Isolation of derivatives from the food-grade probiotic Lactobacillus johnsonii CNCM I-4884 with enhanced anti- Giardia activity

Abstract

Giardiasis, a widespread intestinal parasitosis affecting humans and animals, is a growing concern due to the emergence of drug-resistant strains of G. intestinalis. Probiotics offer a promising alternative for preventing and treating giardiasis. Recent studies have shown that the probiotic Lactobacillus johnsonii CNCM I-4884 inhibits G. intestinalis growth both in vitro and in vivo. This protective effect is largely mediated by bile salt hydrolase (BSH) enzymes, which convert conjugated bile acids (BAs) into free forms that are toxic to the parasite. The objective of this study was to use adaptive evolution to develop stress-resistant derivatives of L. johnsonii CNCM I-4884, with the aim of improving its anti-Giardia activity. Twelve derivatives with enhanced resistance to BAs and reduced autolysis were generated. Among them, derivative M11 exhibited the highest in vitro anti-Giardia effect with enhanced BSH activity. Genomic and proteomic analyses of M11 revealed two SNPs and the upregulation of the global stress response by SigB, which likely contributed to its increased BAs resistance and BSH overproduction. Finally, the anti-Giardia efficacy of M11 was validated in a murine model of giardiasis. In conclusion, our results demonstrate that adaptive evolution is an effective strategy to generate robust food-grade bacteria with improved health benefits.

Keywords: Bile salt hydrolases; Giardia intestinalis; Lactobacillus johnsonii; giardiasis; probiotic.

Figure 1.

Screening and characterization of derivatives of L. johnsonii CNCM I-4884. (a) Schematic representation of the derivatives selection process. Refer to section “whole genome sequencing” for details on mutation identification. (b) Clones resistant to free BAs were selected using CA and DCA gradients. (c) Clones resistant to conjugated BAs were selected on TDCA and GDCA gradients. (d) Clones with altered sedimentation profiles were selected on THY + 0.03% agar; white arrow shows elongated wild-type colonies, red arrow points to slower sedimenting mutants with round colonies. (e) Autolysis was assessed by OD 600 nm in K₂HPO₄/KH₂PO₄ buffer + 0.05% triton X-100; data are shown as mean ± SEM (n = 3). (f) Area under curve of OD 600 nm. Asterisks indicate statistical significance compared to wild-type, as determined with unpaired t-test (***p < 0.001; **p < 0.01; *p < 0.05).

Figure 2.

Growth inhibition of G. intestinalis trophozoites by culture supernatants of L. johnsonii CNCM I-4884 wild-type and derivatives relative to untreated parasite cultures, tested as undiluted (a) and 1/2 diluted (b) bacterial supernatants. Values are mean ± SEM (n = 4). Asterisks indicate statistical significance compared to wild-type, as determined with unpaired t-test (***p < 0.001; **p < 0.01; *p < 0.05).

Figure 3.

Growth rates of L. johnsonii CNCM I-4884 wild-type and derivatives in MRS medium (a) and area under curve (b) or MRS supplemented with a mix of conjugated BAs (1 mg/ml TDCA and GDCA) (c) and area under curve (d). Data are represented as mean ± SEM (n = 3). Asterisks indicate statistical significance compared to wild-type, as determined with unpaired t-test (**p < 0.01; *p < 0.05).

Figure 4.

Adhesion of L. johnsonii CNCM I-4884 wild-type and derivatives to Caco-2 (a), HT-29 (b), HT-29 MTX (c) and T84 cell lines (d). Data are represented as mean ± SEM (n = 12). Asterisks indicate statistical significance compared to LGG, as determined with unpaired t-test (***p < 0.001; **p < 0.01; *p < 0.05).

Figure 5.

Biofilm formation by L. johnsonii CNCM I-4884 wild-type and derivatives. (a) 3D projections and section views of biofilm structure obtained from confocal z-stacks using IMARIS software; white scale bars represent 20 μm (b) quantification of biofilm biovolume. Data are presented as mean ± SEM (n = 6). Asterisks indicate statistical significance compared to wild-type, as determined with unpaired t-test (***p < 0.001).

Figure 6.

TEM observations of L. johnsonii CNCM I-4884 wild-type (a, b), and M3 (c, d), M5 (e, f) and M11 (g, h) derivatives, fixed in exponential growth phase. Inclusions (in M5) are indicated with an arrow. (i) Cell wall thickness. Data are represented as mean ± SEM (n = 15 to 30). Asterisks indicate statistical significance compared to wild-type as determined with unpaired t-test (***p < 0.001; *p < 0.05).

Figure 7.

Bile acids (BAs) deconjugation activity of supernatant samples issued from cultures of L. johnsonii CNCM I-4884 wild-type and derivatives, after various incubation times. Data are represented as mean ± SEM (n = 3). Asterisks indicate statistical significance compared to wild-type, as determined with unpaired t-test (**p < 0.01; *p < 0.05). TUDCA : tauroursodeoxycholic acid; TCDCA : taurochenodeoxycholic acid; TDCA : taurodeoxycholic acid; αTMCA : tauromuricholic acid; TCA : taurocholic acid; TLCA : taurolithocholic acid; GLCA : glycolithocholic acid; GUDCA : glycoursodeoxycholic acid; GCDCA : glycochenodeoxycholic acid; GDCA : glycodeoxycholic acid; GCA : glycocholic acid.

Figure 8.

Proteomic analysis of L. johnsonii CNCM I-4884 wild-type and M11 derivative. (a) Principal component analysis. (b) Global heatmap representation of protein abundances significantly different in wild-type and M11 (n=4, ANOVA, adjusted p<0.05). Each row represents a differentially expressed protein, while each column represents a sample. (c) Functional classification based on GO enrichment analysis of differentially expressed proteins in exponential or (d) stationary growth phase.

Figure 9.

(Continued).

Figure 9.

Fold-change of proteins with significant abundance change (ANOVA, adjusted p<0.05) between L. johnsonii CNCM I-4884 wild-type and M11 derivative, belonging to the main functional categories in (a) exponential and (b) stationary growth phase.

Figure 10.

Reduction of G. intestinalis trophozoite load in the small intestine of OF1 mice treated daily with either PBS, L. johnsonii CNCM I-4884 wild-type or M11, 6 d after Giardia infection challenge. Values are mean ± SEM (n=18). Asterisks indicate statistical significance, as determined with unpaired t-test (****p<0.0001; **p<0.01).