The Transcription Factor DAF-16 is Essential for Increased Longevity in C. elegans Exposed to Bifidobacterium longum BB68

The longevity-promoting benefits of lactobacilli were hypothesized as early as 1907. Although the anti-aging effects of lactic acid bacteria (LAB) have been observed in nematodes, rodents and humans for over a century, the mechanisms underlying the effects of probiotics on aging have rarely been assessed. Using the Caenorhabditis elegans (C. elegans) model, various studies have elucidated the role of different signaling cascades, especially the DAF-16 cascade, on lifespan extension by LAB. In this study, the mechanisms through which Bifidobacterium longum strain BB68 affects the longevity of C. elegans were assessed. The lifespan of nematodes increased by 28% after worms were fed BB68, and this extension of lifespan was completely lost in backgrounds containing a mutated DAF-16 gene. High levels of DAF-16 (in the daf-16 (mu86); muIs61 strain) nuclear accumulation and high expression of the SOD-3 gene (a DAF-16-specific target gene) were observed as a result of BB68 treatment. Immunofluorescence microscopy revealed that TIR-1 and JNK-1 are involved in the phosphorylation and activation of DAF-16. Thus, BB68 increased the longevity of nematodes by activating the TIR-1 – JNK-1 – DAF-16 signaling pathway, and the cell wall component of BB68 contributed to longevity.

increased lifespan in C. elegans by regulating the conserved innate immune signaling mediated by DAF-16. Thus, the cell wall components of BB68 might contribute to the longevity of nematodes.
In C. elegans, mitogen-activated protein kinase (MAPK) signaling and insulin-like signaling (ILS) regulate the process of aging and innate immunity 7,8 . These pathways are well conserved across mammals and nematodes, which designates C. elegans as an ideal model for investigating the process of aging and immune regulation. In mammals, probiotic strains enhance host immunity by regulating the p38/MAPK signaling pathway 9 , suggesting that probiotics might affect the immunity of nematodes and aging through a homologous pathway. We previously reported that Bifidobacterium longum BB68, which was isolated from a centenarian, exerts potent activity in regulating immunity 6 ; however, little is known about its effects on aging.
First, we tested whether BB68 could prolong the lifespan of C. elegans. Survival assays revealed that feeding BB68 to C. elegans could extend the lifespan of wild-type N2 organisms by 28% relative to the lifespan of those fed standard food, Escherichia coli (E. coli) OP50 ( Fig. 1A; Table S1). Moreover, the lifespan extension by BB68 did not affect the nematodes' pharynx pumping, body size, or reproductive ability (Table S2). These results are also supported by a previous study, in which Bifidobacterium infantis did not alter these indices 2 . The bacterial gradient concentration feeding assay (0.1-200 mg/plate) and eat-2 (ad1116) mutant (eating defective mutant with the phenotype of calorie restriction) survival assays indicated that the BB68-mediated lifespan extension was independent of calorie restriction (Table S1; Fig. S1).
To investigate the role of ILS in the BB68-mediated longevity of C. elegans, DAF-16 and DAF-2, the key regulators of ILS, were evaluated. DAF-16, an FOXO transcription factor in C. elegans, controls the transcription of several antioxidant and chaperone genes that delay aging 10 . The lifespan assays showed that mutating DAF-16 (daf-16 (mu86)) caused the BB68-mediated effect of prolonged lifespan to be completely lost ( Fig. 1A; Table S1). This phenomenon indicated that such an extension is DAF-16 dependent. Using a DAF-16-GFP fusion reporter strain (daf-16 (mu86); muIs61), we demonstrated that BB68 significantly increased the nuclear accumulation of DAF-16 by 56% ( Fig. 1B; Table S3) relative to that induced by OP50. Western blotting also showed increased nuclear localization of DAF-16 protein (Fig. 1C). These results suggested the up-regulation of DAF-16 in response to BB68 treatment. To confirm the role of DAF-16 in BB68-induced longevity, the level of gene expression of SOD-3, one of the DAF-16-specific target genes, was determined by qPCR 11 . A significant increase in the expression of SOD-3 (2.27-fold compared to SOD-3 expression in worms fed OP50) was observed in worms fed BB68 for 24 h (Fig. 1D). Thus, we concluded that DAF-16 is essential for BB68-mediated lifespan prolongation.
DAF-2 is a human insulin receptor homolog upstream of DAF-16 in ILS, and mutations in DAF-2 decrease extensions in lifespan and resistance to pathogens by activating the nuclear translocation of DAF-16 8,10 . Our results show that feeding BB68 to worms further prolongs the lifespan of daf-2 (e1368) mutants ( Fig. 1A; Table S1), indicating that the BB68-mediated regulation of DAF-16 is independent of DAF-2.
In addition to functioning in ILS, DAF-16 is downstream of MAPK. To identify the regulator in the MAPK pathway involved in the BB68-induced, DAF-16-mediated regulation of the extension of lifespan, JNK and p38 were assessed. JNK-1 and PMK-1/p38, two members of the MAPK family in C. elegans, can transmit environmental stress signals to DAF-16 and regulate its nuclear localization 12,13 . Our results show that BB68 extends the lifespan of pmk-1 (km25) mutants, but not jnk-1 (gk7) mutants (Table S1; Fig. 1E), and activated JNK-1 was observed in the nerve ring of BB68-treated nematodes (Fig. 2). In addition, the BB68-induced nuclear translocation of DAF-16 was decreased in worms with a mutated JNK-1 gene ( Fig. 1B; Table S3). Thus, JNK appears to be involved in the BB68-mediated regulation of DAF-16. Additionally, we found that the TIR-domain protein TIR-1 participates in the BB68-mediated longevity signaling cascade. BB68 did not increase the lifespan of tir-1 (ok1052) worms ( Fig. 1E; Table S1), and mutations in TIR-1 suspended JNK-1 activation and BB68-induced DAF-16 nuclear accumulation (Table S3; Fig. 2). Therefore, we concluded that BB68 regulates DAF-16 through the TIR-JNK signal transduction pathway, an innate immunity signaling cascade conserved from nematodes to mammals.
To assess the potential active component of BB68 on nematode longevity, the cell wall component (CW) and cell wall-free extracts (CFE) of BB68 were separated and fed to nematodes. Compared to the CF or CFE of OP50, the CW of BB68 significantly increased the lifespan of C. elegans in a dose-response manner (P < 0.05); however, the CFE of BB68 did not exert a similar effect (Fig. 3). Thus, these results indicate that the cell wall components of BB68 contribute to its effects on longevity in nematodes. Our data confirm that Bifidobacterium longum strain BB68 prolongs the lifespan of C. elegans by regulating the activity of DAF-16, which is involved in a conserved immune signaling cascade (Fig. 4). Since these regulators are likely conserved across several species, the possibility of increasing the longevity of humans through the consumption of probiotics is elevated. Nevertheless, future studies are essential to determine the effect of Bifidobacterium longum BB68 on longevity in mammals.

Methods
Bacterial strains and culture conditions. Bifidobacterium longum strain BB68 was isolated from fecal samples of healthy centenarians in Bama County, Guangxi, China, and preserved in the laboratory. E. coli OP50 (OP50), a standard food for nematodes, was obtained from the Caenorhabditis Genetics Center (CGC, USA).
The BB68 strain was cultured at 37 °C in anaerobic conditions with GENbox anaer (bioMérieux, France) using MRS broth (LuQiao, China) for 18 h. OP50 was grown in LB broth (AoBoXing, China) at 37 °C for 18 h with agitation at 220 rpm. Bacteria were harvested by centrifugation at 4,000 × g for 15 min, washed twice with sterile M9 buffer, and centrifuged at 16,000 × g for 15 min at 4 °C to remove the supernatant. Then, the bacteria were adjusted to a final concentration of 0.4 mg/µL (wet weight) in M9 buffer; this slurry was used as concentrated bacteria and preserved at −80 °C. Before use, the concentrated bacteria were transferred into a 95 °C water bath for 30 min and used as heat-killed bacteria.
Lifespan assay. L4 stage nematodes grown on NGM plates were transferred to mNGM plates with a platinum wire. For each lifespan assay, 100 worms for every bacterial species were assayed in ten plates (ten worms/ plate), and plates were cultured at 25 °C. The number of live and dead worms was determined using a dissecting microscope (Chong Qing Optical, China) every 24 h. The lifespan assay was conducted at least three times. The nematode survival rate was calculated using the Kaplan-Meier method, and differences in survival rates were evaluated for significance using the log-rank test (P < 0.05).
Food gradient feeding assay. Heat-killed BB68 and OP50 bacterial suspensions were spread on mNGM plates in serial concentrations ranging from 0.01-200 mg bacterial cells/plate. L4 stage N2 worms were placed on these plates. For each concentration, 100 worms were assayed over ten plates (ten worms/plate). Lifespan was measured as described previously. The test was conducted at least three times.
Measurements of body size, pharynx pumping rate, and reproduction. L4 larvae were placed on NGM plates coated with bacterial lawns. Ten worms/bacterial species were assayed using ten plates (one worm/ plate). Beginning on the first day that C. elegans were transferred to fresh NGM plates, the size of the live worms was examined every 24 h. Images of adult nematodes were captured using an XSP-8CZ digital microscope (Chong Qing Optical), and the projection area of the worms was analyzed as the body size using ImageJ software (National Institutes of Health, USA). To determine the pharynx pumping rate, L4 nematodes were grown on NGM plates in the presence of bacterial lawns for 30 min before counting. The pumping frequency was recorded as the number of contractions in the terminal bulb of the pharynx of an individual worm in a 60-s period. The experiment was performed five times. For the reproduction assay, the worms were transferred daily to fresh NGM plates until reproduction ceased. The offspring of each animal were counted at the L2 or L3 stage. The test was repeated three times.  indicates that no effect was observed in this study. A dashed line indicates that the test was not performed in this study. A "P" indicates a phosphorylation site. *Indicates direct or indirect phosphorylation.
SCIENTIFIC RepoRTs | 7: 7408 | DOI:10.1038/s41598-017-07974-3 Nuclear and cytosolic distribution of DAF-16 (Western blotting). The L4 larvae of DAF-16::GFP worms were collected. Subsequently, the nuclear and cytosolic fractions were separated, and the DAF-16 protein was detected by Western blotting as described by Singh and Aballay 14 . Lamin B1 and GAPDH were used as markers for the nuclear and cytosolic fractions, respectively. Antibodies against GAPDH, DAF-16, and lamin B1 were obtained from Cell Signaling Technology (CST; USA), Santa Cruz Biotechnology (USA), and Abcam (USA), respectively.