Bifidobacterium animalis subsp. lactis BPL1™ and Its Lipoteichoic Acid Modulate Longevity and Improve Age/Stress-Related Behaviors in Caenorhabditis elegans

Abstract

Life expectancy has increased globally in recent decades, driving interest in maintaining a healthy life that includes preservation of physical and mental abilities, particularly in elderly people. The gut microbiome becomes increasingly perturbed with aging so the use of probiotics can be a strategy for maintaining a balanced gut microbiome. A previous report showed that Bifidobacterium animalis subsp. lactis BPL1™ induces through its lipoteichoic acid (LTA) fat reduction activities via the insulin/IGF-1 signaling pathway. Here, we have delved into the mechanism of action, eliminating alternative pathways as putative mechanisms. Furthermore, we have identified that BPL1™, its heat treated form (BPL1™ HT) and its LTA prolong longevity in Caenorhabditis elegans (C. elegans) in an insulin/IGF-1-dependent mechanism, and its consumption improves the oxidative stress response, gut permeability and protection against pathogenic infections. Furthermore, positive effects on C. elegans stress-related behaviors and in the Alzheimer's Disease model were found, highlighting the potential of the strain in improving the cognitive functions and proteotoxicity in the nematode. These results indicate the pivotal role of the IGF-1 pathway in the activity of the strain and pave the way for potential applications of BPL1™, BPL1™ HT and its LTA in the field of longevity and age-related markers.

Keywords: BPL1; Bifidobacterium animalis subsp. lactis; C. elegans; LTA; aging; postbiotic; probiotic.

Figure 1

BPL1™, BPL1™ HT and LTA from BPL1™ exert fat reduction activity through IGF-1. (A) Percentage of nematodes with the different cellular localization of DAF-16 after feeding with BPL1™ (10⁸ cfu/plate), BPL1™ HT (10⁸ cells/plate) and LTA from BPL1™ (10 µg/mL). (B) Representative images of the DAF-16 nucleus translocation of nematodes fed with BPL1™, BPL1™ HT and LTA from BPL1™. Image taken with a Nikon-SMZ18 Fluorescence Stereomicroscope (Scale bar: 250 µm). (C) Percentage of Nile Red fluorescence of N2 wild-type worms of pmk-1(km25) and jnk-1(gk7) mutant strains fed with BPL1™ (10⁸ cfu/plate), BPL1™ HT (10⁸ cells/plate) or LTA from BPL1™ (10 µg/mL). Nile Red staining fluorescence was quantified at young adult stage. Orlistat (6 µg/mL) was used as a positive control. Data are mean ± SD and were calculated from two independent experiments (n = 120/condition for fat deposition, n = 50/condition for DAF-16 nuclear translocation). ** p < 0.01, *** p< 0.001. One-way ANOVA was applied.

Figure 2

Positive effect of BPL1™, BPL1™ HT and LTA from BPL1™ on C. elegans lifespan and other age-related phenotypes. Survival rate of N2 wild-type worms fed with BPL1™ alive cells (A), BPL1™ HT (B) and LTA from BPL1™ (C). Curve comparisons vs. E. coli OP50 are indicated (p-values). Two independent experiments were performed (n = 100–200/condition). Log Rank t-test was applied. (D) Survival rate of C. elegans N2 wild-type strain fed with BPL1™ HT (10⁸ cells/plate) and LTA from BPL1™ (5 µg/mL) after acute oxidative stress. NGM was used as a control feeding condition. * p < 0.05, ** p < 0.01. Data correspond to two independent experiments (n = 100/condition). One-way ANOVA was applied. (E) Percentage of fluorescence of nematodes without intestinal damage (control) or worms with damage provoked with 0.8 mM of H₂O₂. *** p < 0.005. Two independent experiments were performed (n = 60/condition). (F) Percentage of fluorescence of nematodes damaged with 0.8 mM of H₂O_2, fed with BPL1™ (10⁸ cfu/plate), BPL1™ HT (10⁸ cells/plate) or LTA from BPL1™ (10 µg/mL). ** p < 0.01. Values are the average of two independent assays (n = 60/condition). One-way ANOVA was applied. (G) Representative images of fluorescence nematodes treated with 0.8 mM of H₂O₂ and fed with BPL1™, BPL1™ HT or LTA from BPL1™. Image taken with a Nikon-SMZ18 Fluorescence Stereomicroscope (Scale bar: 250 µm).

Figure 3

BPL1™, BPL1™ HT and LTA from BPL1™ exert protective effects against pathogen infection. (A) Survival rate of N2 wild-type nematodes fed with E. coli OP50 strain, S. enterica subsp. enterica serovar Typhimurium ATCC 14028 strain and BPL1™ (10⁸ cfu/plate), BPL1™ HT (10⁸ cells/plate) or LTA from BPL1™ (10 µg/mL) in the presence of the pathogen. (p < 0.0001 in the 3 conditions vs. infection.) (B) Survival rate of N2 wild-type nematodes fed with E. coli OP50 strain, S. aureus ATCC 25923 strain and BPL1™ (10⁸ cfu/plate) (p < 0.0001), BPL1™ HT (10⁸ cells/plate) (p < 0.0001) or LTA from BPL1™ (p > 0.05) in the presence of the pathogen. Data are the average of two independent experiments (n = 100/condition). Log Rank t-test was applied.

Figure 4

BPL1™, BPL1™ HT and LTA from BPL1™ have positive effects on anxiety-related behaviors in C. elegans. (A) Quantification of the time to respond to the octanol after applying starvation during 20 min in N2 wild-type worms fed with BPL1™ (10⁸ cfu/plate), BPL1™ HT (10⁸ cells/plate) or LTA from BPL1™ (10 µg/mL). Data are the average from two independent assays (n = 80/condition). *** p < 0.001, ** p < 0.01. Student’s t-test was applied. (B) Quantification of the number of N2 wild-type worms distributed in each zone after the addition of Denubil, and fed with BPL1™ (10⁸ cfu/plate), BPL1™ HT (10⁸ cells/plate) and LTA from BPL1™ (10 µg/mL) (*** p < 0.001, **** p < 0.0001). (### p < 0.001). Data are the average of two independent assays (n = 60/condition). Two-way ANOVA was applied. (C) Percentage of CL4176 non-paralyzed worms fed with BPL1™ overnight lawn, BPL1™ HT (10⁸ cells/plate) and LTA from BPL1™ (10 µg/mL) after induction by temperature raising. Ginkgo biloba extract (EGb 761) at 100 µg/mL was included as a positive control. Nematodes without temperature induction were also included as a negative control. BPL1™ (p < 0.001), BPL1™ HT and LTA from BPL1™ (p < 0.0001). Data correspond to two independent assays (n = 100/condition). Log Rank t-test was applied.

Figure 5

BPL1™, BPL1™ HT and the LTA from BPL1™ regulate fat deposition and longevity through the IGF-1 pathway. Schematic representation of the core components of the IGF-1, JNK-1/DAF-16 and p38 MAPK pathways.