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The MP65 gene is required for cell wall integrity, adherence to epithelial cells and biofilm formation in Candida albicans.

Sandini S, Stringaro A, Arancia S, Colone M, Mondello F, Murtas S, Girolamo A, Mastrangelo N, De Bernardis F - BMC Microbiol. (2011)

Bottom Line: Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type.All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion.We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious, Parasitic and Immuno-mediated Diseases, Istituto Superiore di Sanità, Rome, 00161, Italy. silvia.sandini@iss.it

ABSTRACT

Background: The MP65 gene of Candida albicans (orf19.1779) encodes a putative β-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation.

Results: The mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p), a high level of expression of two stress-related genes (DDR48 and SOD5), and a modulated expression of β-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion.

Conclusions: We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.

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Adhesion analysis of the mp65Δ mutant. (A) Adhesion of the mp65Δ mutant to BEC. Representative fields randomly selected showing the interaction between yeast cells [wild type (wt), mp65Δ mutant (hom) and revertant (rev) strains] and BEC after 1 h of incubation at 37°C. The magnification bar corresponds to 100 μm. See the Methods section for more details. (B) Adhesion assay data. Histograms showing the adherence of the wild type (wt: black column), mp65Δ mutant (hom: grey column) and revertant (rev: white column) strains to BEC. The bars indicate the standard errors. Significant differences from wild type adhesion (P < 0.05) are indicated by asterisks. (C) Adhesion of the mp65Δ mutant to Caco-2 cell monolayers. Recovery of Candida cells [wild type (wt: black column), mp65Δ mutant (hom: grey column) and revertant (rev: white column) strains] at different time points (30 and 60 min) of incubation with Caco-2 cells. Adherent cells recovered after thorough washing out of the microplate (Panel 1). Non-adherent cells recovered from the supernatant (Panel 2). The results are the mean of 3 independent experiments. The bars indicate the standard deviations.
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Figure 5: Adhesion analysis of the mp65Δ mutant. (A) Adhesion of the mp65Δ mutant to BEC. Representative fields randomly selected showing the interaction between yeast cells [wild type (wt), mp65Δ mutant (hom) and revertant (rev) strains] and BEC after 1 h of incubation at 37°C. The magnification bar corresponds to 100 μm. See the Methods section for more details. (B) Adhesion assay data. Histograms showing the adherence of the wild type (wt: black column), mp65Δ mutant (hom: grey column) and revertant (rev: white column) strains to BEC. The bars indicate the standard errors. Significant differences from wild type adhesion (P < 0.05) are indicated by asterisks. (C) Adhesion of the mp65Δ mutant to Caco-2 cell monolayers. Recovery of Candida cells [wild type (wt: black column), mp65Δ mutant (hom: grey column) and revertant (rev: white column) strains] at different time points (30 and 60 min) of incubation with Caco-2 cells. Adherent cells recovered after thorough washing out of the microplate (Panel 1). Non-adherent cells recovered from the supernatant (Panel 2). The results are the mean of 3 independent experiments. The bars indicate the standard deviations.

Mentions: To examine the differences between wild type and mp65Δ mutant strains in their adherence to epithelial cells, we used two model cell systems: the exfoliated human buccal epithelial cells (BEC) and the Caco-2 cell monolayers. In the first system (visual assay of stained cells), 2 × 107 cells of wild type and mp65Δ mutant strains were incubated with 105 BEC, and the adherence was expressed as the number of yeast cells adhering to 100 epithelial cells ± standard error. The mp65Δ mutant showed significantly reduced adherence to BEC (Figures 5 A and 5B), whereas the revertant strain partially regained the ability to adhere to BEC, reaching a level similar to that of the wild type (C. albicans cells/BEC mean ± S.E.; wild type: 35 ± 2.0 vs. mp65Δ mutant: 10 ± 1.5 vs. revertant: 25 ± 1.0; P < 0.05). In the second system, the number of C. albicans cells adhering to the surface and those remaining in the supernatant were analyzed in a time-dependent manner (Figure 5C). Adhesion of the wild type cells to Caco-2 cells was rapid and efficient: after 30 min, about 65% of the cells recovered had adhered to the Caco-2 cell monolayers, whereas only 35% were recovered from the supernatant. After 60 min the percentage of adhering cells increased to 75%, whereas the percentage of cells in the supernatant decreased to 25%. The mp65Δ mutant cells showed significantly reduced adhesion to the Caco-2 cells: after 30 and 60 min, the percentage of adhering cells was 38% and 43% respectively, whereas the percentage of non-adhering cells was 62% and 57% respectively. In the revertant cells, the efficiency and kinetics of adhesion were similar to those in the wild type.


The MP65 gene is required for cell wall integrity, adherence to epithelial cells and biofilm formation in Candida albicans.

Sandini S, Stringaro A, Arancia S, Colone M, Mondello F, Murtas S, Girolamo A, Mastrangelo N, De Bernardis F - BMC Microbiol. (2011)

Adhesion analysis of the mp65Δ mutant. (A) Adhesion of the mp65Δ mutant to BEC. Representative fields randomly selected showing the interaction between yeast cells [wild type (wt), mp65Δ mutant (hom) and revertant (rev) strains] and BEC after 1 h of incubation at 37°C. The magnification bar corresponds to 100 μm. See the Methods section for more details. (B) Adhesion assay data. Histograms showing the adherence of the wild type (wt: black column), mp65Δ mutant (hom: grey column) and revertant (rev: white column) strains to BEC. The bars indicate the standard errors. Significant differences from wild type adhesion (P < 0.05) are indicated by asterisks. (C) Adhesion of the mp65Δ mutant to Caco-2 cell monolayers. Recovery of Candida cells [wild type (wt: black column), mp65Δ mutant (hom: grey column) and revertant (rev: white column) strains] at different time points (30 and 60 min) of incubation with Caco-2 cells. Adherent cells recovered after thorough washing out of the microplate (Panel 1). Non-adherent cells recovered from the supernatant (Panel 2). The results are the mean of 3 independent experiments. The bars indicate the standard deviations.
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Figure 5: Adhesion analysis of the mp65Δ mutant. (A) Adhesion of the mp65Δ mutant to BEC. Representative fields randomly selected showing the interaction between yeast cells [wild type (wt), mp65Δ mutant (hom) and revertant (rev) strains] and BEC after 1 h of incubation at 37°C. The magnification bar corresponds to 100 μm. See the Methods section for more details. (B) Adhesion assay data. Histograms showing the adherence of the wild type (wt: black column), mp65Δ mutant (hom: grey column) and revertant (rev: white column) strains to BEC. The bars indicate the standard errors. Significant differences from wild type adhesion (P < 0.05) are indicated by asterisks. (C) Adhesion of the mp65Δ mutant to Caco-2 cell monolayers. Recovery of Candida cells [wild type (wt: black column), mp65Δ mutant (hom: grey column) and revertant (rev: white column) strains] at different time points (30 and 60 min) of incubation with Caco-2 cells. Adherent cells recovered after thorough washing out of the microplate (Panel 1). Non-adherent cells recovered from the supernatant (Panel 2). The results are the mean of 3 independent experiments. The bars indicate the standard deviations.
Mentions: To examine the differences between wild type and mp65Δ mutant strains in their adherence to epithelial cells, we used two model cell systems: the exfoliated human buccal epithelial cells (BEC) and the Caco-2 cell monolayers. In the first system (visual assay of stained cells), 2 × 107 cells of wild type and mp65Δ mutant strains were incubated with 105 BEC, and the adherence was expressed as the number of yeast cells adhering to 100 epithelial cells ± standard error. The mp65Δ mutant showed significantly reduced adherence to BEC (Figures 5 A and 5B), whereas the revertant strain partially regained the ability to adhere to BEC, reaching a level similar to that of the wild type (C. albicans cells/BEC mean ± S.E.; wild type: 35 ± 2.0 vs. mp65Δ mutant: 10 ± 1.5 vs. revertant: 25 ± 1.0; P < 0.05). In the second system, the number of C. albicans cells adhering to the surface and those remaining in the supernatant were analyzed in a time-dependent manner (Figure 5C). Adhesion of the wild type cells to Caco-2 cells was rapid and efficient: after 30 min, about 65% of the cells recovered had adhered to the Caco-2 cell monolayers, whereas only 35% were recovered from the supernatant. After 60 min the percentage of adhering cells increased to 75%, whereas the percentage of cells in the supernatant decreased to 25%. The mp65Δ mutant cells showed significantly reduced adhesion to the Caco-2 cells: after 30 and 60 min, the percentage of adhering cells was 38% and 43% respectively, whereas the percentage of non-adhering cells was 62% and 57% respectively. In the revertant cells, the efficiency and kinetics of adhesion were similar to those in the wild type.

Bottom Line: Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type.All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion.We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious, Parasitic and Immuno-mediated Diseases, Istituto Superiore di Sanità, Rome, 00161, Italy. silvia.sandini@iss.it

ABSTRACT

Background: The MP65 gene of Candida albicans (orf19.1779) encodes a putative β-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation.

Results: The mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p), a high level of expression of two stress-related genes (DDR48 and SOD5), and a modulated expression of β-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion.

Conclusions: We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.

Show MeSH
Related in: MedlinePlus