Taxonomic characterization of the strain MKAK9 and its in vitro probiotic properties

The 16S rDNA sequence of the strain MKAK9 was sequenced, and subsequent BLAST analysis revealed significant similarity to the type strain Levilactobacillus brevis strain ATCC 14869 (Accession No. NR 044704.2). To establish phylogenetic relationships, a rooted phylogenetic tree of the strain MKAK9 was constructed alongside sequences of the type strain L. brevis ATCC 14869 and other Levilactobacillus species (Supplementary file 1, Fig. S1). The analysis indicated that the closest relative of strain MKAK9 was Levilactobacillus brevis ATCC 14869, exhibiting a remarkable 99% pairwise sequence similarity. Consequently, strain MKAK9 was officially submitted to the NCBI database as Levilactobacillus brevis strain MKAK9 (Supplementary file 1, Table S1).

Strain MKAK9 underwent rigorous in vitro screening tests for probiotic attributes, following the guidelines set forth by the Department of Biotechnology (DBT) and the Indian Council of Medical Research (ICMR), Government of India [53]. These in vitro tests encompassed evaluations for tolerance to acidic and bile salt conditions, as well as adherence to gastrointestinal epithelium. Notably, MKAK9 demonstrated robust viability, retaining 83.24% and 79.22% viability after 1 h of exposure to simulated gastric juice (SGJ) at pH 3.0 and pH 1.0, respectively (Supplementary file 1, Table S3). Furthermore, exposure to SGJ containing pepsin at pH 2.0 resulted in viability rates of 83.05% after 1 h and 79.08% after 3 h (Supplementary file 1, Table S3). Following treatment with SGJ containing pancreatin (pH 8.0) for 4 h, viability remained high at 81.86% (Supplementary file 1, Table S3). Bile salt tolerance tests demonstrated that strain MKAK9 exhibited remarkable resilience, retaining 85.19% and 79.89% survival rates against bile salt concentrations of 0.3% and 1%, respectively, after 4 h (Supplementary file 1, Table S3).

Given its promising tolerance to simulated gastrointestinal conditions, strain MKAK9 underwent further evaluation for adhesion using the gastrointestinal cell line HT-29. Strain MKAK9 exhibited a substantial adherence rate of 72.17% following a 4 h incubation period (Supplementary file 1, Table S3). Additionally, an antibiotic sensitivity assay revealed that strain MKAK9 was susceptible to tetracycline, ampicillin, sulphatriad, and streptomycin, while demonstrating resistance to penicillin-G and chloramphenicol (Supplementary file 1, Table S4). These findings align closely with the antibiotic sensitivity profiles reported for other Levilactobacillus strains, as described by Costa et al. [54].

Feeding of HK MKAK9 slowed the aging of worms

Treatment with live strain MKAK9 significantly increased the longevity of worms by 25.61% compared to the live standard bacterium E. coli OP50 (***P < 0.0001, log-rank test) (Fig. 1A). Notably, feeding heat-killed MKAK9 (abbreviated as HK MKAK9) also extended the mean lifespan of worms by 24.40% compared to HK OP50 (***P < 0.0001, log-rank test) (Fig. 1A). This suggests that both live and heat-killed MKAK9 can enhance the mean lifespan of worms, prompting us to proceed with HK MKAK9 for further investigation compared to HK OP50. Additionally, the longevity-promoting effect also supported the in vivo investigation in worms, suggesting that strain MKAK9 is beneficial and non-pathogenic to in vivo host.

Fig. 1

Feeding of probiotic heat-killed MKAK9 improves longevity, age-associated biomarkers and resistance of worms against pathogenic infections and abiotic stress in worms. A A lifespan assay on worms was performed after feeding with the bacteria: heat-killed E. coli OP50 (HK OP50), E. coli OP50 (OP50), heat-killed L. brevis MKAK9 (HK MKAK9), L. brevis MKAK9 (MKAK9), and (***P < 0.0001, log-rank test). B-C The pharynx pumps, and locomotory activity of heat-killed bacterium-treated worms were measured on day 14. D-E The accumulation of the aging pigment lipofuscin was measured on day 14 in heat-killed bacterium-treated worms. The lipofuscin levels were observed in worms under a confocal microscope (10X magnification, scale bar, 100 μm). F-G Treatment of HK MKAK9 increased the mean survival of worms against oxidative stress (Paraquat, 10 mM) and heat stress at 35 °C. H-I Pre-treatment of worms on HK reduced the colonization of pathogens S. aureus MTCC3160 and E. coli MTCC 1687 in the intestine of worms. The heat-killed bacterium-treated effects were statistically compared using a Student’s t-test (*P < 0.05 and **P < 0.01). The error bars represent the mean ± SEM

Conducting a developmental rate assay on different bacterial diets revealed that HK MKAK9-fed worms exhibited slower development from eggs to the egg-laying reproductive adult stage compared to HK OP50-treated worms (**P < 0.01) (Supplementary file 1, Fig. S2). However, there were no significant changes observed in the body size of HK MKAK9-fed worms compared to HK OP50-fed worms (P > 0.05 on days 4, 5, 6, and 7) (Supplementary file 1, Fig. S3).

The longevity of organisms often correlates with changes in age-related biomarkers in worms, such as locomotory activity, pharyngeal pumping rate, and the accumulation of the aging pigment lipofuscin [34]. To explore the effect of HK MKAK9, we conducted analyses on these biomarkers. Firstly, we examined the effect of HK MKAK9 on the pharyngeal pumping rate. Our results revealed a notable enhancement in the pharyngeal pumping rate of worms by 52.55% compared to HK OP50-fed worms (*P < 0.05) (Fig. 1B). Subsequently, we evaluated the locomotory activity of worms treated with HK MKAK9 by quantifying the number of body bends per 30 s. Interestingly, we observed a substantial improvement in the frequency of body turns, which increased by 45.20% compared to HK OP50-fed worms (*P < 0.05) (Fig. 1C). Furthermore, we investigated the accumulation of the aging pigment lipofuscin in 14-day worms treated with HK MKAK9. Strikingly, our analysis unveiled a significant reduction of 36.5% in the lipofuscin levels in HK MKAK9-treated worms compared to HK OP50 (*P < 0.05) (Figs. 1D and E). These findings collectively suggest that HK MKAK9 slowed aging and exerted beneficial effects on age-related biomarkers, indicating its potential as a modulator of aging processes in worms.

Feeding of HK MKAK9 provided resistance against abiotic stress and pathogenic infections

The administration of HK MKAK9 significantly enhanced the survival of worms against oxidative stress (Paraquat, 10 mM) by 15.10% compared to HK OP50-treated worms (*P < 0.05) (Fig. 1F). Furthermore, we investigated the protective effects of HK MKAK9 against thermal stress (35 °C) on worms. Our results revealed a notable 17.15% increase in the mean survival rate of HK MKAK9-treated worms compared to those fed with HK OP50 (**P < 0.01) (Fig. 1G).

We investigated the effect of HK MKAK9 on resistance against pathogenic infections in worms. Our findings revealed that HK MKAK9 significantly enhanced worm survival by 23.72% and 16.69% against S. aureus strain MTCC3160 and E. coli strain MTCC1687, respectively (**P < 0.001 for S. aureus and *P < 0.01 for pathogen E. coli, log-rank test) (Supplementary file 1, Figs. S4A and B). Furthermore, we explored whether HK MKAK9 could mitigate pathogenic bacterial colonization, thereby extending worm survival. To investigate this possibility, we pre-cultured young adult worms on either HK OP50 or HK MKAK9 and subsequently transferred them to pathogen-seeded NGM plates. Remarkably, our results demonstrated a significant reduction in the colonization of both S. aureus and E. coli in HK MKAK9-treated worms compared to those fed with HK OP50 (N.S. P > 0.05 for S. aureus on day 1, *P < 0.05 for S. aureus on day 3 and day 5, N.S. P > 0.05 for pathogen E. coli on day 1 and day 3, **P < 0.01 for pathogen E. coli on day 5) (Fig. 1H and I). Notably, the number of pathogenic bacteria exhibited a significant decrease after day 3 of pathogen treatment (*P < 0.05 for S. aureus on day 5 and **P < 0.01 for pathogen E. coli on day 5) (Fig. 1H and I).

Treatment of HK MRKA9 improved cellular and mitochondrial redox states in worms

We aimed to elucidate the alterations in cellular and mitochondrial redox status in HK MKAK9-treated worms. Firstly, employing 2,7-dichlorofluorescein diacetate (H2DCFDA), we assessed cytoplasmic ROS levels in vivo in HK MKAK9- and HK OP50-treated worms. Encouragingly, our results revealed a significant reduction of 45.2% in cytoplasmic ROS levels in HK MKAK9-treated worms compared to those treated with HK OP50 (**P < 0.01) (Fig. 2A and B). Subsequently, we evaluated superoxide dismutase (SOD) activity in worms following HK MKAK9 treatment. HK MKAK9 treatment led to a notable enhancement of 37.5% in SOD activity compared to HK OP50-treated worms (*P < 0.05) (Fig. 2C). Furthermore, we examined the glutathione (GSH) to glutathione disulfide (GSSG) ratio, a crucial indicator of cellular oxidative environment [55]. Intriguingly, HK MKAK9 treatment resulted in approximately a two-fold improvement in the GSH/GSSG ratio in day 14 worms compared to those treated with HK OP50 (*P < 0.05) (Fig. 2D).

Fig. 2

Treatment of HK MKAK9 improved the cellular and mitochondrial redox state in worms. A-D Feeding of HK MKAK9 reduced the accumulation of cytoplasmic ROS levels, improved SOD activity, and the GSH/GSSG ratio in worms. E–G Treatment of HK MKAK9 also reduced mitochondrial ROS levels, thereby improving ATP levels in worms compared to HK OP50-treated worms. The cytoplasmic and mitochondrial ROS levels were observed in worms under a confocal microscope (10X magnification, scale bar, 100 μm). The heat-killed bacterium-treated effects were statistically compared using Student’s t-test (N.S. for P > 0.05, *P < 0.05, and **P < 0.01). The bars represent the mean ± SEM

We employed MitoTracker Red CMXRos to explore the changes in mitochondrial redox state. Our findings demonstrated a substantial reduction of 41.4% in mitochondrial ROS levels in HK MKAK9-treated worms compared to HK OP50-treated worms (**P < 0.01) (Fig. 2E and F). Moreover, we investigated the effect of HK MKAK9 on ATP synthesis levels in worms. HK MKAK9 treatment led to a significant improvement of 38.2% in ATP levels in day 14 worms compared to those treated with HK OP50 (*P < 0.05) (Fig. 2G). These results collectively showed the potent antioxidative effects of HK MKAK9 in mitigating oxidative stress and enhancing mitochondrial function in worms.

Treatment of HK MKAK9 regulates evolutionary conserved insulin-like signaling and the p38 MAPK pathway to promote longevity and immune responses in worms

Diverse conserved signaling mechanisms govern aging. To elucidate the role of the p38 MAPK pathway in HK MKAK9-mediated longevity in worms, we examined its involvement using loss-of-function mutants of the p38 MAPK cascade (nsy-1, sek-1, and pmk-1). HK MKAK9 failed to enhance longevity in these mutants (P > 0.05 for nsy-1, sek-1, and pmk-1, log-rank test) (Table S5) (Fig. 3A, B, and C). Additionally, we investigated whether HK MKAK9-induced upregulation of the p38 MAPK cascade activated downstream gene expression, specifically skn-1. Notably, the skn-1 (zu67) allele mutant failed to exhibit HK MKAK9-induced longevity (P > 0.05, log-rank test) (Table S5) (Fig. 3D). Next, explored the involvement of insulin-like signaling, focusing on daf-2 and daf-16 genes, in mediating HK MKAK9-induced longevity. Both loss-of-function daf-2 and daf-16 mutants failed to extend longevity upon HK MKAK9 treatment (P > 0.05 for daf-2 and daf-16, log-rank test) (Table S5) (Fig. 3E and G). Furthermore, we assessed the role of the TGF-β homolog dbl-1, finding that HK MKAK9 significantly extended longevity in the dbl-1 mutant (***P < 0.0001, log-rank test) (Table S5) (Fig. 3G).

Fig. 3

Effect of HK MKAK9 on the lifespan of C. elegans loss-of-function mutants. Longevity assays were conducted with mutants (A) nsy-1 (ag3), B sek-1 (ag1), C pmk-1 (km25), D skn-1 (zu67), E daf-16 (mgDf50), F daf-2 (e1368), and G dbl-1 (nk3) V. The heat-killed bacterium-treated effects were statistically compared using a log-rank test (N.S. for P > 0.05 and ***P < 0.0001). H qRT-PCR Expression of longevity-promoting genes in HK MKAK9-treated worms compared to HK OP50-fed worms. I A schematic representation showing that HK MKAK9 downregulates insulin-like signaling and upregulates the p38 MAPK pathway to extend longevity in worms. The heat-killed bacterium-treated effects were statistically compared using the Student’s t-test (*P < 0.05 and **P < 0.01). The error bars represent the mean ± SEM

Subsequent qRT-PCR analysis confirmed the modulation of gene expression involved in insulin-like signaling and p38 MAPK pathway in HK MKAK9-induced longevity. Notably, daf-2 expression decreased significantly in HK MKAK9-treated worms compared to HK OP50-treated worms (*P < 0.05 for daf-2) (Fig. 3H). Conversely, the expression of genes involved in the p38 MAPK cascade (nsy-1, sek-1, and pmk-1) increased significantly in HK MKAK9-treated worms (**P < 0.01 for pmk-1 and nsy-1, *P < 0.05 for sek-1) (Fig. 3H). Moreover, the expression of the TGF-β homolog dbl-1 was upregulated approximately two-fold in HK MKAK9-treated worms (*P < 0.05 for dbl-1) (Fig. 3H). However, longevity assays in dbl-1 mutants did not support a significant role for dbl-1 in HK MKAK9-induced longevity.

Furthermore, we confirmed the activation of downstream transcription factors of insulin-like signaling and p38 MAPK cascade, daf-16 and skn-1, by HK MKAK9 treatment. The expression of both daf-16 and skn-1 genes significantly increased by approximately two-fold in HK MKAK9-treated worms compared to HK OP50-treated worms (**P < 0.01 for daf-16 and *P < 0.05 for skn-1) (Fig. 3H). These findings collectively suggest that HK MKAK9 modulates insulin-like signaling and the p38 MAPK pathway to extend longevity in worms (schematically represented in F3g. 3I).

Treatment of HK MKAK9 improves proteostasis by upregulating protein ubiquitination and autophagy receptor expression through insulin-like signaling to extend longevity in worms

We employed mRNA sequencing on young adult wild-type worms following a 24 h treatment with HK MKAK9 and HK OP50 to unravel the downstream pathways of insulin-like signaling and the p38 MAPK pathway modulated by HK MKAK9 in worms. Differential expression analysis of genes revealed a significant transcriptional profile alteration in HK MKAK9-treated worms compared to those treated with HK OP50 (Supplementary file 1, Fig. S5). Subsequently, we generated a heatmap illustrating the differential mRNA expression patterns through hierarchical clustering analyses (Supplementary file 1, Fig. S6). After screening for genes with FDR-adjusted P < 0.05 and fold change across the mean value of HK MKAK9-treated worms versus HK OP50-treated worms > 1, we compiled a list of 229 upregulated and 26 downregulated genes (Supplementary file 2), potentially crucial for HK MKAK9-induced longevity in C. elegans.

Protein ubiquitination is a critical regulatory mechanism in proteostasis, involving the tagging of proteins for degradation. This process becomes increasingly important as organisms age and accumulate non-functional proteins. The ubiquitination process is mediated by Cullin-based E3 ubiquitin ligase complexes, prominently including the SCF (Skp, Cullin, F-box containing) complex [56]. These complexes play a pivotal role in recognizing and targeting proteins for ubiquitination and subsequent degradation. At the core of the SCF complex's functionality are the Skp-1 protein, serving as an adaptor, and various F-box proteins, which are responsible for substrate recognition [56]. Our gene ontology (GO) enrichment analysis revealed upregulation of genes involved in SCF-dependent ubiquitin-mediated protein catabolism, protein ubiquitination, as well as those encoding components of the Cul2-RING ubiquitin ligase complex in HK MKAK9-treated worms compared to HK OP50-treated worms (Fig. 4A, B, and C, Supplementary file 3). Additionally, protein domain enrichment analyses using the DAVID tool by querying the Interpro database identified overexpression of several domain classes related to the SCF complex (SKP1 component (IPR001232), SKP1 component POZ (IPR016073), E3 ubiquitin ligase, SCF complex (IPR016897), SKP1 component dimerization (IPR016072), F-box domain cyclin like (IPR001810), and F-box associated domain type 2 (IPR012885)), further corroborating our findings (Fig. 4D, Supplementary file 3). KEGG analyses highlighted enrichment of critical pathways including ubiquitin-mediated proteolysis, protein processing in the endoplasmic reticulum, and pathways associated with longevity regulation (Fig. 4D, Supplementary file 3). Furthermore, qRT-PCR validation confirmed the upregulated expression of Skp-1-related genes of the SCF complex (skr-7, skr-8, skr-9, skr-14, skr-10, skr-12, and skr-13) involved in protein ubiquitination (**P < 0.01 for skr-8 and skr-9, *P < 0.05 for skr-7, skr-14, skr-10, skr-12, and skr-13) (Fig. 5A).

Fig. 4

A-C Gene ontology (GO) analysis of the upregulated genes involved in molecular function, biological process, and cellular components after treatment of HK MKAK9-treated worms compared to HK OP50-treated worms. D Protein domain enrichment analyses using the DAVID tool by querying the Interpro database and KEGG analysis showing critical pathways

Fig. 5

Treatment of HK MKAK9 regulated insulin-like signaling pathway to promote protein ubiquitination and autophagy-lysosome pathway to promote longevity in worms. A qRT-PCR expression of genes involved in protein ubiquitination (Skp-1-related genes, and autophagy-receptor in wild-type N2 worms. B qRT-PCR of protein ubiquitination representative gene (skr-8) and autophagy gene (sqst-3) in daf-16 (i.e., the downstream target of daf-2/insulin-like signaling) and pmk-1 (i.e., a gene involved in p38 MAPK cascade) mutants (C) qRT-PCR expression of genes involved in innate immunity and antioxidation. D qRT-PCR expression confirmed proteomic analysis results of the genes involved in protein synthesis, RNA metabolism, and the lysosome-associated membrane protein gene lmp-1 (lmp-1). E qRT-PCR of protein synthesis, RNA metabolism, and the lysosomal protein gene (lmp-1) in daf-16 and pmk-1 mutants. F The lifespan assay on loss-of-function lmp-1 mutants confirms their role in HK MKAK9-induced longevity in worms. The lifespan assays were statistically compared using a log-rank test (N.S. for P > 0.05 and ***P < 0.0001). The heat-killed bacterium-treated effects were statistically compared using Student’s t-test (N.S. for P > 0.05, *P < 0.05, and **P < 0.01). The error bars represent the mean ± SEM

Additionally, our gene ontology (GO) enrichment analysis and qPCR results showed upregulation of the sqst-3 gene encoding an autophagy receptor (**P < 0.01 for sqst-3 and N.S. P > 0.05 for sepa-1, vet-2, and vet-6), suggesting the involvement of autophagy in HK MKAK9-induced longevity (Fig. 5A, Supplementary file 3).

Further investigation using qRT-PCR on daf-16 (downstream target of daf-2/insulin-like signaling) and pmk-1 (a gene involved in the p38 MAPK cascade) mutants revealed that HK MKAK9 regulates protein ubiquitination and autophagy. The results confirmed that HK MKAK9 treatment to daf-16 mutant did not change the expression of skr-8 (a protein ubiquitination representative gene) as well as sqst-3 (an autophagy-receptor) (Fig. 5B) (N.S. P > 0.05 for skr-8 and sqst-3 in the daf-16 mutant). In contrast, there was a significant increase in the expression of skr-8 and sqst-3 in pmk-1 mutants (*P < 0.05 for skr-8 and **P < 0.01 for sqst-3 in pmk-1 mutant) (Fig. 5B). Thus, it was confirmed that HK MKAK9 regulated the insulin-like signaling pathway in a DAF-16-dependent manner, not the p38 MAPK pathway, to promote protein ubiquitination and autophagy.

Administration of HK MKAK9 enhances immune responses in worms

GO analysis highlighted upregulation of genes associated with innate immune responses, defense responses to pathogens, as well as cellular responses to oxidative stress and heat (Fig. 4A). Protein domain enrichment analysis indicated increased domain proteins related to immunity, including F-box protein, saponins, and cytochrome P450 in HK MKAK9-treated worms (Fig. 4D). qRT-PCR validation confirmed the upregulated expression of genes involved in innate immunity (thn-1, ilys-1, cnc-2, spp-9, spp-21, clec-47, and clec-266) and antioxidative genes (gst-44 and sod-3) (**P < 0.01 for ilys-1 and clec-47, *P < 0.05 for thn-1, cnc-2, spp-9, spp-21, clec-266, gst-44, and sod-3) (Fig. 5C). These findings shed light on the multifaceted molecular mechanisms underlying HK MKAK9-induced longevity and its potential implications in immunity and antioxidative machinery in C. elegans.

Treatment of HK MKAK9 enhanced lysosome-Associated Membrane Protein (LAMP) family protein lmp-1 expression through insulin-like signaling to promote longevity in worms

In our investigation of the effects of HK MKAK9 on proteome dynamics, proteomics analysis was conducted on young adult wild-type N2 worms post 24 h exposure to HK MKAK9 and HK OP50 at 20 °C. This analysis revealed the presence of 2768 proteins across both conditions. Focusing on proteins with significant changes in abundance (based on the median log2-fold change of HK MKAK9/HK OP50), we identified 26 proteins with significant differential expression in the HK MKAK9-treated group (Supplementary file 4). Notably, this group exhibited a marked increase in the abundance of lysosome-associated membrane protein (LAMP) family member lmp-1, a protein synonymous with mammalian lysosomal markers (Supplementary file 4). Subsequent qRT-PCR analysis corroborated the upregulation of lmp-1 gene expression (***P < 0.001 for lmp-1) (Fig. 5D).

Furthermore, the proteomics analysis revealed a decrease in proteins associated with the translational machinery and RNA metabolism in HK MKAK9-treated worms compared to HK OP50-tretaed. This includes proteins such as 26S rRNA -methyltransferase (nsun-1), RNA helicase (ddx-17), and a putative H/ACA ribonucleoprotein complex subunit 2-like protein (Y48A6B.3) (Fig. 5D, Supplementary file 4). qRT-PCR analysis confirmed significant downregulation of genes Y48A6B.3 and ddx-17 in HK MKAK9-treated worms, whereas nsun-1 levels did not show substantial alteration (N.S. P > 0.05 for nsun-1, *P < 0.05 for Y48A6B.3 and ddx-17) (Fig. 5D).

To further delineate the regulatory pathways involved, qRT-PCR was performed on daf-16 and pmk-1 mutants subjected to HK MKAK9 treatment, aiming to verify its role on the expression of genes linked to protein lmp-1 expression, translational machinery, and RNA metabolism. The analysis revealed no significant changes in the expression of lmp-1, Y48A6B.3, and ddx-17 in daf-16 mutant, whereas pmk-1 mutant exhibited increased expression of these genes (N.S. P > 0.05 for lmp-1, ddx-17, and Y48A6B.3 in daf-16 mutant, *P < 0.05 for ddx-17, Y48A6B.3 in pmk-1 mutant, and **P < 0.01 for lmp-1 in pmk-1 mutant) (Fig. 5E). These findings highlight the pivotal role of the insulin-like signaling pathway, particularly through DAF-16, in regulating the expression of proteins involved in lysosome integrity, protein synthesis, and RNA metabolism in HK MKAK9-treated worms.

Additionally, lifespan assays were conducted on lmp-1 loss-of-function mutant fed with HK MKAK9 to confirm its role in HK MKAK9-induced longevity. The result indicated that HK MKAK9 treatment did not extend longevity in the lmp-1 mutant (N.S. P > 0.05, log-rank test) (Fig. 5F). In sum, our findings substantiate the hypothesis that HK MKAK9 treatment regulates insulin-like signaling pathways that increase the expression of polyubiquitinated proteins, tagging damaged or aggregated proteins for degradation by activating the autophagy-lysosome pathway to promote longevity in worms.

mir-243 partially lengthens the worm’s lifespan by regulating insulin-like signaling and its downstream genes involved in protein ubiquitination, autophagy receptor Sqst-3, and lysosomal LAMP family protein lmp-1

We next asked whether observed differential gene expression patterns and altered proteomic profiles might be associated with the changes in the microRNA (miRNA) profiles. Through microRNA sequencing, we identified a specific pattern of miRNA expression in HK MKAK9-treated worms: three miRNAs (mir-243, mir-253, and mir-78) were upregulated, while mir-1818 was downregulated (Supplementary file 5). Subsequent lifespan assays on worms with mutations in these differentially expressed miRNAs revealed that HK MKAK9 treatment significantly enhanced the lifespan in mir-78, mir-253, and mir-1818 mutants (Fig. 6B, C, and D) (***P < 0.0001 for mir-78, mir-253, and mir-1818, log-rank test). Interestingly, while mir-1818 mutants showed a higher mortality rate than the other miRNA mutants, their lifespan still increased in HK MKAK9-treated worms (Fig. 6D) (***P < 0.0001, log-rank test). However, mir-243 mutant stands out as a distinctive loss-of-function miRNA mutant, exhibiting a noteworthy alteration in the response to HK MKAK9 treatment. Specifically, we found that HK MKAK9-induced longevity was partially declined in the mir-243 mutant and increased only 12.16% in HK MKAK9-treated worms compared to the HK OP50-treated worms (**P < 0.001, log-rank test) (Fig. 6A). This observation underlines the partial involvement of mir-243 in mediating the longevity-promoting effects of HK MKAK9.

Fig. 6

Treatment of HK MKAK9 and its exopolysaccharide (EPS) partially extend longevity by improving the expression of mir-243 in worms. A-D Lifespan assays on differentially expressed loss-of-function miRNA mutants. E qRT-PCR expression of genes involved in insulin-like signaling (daf-16), protein ubiquitination representative gene (skr-8), autophagy-receptor (sqst-3), lysosomal protein lmp-1 (lmp-1), and the p38 MAPK pathway gene (pmk-1) in mir-243 mutant compared to their expression in wild-type N2 worms (Data from Figs. 3H, 5A and D). F The lifespan assay showing the effect of cellular components and derived EPS component of HK MKAK9 in improving longevity. G qRT-PCR expression of daf-16 gene in worms treated with cellular components of HK MKAK9 and compared with daf-16 expression in HK OP50-treated worms. H qRT-PCR expression showing the effect of EPS in regulating expression of genes involved in p38 MAPK pathway (pmk-1), protein ubiquitination (skr-8), autophagy-receptor (sqst-3), and lysosomal protein lmp-1 (lmp-1). I EPS of HK MKAK9 partially decreased longevity in the mir-243 mutant. The lifespan assays were statistically compared using a log-rank test (N.S. for P > 0.05, (**P < 0.001, and ***P < 0.0001). The heat-killed bacterial effects were significantly compared using Student’s t-test (N.S. for P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001). The error bars represent the mean ± SEM

To further dissect mir-243's involvement, we conducted qRT-PCR analysis on mir-243 mutants to elucidate its role in modulating the insulin-signaling pathway and p38 MAPK signaling, alongside its impact on genes associated with protein ubiquitination (skr-8), the autophagy receptor (sqst-3), and the lysosomal LAMP protein lmp-1 (lmp-1) in worms fed with HK MKAK9 compared to those fed with HK OP50. Additionally, we compared the gene expression profiles between the mir-243 mutant and wild-type N2 worms treated with HK MKAK9 and HK OP50 (Data from Figs. 3H, 5A, and D). Our analysis revealed a significant reduction in the expression of daf-16, a downstream target of the DAF-2/insulin-like signaling pathway, in HK MKAK9-treated mir-243 mutants compared to daf-16 expression in wild-type N2 worms (*P < 0.05 for daf-16 in mir-243 mutant and **P < 0.01 for daf-16 in N2 worms) (Fig. 6E). Additionally, we observed a notable decrease in the upregulation of representative genes involved in protein ubiquitination (skr-8), the autophagy receptor sqst-3 (sqst-3), and the lysosomal protein lmp-1 (lmp-1) in HK MKAK9-treated mir-243 mutants compared to their expression in HK MKAK9-treated N2 worms (*P < 0.05 for lmp-1 in mir-243 mutant and **P < 0.01 for lmp-1 in N2 worms, *P < 0.05 for skr-8 in mir-243 mutants and **P < 0.01 for skr-8 in N2 worms, and *P < 0.05 for sqst-3 in mir-243 mutants and **P < 0.01 for sqst-3 in N2 worms) (Fig. 6E). Furthermore, there was no significant change in the expression of pmk-1, a gene associated with the p38 MAPK pathway, between mir-243 mutants and N2 worms (**P < 0.01 for pmk-1 in both mir-243 mutants and N2 worms) (Fig. 6E). Taken together, our findings from the lifespan screening of differentially regulated miRNA mutants and qRT-PCR analysis indicate that HK MKAK9 upregulates the expression of mir-243, which in turn partially regulates the insulin-like signaling pathway, possibly directly or indirectly modulating protein ubiquitination and their degradation by activating the autophagy-lysosomal pathway to enhance longevity in worms.

Exopolysaccharide (EPS) from HK MKAK9 increases longevity by regulating insulin-like signaling, mir-243 expression, and its downstream genes involved in protein ubiquitination, autophagy receptor (Sqst-3), and lysosomal LAMP family protein lmp-1

We investigated the role of specific cellular components of HK MKAK9 on the longevity of C. elegans. Initially, we fractionated various cellular components, including the cell wall, peptidoglycan-wall teichoic acid (PGN-WTA), lipoteichoic acid (LTA), S-layer protein, and extracellular polysaccharides (EPS). We performed lifespan assays with synchronized adult-stage wild-type N2 worms treated with different fractions of HK MKAK9. Specifically, the cell wall and its associated EPS component of HK MKAK9 significantly increased worm longevity by 20.95% and 28%, respectively (***P < 0.0001 for cell wall and EPS, log-rank test) (Fig. 6F). Conversely, treatment with LTA derived from HK MKAK9 did not show a significant effect on worm longevity (P > 0.05 for LTA, log-rank test) (Fig. 6F). Additionally, adult wild-type N2 worms cultured on S-layer and PGN-WTA exhibited developmental arrest (Supplementary file 1: Fig. S7). Subsequently, we treated synchronized adult wild-type N2 worms with specific cellular components of HK MKAK9 for 24 h and analyzed the expression of daf-16 through qRT-PCR. Remarkably, only the cell wall and EPS components of HK MKAK9 induced the upregulation of daf-16 expression in N2 worms (*P < 0.05 for cell wall, **P < 0.01 for EPS) (Fig. 6G), confirming their role in modulating the insulin-like signaling pathway in a DAF-16-dependent manner. Further analysis revealed that the EPS component of HK MKAK9 also upregulated the expression of pmk-1 (a gene involved in the p38 MAPK pathway), skr-8 (representative of protein ubiquitination), sqst-3 (autophagy receptor), and lysosomal LAMP protein lmp-1 (*P < 0.05 for pmk-1 and skr-8, **P < 0.01 for sqst-3, and ***P < 0.001 for lmp-1) (Fig. 6H). Lastly, we conducted a lifespan assay using EPS from HK MKAK9 in the mir-243 mutant. Interestingly, EPS only partially increased longevity in the mir-243 mutant, suggesting the involvement of mir-243 in extending the lifespan of wild-type N2 worms (Fig. 6I) (**P < 0.001, log-rank test). In conclusion, EPS derived from HK MKAK9 promotes longevity by regulating mir-243 expression, which activates insulin-like signaling in a DAF-16-dependent manner. This activation further enhances protein ubiquitination and its degradation by stimulating the autophagy-lysosomal pathway.