Abstract
Lactobacillus crispatus is a dominant member of the healthy vaginal microbiota, yet the mechanisms by which it modulates host immunity remain poorly defined, in part due to the lack of tractable in vivo models. Here, we integrate bacterial genetics, in vitro epithelial systems, human-derived data and proteomic approach (Olink®) to uncover a critical role for L. crispatus exopolysaccharides (EPS) in shaping the bacteria-vagina interactions. Comparative genomics identified a conserved EPS biosynthetic locus, with the priming glycosyltransferase gene epsE emerging as a regulatory node, in line with its distinct expression in human vaginal samples. Functional disruption of epsE abrogated L. crispatus EPS production and revealed its role for immune modulation. In human vaginal epithelial monolayers, EPS presence enhanced immune-regulatory (LAP TGF-beta-1) and anti-inflammatory (CST5) responses, whereas its absence triggered elevated pro-inflammatory cytokines (IL1β, IL6, IL8) and matrix metalloproteinase (MMP10). In a 3D vaginal organotypic model, EPS increased chemokines (CXCL5, CXCL6) linked to immune surveillance and the presence of the markers was validated in vaginal samples of healthy volunteers. These findings position EPS as a key immunomodulatory structure of L. crispatus, advancing our mechanistic understanding of host-commensal interactions and informing microbiome-based strategies to promote vaginal health.
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Data availability
The datasets supporting the findings of this study are available in the European open-access repository Zenodo (https://zenodo.org/) under the accession number https://doi.org/10.5281/zenodo.15388380.
Code availability
The analysis code supporting the findings of this study are available in the European open-access repository Zenodo (https://zenodo.org/) under the accession number https://doi.org/10.5281/zenodo.15388380.
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Acknowledgements
Sincere thanks to the Laboratory of Applied Microbiology and Biotechnology team in Belgium, as well as the Benefi cial Microbes lab team in Italy. The following colleagues have provided important support for the study, such as contributing to the Isala project management, strain collection and the logistics of lab, general genetic engineering advise and biosafety: Sarah Ahannach, Isabel Erreygers, Camille Gepts, Ines Tuyaerts, Nele Van Vliet and Max Dekeukeleire. All the icons and schematic images were created with BioRender.com (full license). This work was supported by the European Research Council grant Lacto-Be (grant ID 852600) (awarded to SL and supports TE, JD) and proof-of-concept VALERIE (Horizon) (grant ID 101213306) (awarded to SL, supports JD) and ERC Runner-up ELLA (grant ID G0ATZ25N), FWO (G049022N, G031222N, SBO DeVeniR S006424N), the Inter-University Special Research Fund of Flanders (iBOF) for the POSSIBL project and the industrial research fund UAntwerpen for IOF POC project CRUCIAL. In addition, IS was supported by the University of Antwerp BOF-KP grant 53399, SW by a FWO postdoctoral grant 12AZ624N and CD by FWO doctoral grant 1S28622N. VC doctoral scholarship and part of the experimental activities were carried out in the Benefi cial Microbes laboratory of the University of Bologna (Italy) and supported by the Italian Ministry of University and Research. The microscopes used in this publication, i.e. Tecnai Spirit G2 Biotwin (AUHA-08-004), Nikon SoRA (I003420N), were funded by Medium-scale research infrastructure grants of the FWO.
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Conceived and designed the experiments: V.C., C.D., T.E., D.V., C.A., I.P., S.T., W.M., M.N., P.A.B., I.S., C.P., B.V., and S.L. Performed the experiments: V.C., C.D., S.B., I.P., S.T., W.M., and M.N. Analyzed the data: V.C., C.D., T.E., J.D., T.V.R., D.V., I.P., S.T., W.M., M.N., I.S., C.P., B.V., and S.L. Contributed materials/analysis tools: V.C., C.D., T.E., J.D., T.V.R., E.C., I.V.T., I.P., S.T., S.W., W.M., M.N., P.A.B., I.S., C.P., B.V., and S.L. Wrote the paper: V.C., T.E., T.V.R., I.V.T., I.P., S.T., W.M., M.N., P.A.B., and S.L. Revised the paper: V.C., C.D., T.E., J.D., T.V.R., E.C., I.V.T., S.B., D.V., C.A., I.P., S.T., W.M., M.N., P.A.B., S.W., I.S., C.P., B.V., and S.L. Funding acquisition and project management: B.V. and S.L.
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S.L. serves as Guest Editor of npj Biofilms and Microbiomes but had no involvement in the peer review process or the decision to publish this paper. S.L. declares no financial competing interests. She is a voluntary academic board member of the International Scientific Association on Probiotics and Prebiotics (ISAPP, www.isappscience.org) and scientific advisor for Freya Biosciences, and declares research funding from YUN, Bioorg, Puratos, Lesaffre/Gnosis, DSM i-Health & dsm-firmenich (not directly involved in the content of this work). DSM i-Health & br/dsm-firmenich provided funding for the collection of human samples used as a reference in this work. I.S. has received funding from ISAPP and the International Probiotics Association (IPA) to attend conferences. P.A.B. is an independent consultant for several companies in the food and pharmaceutical industry bound by confidentiality agreements. B.V. has scientific collaborations with SACCO System and DEPOFARMA and is a scientific consultant for EOS2021. J.D. and S.L. are co-inventors on a patent application on L. crispatus AMBV-0815 related to its unique antimicrobial peptide production. This patent application is not directly linked to the content of this paper, with EPS being a more conserved immunomodulatory property.
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Croatti, V., Dricot, C., Eilers, T. et al. Exopolysaccharides of Lactobacillus crispatus mediate key balancing interactions with the vaginal mucosa. npj Biofilms Microbiomes (2026). https://doi.org/10.1038/s41522-026-00937-5
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DOI: https://doi.org/10.1038/s41522-026-00937-5
