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Mucosal tissue invasion by Candida albicans is associated with E-cadherin degradation, mediated by transcription factor Rim101p and protease Sap5p - PubMed

Mucosal tissue invasion by Candida albicans is associated with E-cadherin degradation, mediated by transcription factor Rim101p and protease Sap5p

C C Villar et al. Infect Immun. 2007 May.

Abstract

The ability of Candida albicans to invade mucosal tissues is a major virulence determinant of this organism; however, the mechanism of invasion is not understood in detail. Proteolytic breakdown of E-cadherin, the major protein in epithelial cell junctions, has been proposed as a mechanism of invasion of certain bacteria in the oral mucosa. The objectives of this study were (i) to assess whether C. albicans degrades E-cadherin expressed by oral epithelial cells in vitro; (ii) to compare the abilities of strains with different invasive potentials to degrade this protein; and (iii) to investigate fungal virulence factors responsible for E-cadherin degradation. We found that while E-cadherin gene expression was not altered, E-cadherin was proteolytically degraded during the interaction of oral epithelial cells with C. albicans. Moreover, C. albicans-mediated degradation of E-cadherin was completely inhibited in the presence of protease inhibitors. Using a three-dimensional model of the human oral mucosa, we found that E-cadherin was degraded in localized areas of tissue invasion by C. albicans. An invasion-deficient rim101-/rim101- strain was deficient in degradation of E-cadherin, and this finding suggested that proteases may depend on Rim101p for expression. Indeed, reverse transcription-PCR data indicated that expression of the SAP4, SAP5, and SAP6 genes is severely reduced in the rim101-/rim101- mutant. These SAP genes are functional Rim101p targets, because engineered expression of SAP5 in the rim101-/rim101- strain restored E-cadherin degradation and invasion in the mucosal model. Our data support the hypothesis that there is a mechanism by which C. albicans invades mucosal tissues by promoting the proteolytic degradation of E-cadherin in epithelial adherens junctions.

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Figures

FIG. 1.
FIG. 1.

Time-dependent degradation of E-cadherin during incubation of oral epithelial cells with C. albicans. SCC15 cells were exposed to C. albicans strain SC5314 for 2 h (lane 2), 4 h (lane 4), 6 h (lane 6), 8 h (lane 9), 10 h (lane 11), 12 h (lane 13), and 14 h (lane 15), and E-cadherin expression was analyzed by Western blotting. Membranes were probed with β-actin to control for sample loading. Cell lysates of C. albicans alone were analyzed to determine E-cadherin and β-actin contents (lane 7). Densitometry values (in optical density [OD] units) are indicated below the bands. The positions of the molecular mass markers are indicated on the left. The results of one of three independent experiments are shown in each panel.

FIG. 2.
FIG. 2.

Intercellular location of C. albicans in oral epithelial cell monolayers. Oral epithelial cell monolayers were incubated with C. albicans strain SC5314 for 4 h and examined by transmission electron microscopy. C. albicans (black arrow) gained access to the intercellular spaces of the confluent monolayer. Oral epithelial cells are indicated by white arrows. Bar = 1 μm.

FIG. 3.
FIG. 3.

Analysis of E-cadherin gene expression during incubation of oral epithelial cells with C. albicans. SCC15 cells were exposed to C. albicans strain SC5314 for up to 14 h, and E-cadherin gene expression was analyzed by quantitative RT-PCR. Data were expressed as fold induction values obtained by dividing the relative expression value for E-cadherin in epithelial cells cocultured with C. albicans by the relative expression value for E-cadherin in epithelial cells alone. A fold induction value of 1.0 indicates that there was no induction of E-cadherin expression in epithelial cells by C. albicans, while values less than 0.5 and greater that 2.0 indicate that there was decreased and increased E-cadherin expression in epithelial cells cocultured with C. albicans. The error bars indicate one standard deviation of the mean of triplicate experiments.

FIG. 4.
FIG. 4.

E-cadherin is proteolytically degraded by C. albicans. (A) Effect of inhibitors of proteinases on E-cadherin degradation during incubation of SCC15 cells with C. albicans strain SC5314. (B) Effect of C. albicans strain SC5314 and C. albicans-conditioned medium on the integrity of E-cadherin expressed on epithelial cell-derived membranes. Trypsin was used as a positive control. (C) Effect of C. albicans strain SC5314 on the integrity of immunoprecipitated E-cadherin in the presence or absence of a cocktail of protease inhibitors or pepstatin A. The protease inhibitor cocktail contained aprotinin, bestatin, leupeptin, E-64, and pepstatin A. E-cadherin integrity was analyzed by Western blotting. The positions of the molecular mass markers are indicated on the left. The results of one of three independent experiments are shown in each panel.

FIG. 5.
FIG. 5.

Intercellular location of C. albicans in the three-dimensional model of oral mucosa. The oral mucosal model was challenged with C. albicans strain SC5314 for 8 h. Paraffin sections (thickness, 5 μm) of the three-dimensional culture were stained with fluorescein isothiocyanate-conjugated antibody against C. albicans, tetramethyl rhodamine isocyante-conjugated phalloidin, and Hoechst 33258 and analyzed by confocal laser scanning microscopy. C. albicans invaded the three-dimensional model of the human oral mucosa both directly by entry into keratinocytes (keratinocytes are red, indicated by yellow fungi [white arrows]) and by gaining access to intercellular spaces (indicated by green fungi [blue arrows]). Bar = 20 μm.

FIG. 6.
FIG. 6.

Immunohistochemistry analysis of E-cadherin expression in a three-dimensional model of oral mucosa. (A) Paraffin section (thickness, 5 μm) of three-dimensional culture alone stained with periodic acid-Schiff stain and antibody specific for E-cadherin (brown). (B) Paraffin section of three-dimensional culture incubated with DAY185 and stained with periodic acid-Schiff stain and antibody specific for E-cadherin (brown). (C) Paraffin section of three-dimensional culture incubated with invasion-deficient rim101−/rim101−mutant CJN793 and stained with periodic acid-Schiff stain and antibody specific for E-cadherin (brown). (D) Paraffin section of three-dimensional culture incubated with the rim101−/rim101− mutant strain expressing SAP5 (strain CJN1111) and stained with periodic acid-Schiff stain and antibody specific for E-cadherin (brown). Sections were counterstained with hematoxylin and eosin (blue). The oral mucosal model was challenged with C. albicans added on the apical surface. Bars = 65 μm.

FIG. 7.
FIG. 7.

Effect of C. albicans invasion-deficient mutant DAY25, complemented strain DAY44, and reference strain DAY185 on the integrity of E-cadherin. Immunoprecipitated E-cadherin was incubated with C. albicans for 4 and 6 h, and the protein integrity was analyzed by Western blotting. The positions of the molecular mass markers are indicated on the left. The results of one of three independent experiments in which similar data were obtained are shown.

FIG. 8.
FIG. 8.

Effect of secreted aspartyl proteinases on the integrity of E-cadherin. (A) Role of SAP5 expression in the RIM101 requirement for E-cadherin degradation. Immunoprecipitated E-cadherin was incubated with rim101−/rim101− mutant strain CJN793, rim101−/rim101−mutant strain CJN1111 expressing SAP5, or reference strain DAY185 for 4 and 6 h, and the protein integrity was analyzed by Western blotting. The positions of the molecular mass markers are indicated on the left. The results of one of two independent experiments in which similar data were obtained are shown. (B) Effect of loss of SAP5 and loss of SAP4, SAP5, and SAP6 on the integrity of E-cadherin. Immunoprecipitated E-cadherin was incubated with SAP5 gene knockout strain DSY 452, SAP4-SAP5-SAP6 gene knockout strain DSY 459, or reference strain CAI4 for 12 h, and the protein integrity was analyzed by Western blotting. The results of one of two independent experiments in which similar data were obtained are shown. The positions of the molecular mass markers are indicated on the left.

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References

    1. Bernhardt, J., K. Zimmermann, K. Schulz, M. Knoke, and H. Bernhardt. 2000. Oesophageal candidosis in intensive care patients. Mycoses 43:377-379. - PubMed
    1. Bernhardt, J., D. Herman, M. Sheridan, and R. Calderone. 2001. Adherence and invasion studies of Candida albicans strains, using in vitro models of esophageal candidiasis. J. Infect. Dis. 184:1170-1175. - PubMed
    1. Birkness, K. A., M. Deslauriers, J. H. Bartlett, E. H. White, C. H. King, and F. D. Quinn. 1999. An in vitro tissue culture bilayer model to examine early events in Mycobacterium tuberculosis infection. Infect. Immun. 67:653-658. - PMC - PubMed
    1. Birse, C. E., M. Y. Irwin, W. A. Fonzi, and P. S. Sypherd. 1993. Cloning and characterization of ECE1, a gene expressed in association with cell elongation of the dimorphic pathogen Candida albicans. Infect. Immun. 61:3648-3655. - PMC - PubMed
    1. Brown, A. J. P. 2001. Morphogenetic signaling pathways in Candida albicans, p. 95-106. In R. A. Calderone (ed.), Candida and candidiasis. American Society for Microbiology, Washington, DC.

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