IDCC 3201 in C. elegans.

The adhesive ability of OP50, 3201, or LGG in C. elegans strain fer-15;fem-1 after 48 h conditioning period. OP50, E. coli; 3201, L. rhamnosus IDCC 3201; LGG, L. rhamnosus GG. Statistical analysis was performed using one-way ANOVA, and statistical significance was considered when the p values were below 0.05 (*), 0.01 (**), 0.001 (***), and 0.0001 (****). Statistics compared with 3201: p < 0.0001 and p = 0.3699 for OP50 and LGG, respectively. Data are expressed as means ± SEM.

Fig. 2. Evaluation of longevity and immune response of Lacticaseibacillus rhamnosus IDCC 3201 using C. elegans.

The lifespan of C. elegans strain fer-15;fem-1 using OP50, 3201, and LGG. The killing assay of C. elegans strain fer- 15;fem-1 preconditioned with OP50, 3201, or LGG for 48 h then infected with each of four foodborne pathogenic bacteria (two of each gram-negative and positive bacteria). (A) Lifespan assay. (B) Killing assay using Escherichia coli O157:H7 EDL933. (C) Killing assay using Salmonella Typhimurium SL1344. (D) Killing assay using Staphylococcus aureus Newman. (E) Killing assay using Listeria monocytogenes EGD-e. OP50, E. coli; 3201, L. rhamnosus; LGG, L. rhamnosus. Statistical analysis was performed using the Kaplan–Meier method, and differences were considered significant when the p value was below 0.05 (*) and 0.01 (**). Survival statistic in lifespan assay compared with 3201: p = 0.0000 and p = 0.0000 for OP50 and LGG, respectively. Survival statistics in killing assay compared with 3201: Escherichia coli O157:H7 EDL933, p = 0.0114 and p = 0.5001 for OP50 and LGG, respectively; Salmonella Typhimurium SL1344, p = 0.0000 and p = 0.2732 for OP50 and LGG, respectively; Staphylococcus aureus Newman, p = 0.0000 and p = 0.0000 for OP50 and LGG, respectively; Listeria monocytogenes EGD-e, p = 0.0000 and p = 0.0496 for OP50 and LGG, respectively. Data are expressed as means ± SEM.

Fig. 3. Evaluation of phenotypic change of C. elegans after exposure to Lacticaseibacillus rhamnosus IDCC 3201.

Worm size and locomotive activity of C. elegans strain fer-15;fem-1 after 48 h exposure period with OP50, 3201, or LGG. (A) Length, (B) Width, (C) Peristaltic speed, and (D) Pumping rate. OP50, E. coli; 3201, L. rhamnosus; LGG, L. rhamnosus. Statistical analysis was performed using one-way ANOVA, and statistical significance was considered when the p values were below 0.05 (*), 0.01 (**), 0.001 (***), and 0.0001 (****). Statistics compared with 3201: length, p < 0.0001 and p < 0.0001 for OP50 and LGG, respectively; width, p < 0.0001 and p = 0.0028 for OP50 and LGG, respectively; peristaltic speed, p = 0.5366 and p = 0.6957 for OP50 and LGG, respectively; pumping rate, p < 0.0001 and p = 0.0002 for OP50 and LGG, respectively. Data are expressed as means ± SEM.

Fig. 4. Transcriptomic analysis of C. elegans after exposure to Lacticaseibacillus rhamnosus IDCC 3201.

The identification of KEGG pathways related to genes significantly upregulated by more than 3-fold in C. elegans when fed with 3201 compared with OP50. We utilized Cytoscape for the analysis.

Fig. 5. Metabolomic analysis of C. elegans after exposure to Lacticaseibacillus rhamnosus IDCC 3201.

Change of metabolites composition in C. elegans strain fer-15;fem-1 after 48 h exposure period with OP50 or 3201. (A) PLS-DA. (B) Volcano plot. (C) Top 25 enriched heatmap. (D) The quantitative graph using the metabolites which changed more than 4.0 folds.