HAL Id: hal-02741302
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Submitted on 3 Jun 2020
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Candida albicans interaction with enterocytes : impact on tight junction integrity
Alicia Loiselet, Fabienne Bon, Patrick Ducoroy, Frédéric Dalle
To cite this version:
Alicia Loiselet, Fabienne Bon, Patrick Ducoroy, Frédéric Dalle. Candida albicans interaction with enterocytes : impact on tight junction integrity. 4. Journées des Doctorants, Mar 2015, Dijon, France.
2015. �hal-02741302�
Candida albicans interaction with enterocytes: impact on tight junction integrity
Alicia Loiselet1, Fabienne Bon1, Patrick Ducoroy2, Frédéric Dalle1
1 UMR 1347 Agroécologie Pôle MERS Université de Bourgogne Dijon 2 Plateforme protéomique CLIPP Dijon
Scale bar: 10 μm
Candida albicans is responsible for disseminated infections (candidemia and deep seated infections) mainly originating from the gastro-intestinal tract where the fungus resides initially as a commensal. In healthy humans, the gut mucosa has a protective role in limiting invasiveness of pathogens. The integrity and impermability of the intestinal epithelium is partly due to specific cell-cell junctions : "tight junctions" (TJ). The aim of this work was to determine whether invasion of C. albicans into intestinal cells resulted from a direct modulatory effect of the yeast upon the TJ integrity.
Enterocyte cell line : Caco-2 cells from colic adenocarcinoma used in transwells or in 24-well plates ;
Strain : C. albicans SC5314 used according to two inoculi : 105/ml and 107/ml.
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Apical chamber
Basal chamber
Lucifer Yellow: fluorescent molecule
Caco-2 cell
Paracellular crossing
Candida albicans SC 5314
% fluorescence intensity of Lucifer Yellow
Figure 2. Variation of fluorescence intensity compared with time zero for each experimental condition
JAM: Jonctional Adhesion Molecule ZO: Zonula Occludens
CLDN: Claudin OCLN: Occludin
Intercellular space
Cytoplasm
Membrane cell
1: Cells infected with C. albicans at 107/ml 2: Cells infected with C. albicans at 105/ml 3: Cells non infected
Figure 3. Organization chart of tight junction protein expression in infected Caco-2 cell
Figure1. Experimental protocol
Experimental protocol used to measure cell layer permeability: paracellular crossing of Lucifer Yellow through Caco-2 cells infected or not with C. albicans was evaluated by quantifying the fluorescence intensity of the basal solution at different time points, using a spectrophotometer (*p < 0.05; ** p < 0.01 (ANOVA test))
Figure 4. Analysis of tight junction protein at the membrane cell by western blotting
Quantification of tight junction proteins of fungal infected cells (4h, C. albicans107/ml) according to non infected cells proteins by NanoLC-MSMS (Progenesis software, in green: Anova test, p< 0.05)
Quantification of tight junction proteins of fungal infected cells (4h, C. albicans 105/ml or 107/ml) according to non infected cells proteins by western blotting (three replicats, Anova test, * p<0.05)
Occludin
β-actine
1 2 3
JAM-A
*
NS
Figure 5. Occludin staining after four-hours infection
A
GX200
C
GX200
B
GX400
D
GX400
Arrows head indicated membrane splitting of adjacent enterocytes
Non infected cells Infected cells with C. albicans (107/ml)
High inocula of C. albicans increase in the paracellular permeability of Caco-2 cell monolayers, as a result of (1) a decrease in membrane TJs proteins’
expression and consequently (2) a desorganization of the TJs’ structure.
The molecular mechanisms of the regulation of TJs’ integrity are under investigation. In addition, C. albicans proteins responsible for TJs’ trafficking will be investigated using banks of C. albicans’ mutants.
