Lactobacillus rhamnosus Lcr35® as an effective treatment for preventing Candida albicans infection in preclinical models: first mechanistical insights

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 the 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-Candida albicans properties of Lactobacillus rhamnosus Lcr35® using the in vitro Caco-2 cells model and the in vivo Caenorhabditis elegans model. On Caco-2 cells, we showed that the strain Lcr35® significantly inhibited the growth of the pathogen (~2 log CFU.mL−1) and its adhesion (150 to 6,300 times less). Moreover, on the top of having a prolongevity activity in the nematode, Lcr35® protects the animal from the fungal infection even if the yeast is still detectable in its intestine. At the mechanistic level, we noticed the repression of genes of the p38 MAPK signaling 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.

157 Individual adult worms were photographed using an Evos FL microscope (Invitrogen, 10X 158 magnification). After reaching L4 stage, they were transferred on NGMY plates previously seeded 159 with the probiotic strain Lcr35 ® and their size were measured daily for three days. Length of worm 160 body was determined by using ImageJ software as described by Mörck and Pilon (2006) (41) and 161 compared to OP50-fed worms. At least 10 nematodes per experiment were imaged on at least three 162 independent experiments. 163 2.6 Caenorhabditis elegans lifespan assay 164 Synchronous L4 worms were transferred on NGMY with 0.12 mM 5-fluorodeoxyuridine FUdR 165 (Sigma, Saint-Louis, USA) and seeded with 100 µL of the 100 mg.mL -1 microbial strain (~50 worms 166 per plate). The plates were kept at 20 °C and live worms were scored each day until the death of all 167 animals. An animal was scored as dead when it did not respond to a gentle mechanical stimulation.  245 In the presence of Caco-2 cells, regardless of the concentration of the inoculum (from 10 2 to 10 7 246 CFU.mL -1 ), C. albicans grew to concentrations that ranged from 7.48 ± 0.39 to 7.83 ± 0.34 log 247 CFU.mL -1 after 48 hours of incubation. Similar C. albicans growth was measured in the absence of 248 human cells (data not shown). When prophylactic treatment was used, i.e. when the Caco-2 cells were 249 pre-incubated with the probiotic Lcr35 ® , we observed an antifungal activity against C. albicans.
250 Indeed, the bacterium induced a significant inhibition of the yeast of 2 log CFU.mL -1 which then 251 reached a concentration ranging from 5.40 ± 0.07 to 6.05 ± 0.25 log CFU.mL -1 . Two different inhibition 252 profiles were observed after 48 h. On one hand, when the inoculum was highly concentrated (7 log 255 able to grow although its growth seemed to stop between 5.32 ± 0.36 and 5.51 ± 0.14 log CFU.mL -1 256 (

3.2.1Lcr35 ® extends C. elegans lifespan
282 We investigated the effects on C. elegans lifespan induced by either the pathogenic yeast C. albicans 283 or the probiotic Lcr35 ® . Feeding adult nematodes with the probiotic strain resulted in a significant 284 increase of the mean lifespan compared to OP50-fed worms (p = 3.56 .10 -6 ) evolving from 7 to 10 days 285 (+ 42.9%) whereas C. albicans had no impact on C. elegans mean lifespan. On the other hand, when 286 C. albicans was used as a feeding source, worms displayed a significant reduced lifespan (p = 1.27 .10 -287 5 ) which dropped from 16 to 14 days (-12.5%). Lcr35 ® did not increase the worm's longevity compared 288 to OP50 (Fig 2). These results showed that the probiotic strain ameliorated the mean lifespan without 289 increasing the life expectancy of the worm.

3.2.2Lcr35 ® does not modify C. elegans growth
297 The body size of Lcr35 ® fed nematodes were compared to OP50-fed worms. Feeding worms with the 298 probiotic strain did not significantly change in growth rate nor body size as they all reached their 299 maximal length after three days (Fig 3). 3.3.1Effect of Lcr35 ® on C. elegans survival after C. albicans exposure 305 When C. elegans was sequentially exposed for 2 h to Lcr35 ® prior being infected by C. albicans, the 306 survival of the nematodes was increased significantly as the mean lifespan rised from 3 to 11 days 307 (267% increase in survival) compared with that observed with C. albicans infection alone (p < 2.10 -308 16 ). There was no significant difference between worms sequentially exposed to Lcr35 ® and C. albicans 309 and those exposed to Lcr35 ® only (Fig 4) (p = 1). Similar results were obtained when the nematodes 310 were exposed to the probiotic for 4 hours. In that case, we observed that Lcr35 ® completely protected  (Fig 5A). However, it turned out that this strain of 336 C. albicans was not able to form hyphae within the worm. We subsequently applied prophylactic 337 treatment to the worms for 4 hours before infecting them with yeast. We observed that after a 338 preventive treatment with the control OP50 (Fig 5B) or the probiotic Lcr35 ® (Fig 5C), the yeast C.
339 albicans was still detected in the digestive tract of the host.

3.4.1Modulation of C. elegans genes expression induced by Lcr35 ® and C.
347 albicans 348 To elucidate the mechanisms involved in the action of Lcr35 ® against C. albicans, we studied the 349 expression of seven C. elegans genes (Table 3). We targeted three groups of genes: daf-2 and daf-16 350 (insulin signaling pathway) involved in host longevity and antipathogenicity, sek-1 and pmk-1 (p38 351 MAPK signaling pathway) which concern the immunity response as well as abf-2, cnc-4 and fipr-22 / 352 fipr-23 which encode for antimicrobial proteins. We noted that Lcr35 ® tended to induce an 353 overexpression of daf-16 (p = 0.1635) while having no effect on daf-2 (p = 0.2536) when C. albicans