Lactobacillus rhamnosus Lcr35 as an effective treatment for preventing Candida albicans infection in the invertebrate model Caenorhabditis elegans: First mechanistic insights

Abstract

The increased recurrence of Candida albicans infections is associated with greater resistance to antifungal drugs. This involves the establishment of alternative therapeutic protocols, such as probiotic microorganisms whose antifungal potential has already been demonstrated using preclinical models (cell cultures, laboratory animals). Understanding the mechanisms of action of probiotic microorganisms has become a strategic need for the development of new therapeutics for humans. In this study, we investigated the prophylactic anti-C. albicans properties of Lactobacillus rhamnosus Lcr35® using the in vitro Caco-2 cell model and the in vivo Caenorhabditis elegans model. In Caco-2 cells, we showed that the strain Lcr35® significantly inhibited the growth (~2 log CFU.mL-1) and adhesion (150 to 6,300 times less) of the pathogen. Moreover, in addition to having a pro-longevity activity in the nematode (+42.9%, p = 3.56.10-6), Lcr35® protects the animal from the fungal infection (+267% of survival, p < 2.10-16) even if the yeast is still detectable in its intestine. At the mechanistic level, we noticed the repression of genes of the p38 MAPK signalling pathway and genes involved in the antifungal response induced by Lcr35®, suggesting that the pathogen no longer appears to be detected by the worm immune system. However, the DAF-16/FOXO transcription factor, implicated in the longevity and antipathogenic response of C. elegans, is activated by Lcr35®. These results suggest that the probiotic strain acts by stimulating its host via DAF-16 but also by suppressing the virulence of the pathogen.

Fig 1. Determination of the C. albicans concentration in the biofilm in the presence or absence of Lcr35^® (10⁸ CFU.mL^-1) on the Caco-2 cell monolayer (mean ± standard deviation).

Different concentrations of yeast were tested, and the amount present in the biofilm was evaluated after 48 h of incubation. Comparison between conditions with and without Lcr35^® was performed using a two-way ANOVA followed by a Fisher’s LSD post hoc test (p < 0.05: *; p < 0.01: **; p < 0.001: ***; p < 0.0001: ****).

Fig 2. Influence of Lactobacillus rhamnosus Lcr35^® and C. albicans on the lifespan of the C. elegans wild-type N2 strain.

Worms were fed E. coli OP50 (n = 285), C. albicans ATCC 10231 (n = 242), and Lcr35^® (n = 278). The mean lifespan, where half of the population was dead, is represented on the abscissa. The asterisks indicate the p-values (log-rank test) with E. coli OP50 as a control (p < 0.05: *; p < 0.01: **; p < 0.001: ***).

Fig 3. Growth of C. elegans (adult) on E. coli OP50 and on Lcr35^®.

All results are represented as means +/- standard deviations (ns: statistically not significant).

Fig 4. Preventive effects of Lcr35^® against C. albicans ATCC 10231.

Mean survival, where half of the population was dead, is represented on the abscissa. The asterisks indicate the p-values (log-rank test) against E. coli OP50 (p < 0.05: *; p < 0.01: **; p < 0.001: ***). Infection duration: 2 hour;2-hour preventive treatment (E. coli OP50 (OP50, n = 126); C. albicans ATCC 10231 (CA, n = 424); Lcr35^® (Lcr35, n = 93); E. coli OP50 + C. albicans (OP50 + CA, n = 287); Lcr35^® + C. albicans (Lcr35 + CA, n = 224));4-hour preventive treatment (E. coli OP50 (OP50, n = 313); C. albicans ATCC 10231 (CA, n = 424); Lcr35^® (Lcr35, n = 259); E. coli OP50 + C. albicans (OP50 + CA, n = 120); Lcr35^® + C. albicans (Lcr35 + CA, n = 164)); 6-hour preventive treatment (E. coli OP50 (OP50, n = 222); C. albicans ATCC 10231 (CA, n = 424); Lcr35^® (Lcr35, n = 165); E. coli OP50 + C. albicans (OP50 + CA, n = 339); Lcr35^® + C. albicans (Lcr35 + CA, n = 300)); 24-hour preventive treatment (E.treatment coli OP50 (OP50, n = 248); C. albicans ATCC 10231 (CA, n = 424); Lcr35^® (n = 170); E. coli OP50 + C. albicans (OP50 + CA, n = 220); Lcr35^® + C. albicans (Lcr35 + CA, n = 183)).

Fig 5. C. albicans colonization of the C. elegans gut after 72 h (A) and after a 4-hour prophylactic treatment with E. coli OP50 (B) or Lcr35^® (C).

The green colour represents yeast labelled with rhodamine 123. Scale bar, 10 μm.

Fig 6. DAF-16 cellular localization in C. elegans transgenic strain TJ-356 (daf-16p::daf-16a/b::GFP + rol-6(su1006)) expressing DAF-16::GFP.

Worms fed on E. coli OP50 (A), on Lcr35^® (B) and on C. albicans ATCC 10231 (C). Scale bar, 100 μm.

Fig 7. Impact of preventive Lcr35^® treatment on DAF-16 cellular localization in the C. elegans transgenic strain TJ-356 expressing DAF-16::GFP.

Worms fed with E. coli OP50 + C. albicans (A) and on Lcr35^® + C. albicans (B). Scale bar, 100 μm.