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. 2024 Nov 12;10:124. doi: 10.1038/s41522-024-00600-x

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

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Fig. 1 — A Schematic diagram of interactions between bacteria and cells in the intestinal microenvironment. B Schematic diagram of an in vitro model of bacterial interaction with epithelial cells under different osmotic pressures. The host cells were cultured for 12 h in regular culture medium and then stimulated for 1 h in a specific osmotic mixture. Then, they were allowed to interact with GFP-expressing S. aureus or E. coli for another 2 h and rinsed three times with Dulbecco’s phosphate-buffered saline (DPBS) solution. Finally, the osmoregulated bacterium‒cell interactions were quantified. C Images of GFP-expressing S. aureus adherent/internalized to IEC-6 cells after the host cells were prestimulated with hypotonic (0.5x), isotonic (1x), and hypertonic (2x) solutions, which were acquired with a laser scanning confocal microscope. The F-actin cytoskeleton was labeled in red by rhodamine phalloidin, and the nuclei were labeled in blue by DAPI. D and E Statistical results of the number of bacteria adherent/internalized to host cells (N_adB) and the number of host cells harboring adherent/internalized bacteria (N_adC) in microscopy images with an imaging field of view of 337 × 337 μm², where the host cells were prestimulated with hypotonic (0.5x), isotonic (1x), and hypertonic (2x) solutions, respectively. F and G Fold changes in N_adB and N_adC under hypertonic and hypotonic conditions relative to the indices in the isotonic situation. (H) Statistical results of the number of host cells harboring adherent/internalized bacteria (N_adC (%)) quantified through flow cytometry. I–N are similar to C–H, respectively, except that the host cells were replaced with the HaCat cell line. All the statistical data (mean ± SD) originated from at least three independent experiments for each specific condition. Statistical analyses based on one-way ANOVA were used in the experiments, and **, *** and **** denote P < 0.01, P < 0.001 and P < 0.0001, respectively. Scale bar: 50 μm.