The TGF-β ligand DBL-1 is a key player in a multifaceted probiotic protection against MRSA in C. elegans

Abstract

Worldwide the increase in multi-resistant bacteria due to misuse of traditional antibiotics is a growing threat for our health. Finding alternatives to traditional antibiotics is thus timely. Probiotic bacteria have numerous beneficial effects and could offer safer alternatives to traditional antibiotics. Here, we use the nematode Caenorhabditis elegans (C. elegans) to screen a library of different lactobacilli to identify potential probiotic bacteria and characterize their mechanisms of action. We show that pretreatment with the Lactobacillus spp. Lb21 increases lifespan of C. elegans and results in resistance towards pathogenic methicillin-resistant Staphylococcus aureus (MRSA). Using genetic analysis, we find that Lb21-mediated MRSA resistance is dependent on the DBL-1 ligand of the TGF-β signaling pathway in C. elegans. This response is evolutionarily conserved as we find that Lb21 also induces the TGF-β pathway in porcine epithelial cells. We further characterize the host responses in an unbiased proteome analysis and identify 474 proteins regulated in worms fed Lb21 compared to control food. These include fatty acid CoA synthetase ACS-22, aspartic protease ASP-6 and vitellogenin VIT-2 which are important for Lb21-mediated MRSA resistance. Thus, Lb21 exerts its probiotic effect on C. elegans in a multifactorial manner. In summary, our study establishes a mechanistic basis for the antimicrobial potential of lactobacilli.

Figure 1

Identifying probiotic lactobacilli strains using C. elegans. (a) Outline of screening setup to detect bacteria that extend nematode lifespan compared to control bacteria, E. coli OP50 and strains isolated in the screen. (b) Lifespan of rrf-3(pk1426) mutants feeding on Lactobacillus spp. Lb21 compared to control bacteria, OP50. (c) Lifespan of rrf-3(pk1426) mutants feeding on Lactobacillus strain Lb23 compared to control bacteria, OP50. (d) CFU levels on day 5 of animals fed OP50 expressing GFP (OP50-GFP) until adulthood (day 3) and then shifted to Lb21, Lb23 or OP50 for 48 h (n = 10). (e) Localization and level of OP50-GFP after 5 days ± UV treatment of plates seeded with OP50-GFP. Scale bar = 100 μm. Graphs show a representative experiment from several independent experiments. See Table S1 for mean ± SD and log-rank test significance.

Figure 2

Lb21 but not Lb23 increases MRSA tolerance in C. elegans. (a) Survival of rrf-3(pk1426) mutants pretreated with Lb21, Lb23 and OP50 for 48 h before being shifted to MRSA strain 43484. (b) Survival of rrf-3(pk1426) mutants pretreated with Lb21, Lb23 and OP50 for 48 h before being shifted to MRSA strain 64. (c) Survival of rrf-3(pk1426) mutants after pretreatment with Lb21, Lb23 and OP50 for 48 h before being challenged with the pathogen ETEC F18. Graphs show a representative experiment from several independent experiments. See Table S1 for mean ± SD and log-rank test significance. (d) CFU level on day 6 of Lb21, Lb23 or OP50 and MRSA 43484 in rrf-3(pk1426) mutants pretreated for 48 h with Lb21, LB23 or OP50, respectively (3 technical replicates, n = 10 animals pr. group).

Figure 3

DBL-1 is essential for Lb21-mediated MRSA tolerance. (a) Double mutant daf-16(mu86);rrf-3(pk1426) survival compared to survival of rrf-3(pk1426) pretreated with either OP50 or Lb21 for 48 h before being shifted to MRSA 43484. (b) Double mutant rrf-3(pk1426);pmk-1(km25) survival compared to survival of rrf-3(pk1426) pretreated with either OP50 or Lb21 before being shifted to MRSA 43484. (c) Double mutant tol-1(nr2033);rrf-3(pk1426) survival compared to survival of rrf-3(pk1426) pretreated with either OP50 or Lb21 before being shifted to MRSA 43484. (d) Double mutant rrf-3(pk1426);dbl-1(nk3) survival compared to survival of rrf-3(pk1426) pretreated with either OP50 or Lb21 before being shifted to MRSA 43484. Graphs show a representative experiment from several independent experiments. See Table S1 for mean ± SD and log-rank test significance.

Figure 4

C. elegans proteome response to Lb21, Lb23 and OP50. (a) Heat map showing hierarchical clustering of the C. elegans proteome response to Lb21, Lb23 and OP50, based on log(2) transformed intensities of all quantifiable proteins. (b) Venn diagram showing the number of proteins with regulated abundance when two of the three food sources are compared pairwise. A total of 474 proteins showed regulated abundance in either of the comparisons. (c) A subset of regulated proteins between Lb21 and Lb23. (d) A subset of regulated proteins between Lb21 and OP50. See Table S3 for list of all regulated proteins.

Figure 5

acs-22, vit-2 and asp-6 are important for the host response to Lb21. (a) Double mutant rrf-3(pk1426); acs-22(tm3236) pretreated with Lb21 before MRSA challenge compared to rrf-3(pk1426) pretreated with Lb21 before MRSA challenge. (b) Double mutant rrf-3(pk1426);vit-2(ok3211) pretreated with Lb21 before MRSA challenge compared to rrf-3(pk1426) pretreated with Lb21 before MRSA challenge. (c) Double mutant rrf-3(pk1426);asp-6(tm2213) pretreated with Lb21 before MRSA challenge compared to rrf-3(pk1426) pretreated with Lb21 before MRSA challenge. See Table S1 for additional data.

Figure 6

Lb21 but not Lb23 enhanced TGF-β expression of pig epithelial cells (IPEC-J2). Pig epithelial cells (IPEC-J2) were cultured with bacterial supernatant (Lb21 or Lb23, respectively) for 6 h. Cells were then collected and TGF-β mRNA was measured by RT-PCR. The experiments were performed twice with a total of 6 replicates. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; n.s. not statistically significant. (a) The TSB media did not increase TGF-β mRNA. However, the Lb21 supernatant at 10⁷ CFU/mL significantly increased TGF-β expression in IPEC-J2 cells. (b) TGF-β induction in IPEC-J2 cells by the Lb21 supernatant is dose-dependent.

Figure 7

Lb21 affects C. elegans in a multifaceted manner. The protection against MRSA mediated by Lb21 is dependent on DBL-1 and the effect could likely be mediated by TGF-β controlled immune genes. MRSA tolerance is also dependent on ACS-22 and the underlying mechanism could be both improved intestinal barrier function and altered fat metabolism. VIT-2 and ASP-6 are also required for the protective effect of Lb21 towards MRSA infection. Black lines indicate interactions shown in this study and grey lines indicate possible mechanisms of action based on published studies.