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. 2023 Apr 14;13(1):6114.
doi: 10.1038/s41598-023-32116-3.

Over-production of exopolysaccharide by Lacticaseibacillus rhamnosus CNCM I-3690 strain cutbacks its beneficial effect on the host

R Martín  1 , A Benítez-Cabello  2 , S Kulakauskas  3 , M V C Viana  2   4 , C Chamignon  2 , P Courtin  3 , C Carbonne  2 , F Chain  2 , H P Pham  5 , Muriel Derrien  6 , L G Bermúdez-Humarán  2 , M P Chapot-Chartier  3 , T Smokvina  6 , P Langella  2
Affiliations

Over-production of exopolysaccharide by Lacticaseibacillus rhamnosus CNCM I-3690 strain cutbacks its beneficial effect on the host

R Martín et al. Sci Rep. .

Abstract

Most lactobacilli produce extracellular polysaccharides that are considered to contribute to the probiotic effect of many strains. Lacticaseibacillus rhamnosus CNCM I-3690 is an anti-inflammatory strain able to counterbalance gut barrier dysfunction. In this study ten spontaneous variants of CNCM I-3690 with different EPS-production were generated and characterized by their ropy phenotype, the quantification of the secreted EPS and genetic analysis. Amongst them, two were further analysed in vitro and in vivo: an EPS over-producer (7292) and a low-producer derivative of 7292 (7358, with similar EPS levels than the wild type (WT) strain). Our results showed that 7292 does not have anti-inflammatory profile in vitro, and lost the capacity to adhere to the colonic epithelial cells as well as the protective effect on the permeability. Finally, 7292 lost the protective effects of the WT strain in a murine model of gut dysfunction. Notably, strain 7292 was unable to stimulate goblet cell mucus production and colonic IL-10 production, all key features for the beneficial effect of the WT strain. Furthermore, transcriptome analysis of colonic samples from 7292-treated mice showed a down-regulation of anti-inflammatory genes. Altogether, our results point out that the increase of EPS production in CNCM I-3690 impairs its protective effects and highlight the importance of the correct EPS synthesis for the beneficial effects of this strain.

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Conflict of interest statement

M.D., T.S are employees of Danone Nutricia Research. The funder was involved in the design of the study, in the collection, analyses or interpretation of data and in the writing of the manuscript. The rest of authors do not have conflict of interest.

Figures

Figure 1
Figure 1
Scheme of isolation of L. rhamnosus CNCM I-3690 derivatives with affected EPS production.
Figure 2
Figure 2
Immunomodulatory (A) and protective (B) activity of L. rhamnosus CNCM I-3690 and its variants. The immunomodulatory property is measured by the ability of the strains to regulate the production of interleukin 8 (IL-8) (%) by HT-29 cells stimulated with TNF-α. The protective effect is evaluated by measuring the trans-epithelial resistance in Caco-2 cells in the presence of the bacterial strains (b). Significance: * p < 0.05 (n = 3 × 3).
Figure 3
Figure 3
Relative EPS production of L. rhamnosus CNCM I-3690 and its derivatives.
Figure 4
Figure 4
Percentage of adhesion (%) to HT-29-MTX cells of the EPS-overproducer (7292) and underproducer (7358) variants in comparation with the wild-type L. rhamnosus CNCM I-3690. Significance: * p < 0.05 (n = 3 × 3).
Figure 5
Figure 5
Evaluation of the macroscopic score in a dinitro-benzene sulfonic acid (DNBS) low grade colitis model after treatment with the EPS variants 7292 and 7358 in comparation with the wild-type L. rhamnosus CNCM I-3690. Significance: * p < 0.05.
Figure 6
Figure 6
Colonic interleukin levels in a low grade dinitro-benzene sulfonic acid (DNBS) colitis model after treatment with the the EPS variants 7292 and 7358 in comparation with the wild-type L. rhamnosus CNCM I-3690. Significance: * p < 0.05.
Figure 7
Figure 7
Measure of the in vivo gut permeability in a DNBS low grade murine model after treatment with the EPS variants 7292 and 7358 in comparation with the wild-type L. rhamnosus CNCM I-3690. Significance: * p < 0.05.
Figure 8
Figure 8
Effects of the treatment with the EPS-overproducer (7292) and underproducer (7358) variants in comparation with the wild-type L. rhamnosus CNCM I-3690 on the mucus layer (A), and globet cell abundance through the AB-staining (B) and PAS (C) test in the DNBS low grade murine model. Significance: * p < 0.05.
Figure 9
Figure 9
Immunological activity in MLN cells. MLN cells positive for CD3 + , CD4 + , T-bet, or GATA-3 as detected using Flow cytometry. Significance: * p < 0.05.
Figure 10
Figure 10
Venn diagram showing the number of unique and shared upregulated (A) and downregulated (B) genes as identified via IPA in the murine model after treatment with the variant 7292. Gene number were compared with those modulated after treatment with the wild-type L. rhamnosus CNCM I-3690, not-treated, and mice administered with the vehicule. Significance: * p < 0.05.
Figure 11
Figure 11
Effect of DNBS and EPS variation on gut microbiota. A) Genus-based Shannon index (Mann–Whitney test; B) Within-mice Bray–Curtis dissimilarity to D13 (Mann–Whitney test) C) Differentially abundant genera were identified using Deseq2 based and between-group effects with adjustment for D13. *FDR < 0.1. Red indicates higher abundance in the first group at D23 versus D13 compared to second group at D23 versus D13.
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