<i>Lactobacillus</i> spp. prolongs the survival of <i>G</i>. <i>mellonella</i> larvae infected with <i>C</i>. <i>albicans</i>.

<p>There was a significant difference between the “<i>Lactobacillus</i> strain + <i>C</i>. <i>albicans</i> group” and “PBS + <i>C</i>. <i>albicans</i> control group”: <b>A.</b> <i>p</i> = 0.0097; <b>B.</b> p = 0.0013; <b>C.</b> <i>p</i> = 0.0044; <b>D.</b> p = 0.0001; <b>E.</b> <i>p</i> = 0.0245; <b>F.</b> p = 0.0001; <b>G.</b> <i>p</i> = 0.0075; <b>H.</b> p = 0.0003 and <b>I.</b> <i>p</i> = 0.0733. Kaplan-Meier test, <i>p</i>≤ 0.05.</p

<i>L</i>. <i>paracasei</i> strain 28.4 prolonged survival of <i>G</i>. <i>mellonella</i> larvae infected with <i>C</i>. <i>albicans</i>.

<p><b>A.</b> Survival rate for different concentrations of <i>L</i>. <i>paracasei</i>: significant differences were observed for the groups 10⁵ cells of 28.4 + <i>Candida</i> (<i>p</i> = 0.0166), 10⁶ cells of 28.4 + <i>Candida</i> (<i>p</i> = 0.0003) and 10⁷ cells of 28.4 + <i>Candida</i> (<i>p</i> = 0.0001) in relation to the control group (PBS + 10⁶ cells of <i>Candida</i>). <b>B.</b> Survival rate for Heat-Killed (HK) <i>L</i>. <i>paracasei</i>: no significant statistically differences were observed for the groups 10⁵ cells of HK28.4 + <i>Candida</i> (<i>p</i> = 1.000), 10⁶ cells of HK28.4 + <i>Candida</i> (<i>p</i> = 1.000) and 10⁷ cells of HK28.4 + <i>Candida</i> (<i>p</i> = 0.0733) when compared to the control group PBS + <i>Candida</i>. Kaplan-Meier test, p≤ 0.05.</p

<i>L</i>. <i>paracasei</i> strain 28.4 increased the expression of antifungal peptides of <i>G</i>. <i>mellonella</i>.

<p>Relative quantification (log) of Galiomicin (<b>A</b>) and Gallerymicin (<b>B</b>) for the groups treated with only PBS (Control), pre-treated with PBS and infected with <i>C</i>. <i>albicans</i>, only treated with <i>L</i>. <i>paracasei</i>, and pre-treated with <i>L</i>. <i>paracasei</i> and infected with <i>C</i>. <i>albicans</i>. The units in the Y-axis were calculated based on the 2^-ΔΔCT method, and they are expressed as the means and standard deviation. Each gene was normalized and compared with the expression of insects exposed to the control (PBS) using the reference gene β-actin. Different letters (A, B, and C) represent statistically significant differences among the groups. ANOVA and Tukey Tests (<i>p</i>≤0.05). ***<i>p</i> ≤ 0.001.</p

Clinical strains of <i>Lactobacillus</i> used in this study.

<p>Clinical strains of <i>Lactobacillus</i> used in this study.</p

Susceptibility of <i>G</i>. <i>mellonella</i> to infection with <i>Lactobacillus</i> spp. using larvae not infected by <i>C</i>. <i>albicans</i>.

<p><i>G</i>. <i>mellonella</i> larvae were treated with serial concentrations of different <i>Lactobacillus</i> strains (CFU/larva). The control group was composed of untreated <i>G</i>. <i>mellonella</i> larvae that received only PBS injection.</p

<i>Lactobacillus paracasei</i> modulates the immune system of <i>Galleria mellonella</i> and protects against <i>Candida albicans</i> infection

<div><p>Probiotics have been described as a potential strategy to control opportunistic infections due to their ability to stimulate the immune system. Using the non-vertebrate model host <i>Galleria mellonella</i>, we evaluated whether clinical isolates of <i>Lactobacillus</i> spp. are able to provide protection against <i>Candida albicans</i> infection. Among different strains of <i>Lactobacillus paracasei</i>, <i>Lactobacillus rhamnosus</i> and <i>Lactobacillus fermentum</i>, we verified that <i>L</i>. <i>paracasei</i> 28.4 strain had the greatest ability to prolong the survival of larvae infected with a lethal dose of <i>C</i>. <i>albicans</i>. We found that the injection of 10⁷ cells/larvae of <i>L</i>. <i>paracasei</i> into <i>G</i>. <i>mellonella</i> larvae infected by <i>C</i>. <i>albicans</i> increased the survival of these insects compared to the control group (<i>P</i> = 0.0001). After that, we investigated the immune mechanisms involved in the protection against <i>C</i>. <i>albicans</i> infection, evaluating the number of hemocytes and the gene expression of antifungal peptides. We found that <i>L</i>. <i>paracasei</i> increased the hemocyte quantity (2.38 x 10⁶ cells/mL) in relation to the control group (1.29 x 10⁶ cells/mL), indicating that this strain is capable of raising the number of circulating hemocytes into the <i>G</i>. <i>mellonella</i> hemolymph. Further, we found that <i>L</i>. <i>paracasei</i> 28.4 upregulated genes that encode the antifungal peptides galiomicin and gallerymicin. In relation to the control group, <i>L</i>. <i>paracasei</i> 28.4 increased gene expression of galiomicin by 6.67-fold and 17.29-fold for gallerymicin. Finally, we verified that the prophylactic provision of probiotic led to a significant reduction of the number of fungal cells in <i>G</i>. <i>mellonella</i> hemolymph. In conclusion, <i>L</i>. <i>paracasei</i> 28.4 can modulate the immune system of <i>G</i>. <i>mellonella</i> and protect against candidiasis.</p></div

<i>L</i>. <i>paracasei</i> strain 28.4 decreased the number of fungal cells in <i>G</i>. <i>mellonella</i> hemolymph.

<p>Mean and standard deviation of <i>C</i>. <i>albicans</i> counts (CFU/larvae) in the hemolymph of <i>Galleria mellonella</i> after 4, 8 and 24 h of experimental infection. The following groups were compared at each time of infection: PBS + <i>C</i>. <i>albicans</i> (control) and <i>L</i>. <i>paracasei</i> + <i>C</i>. <i>albicans</i>. A significant difference between groups was only observed after 24 h of infection, with a larger number of CFU/larvae in the control group compared to the <i>L</i>. <i>paracasei</i> + <i>C</i>. <i>albicans</i> (<i>p</i> = 0.0199). Student <i>t</i> test, <i>p</i> ≤ 0.05.</p

PCR primer pairs used to amplify regions of the genes involved in the immune system of <i>G</i>. <i>mellonella</i> and a reference gene.

<p>PCR primer pairs used to amplify regions of the genes involved in the immune system of <i>G</i>. <i>mellonella</i> and a reference gene.</p

<i>G</i>. <i>mellonella</i> hemocyte number increased with the injection of <i>L</i>. <i>paracasei</i> strain 28.4.

<p>The group of <i>L</i>. <i>paracasei</i> 28.4 + PBS increased the hemocyte number compared to a PBS control (PBS + PBS) at all different time points studied. The group <i>L</i>. <i>paracasei</i> + <i>C</i>. <i>albicans</i> also increased the hemocyte quantity compared to <i>C</i>. <i>albicans</i> group (PBS + <i>C</i>. <i>albicans</i>). PBS + <i>C</i>. <i>albicans</i> group showed a reduction of hemocyte quantity in relation to the PBS control group, but when the larvae were pretreated with <i>Lactobacillus</i> (<i>L</i>. <i>paracasei</i> + <i>C</i>. <i>albicans</i> group) the hemocyte quantity was very similar to the values found in the PBS control group. The four groups were compared in each time point studied by ANOVA test (4h: <i>p</i> = 0.0001, 8h: <i>p</i> = 0.0003, 24h: <i>p</i> = 0.0006). The results of Tukey test are indicated by letters: different letters (A, B, and C) represent statistically significant differences among the groups for each time point studied. A p ≤ 0.05 value was considered significant.</p

Experimental groups used to evaluate the effects of <i>Lactobacillus</i> strains on <i>Candida</i> infections.

<p>Experimental groups used to evaluate the effects of <i>Lactobacillus</i> strains on <i>Candida</i> infections.</p