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Fibronectin-, vitronectin- and laminin-binding proteins at the cell walls of Candida parapsilosis and Candida tropicalis pathogenic yeasts.

Kozik A, Karkowska-Kuleta J, Zajac D, Bochenska O, Kedracka-Krok S, Jankowska U, Rapala-Kozik M - BMC Microbiol. (2015)

Bottom Line: The major individual compounds of the fungal cell wall that bound fibronectin, vitronectin and laminin were found to comprise two groups: (1) true cell wall components similar to C. albicans adhesins from the Als, Hwp and Iff/Hyr families; and (2) atypical (cytoplasm-derived) surface-exposed proteins, including malate synthase, glucose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, enolase, fructose-1,6-bisphosphatase, transketolase, transaldolase and elongation factor 2.The adhesive abilities of two investigated non-albicans Candida species toward extracellular matrix proteins were comparable to those of C. albicans suggesting an important role of this particular virulence attribute in the pathogenesis of infections caused by C. tropicalis and C. parapsilosis.Our results reveal new insight into host-pathogen interactions during infections by two important, recently emerging, fungal pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, Gronostajowa 7, 30-387, Krakow, Poland. andrzej.kozik@uj.edu.pl.

ABSTRACT

Background: Candida parapsilosis and C. tropicalis increasingly compete with C. albicans-the most common fungal pathogen in humans-as causative agents of severe candidiasis in immunocompromised patients. In contrast to C. albicans, the pathogenic mechanisms of these two non-albicans Candida species are poorly understood. Adhesion of Candida yeast to host cells and the extracellular matrix is critical for fungal invasion of hosts.

Methods: The fungal proteins involved in interactions with extracellular matrix proteins were isolated from mixtures of β-1,3-glucanase- or β-1,6-glucanase-extractable cell wall-associated proteins by use of affinity chromatography and chemical cross-linking methods, and were further identified by liquid chromatography-coupled tandem mass spectrometry.

Results: In the present study, we characterized the binding of three major extracellular matrix proteins-fibronectin, vitronectin and laminin-to C. parapsilosis and C. tropicalis pseudohyphae. The major individual compounds of the fungal cell wall that bound fibronectin, vitronectin and laminin were found to comprise two groups: (1) true cell wall components similar to C. albicans adhesins from the Als, Hwp and Iff/Hyr families; and (2) atypical (cytoplasm-derived) surface-exposed proteins, including malate synthase, glucose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, enolase, fructose-1,6-bisphosphatase, transketolase, transaldolase and elongation factor 2.

Discussion: The adhesive abilities of two investigated non-albicans Candida species toward extracellular matrix proteins were comparable to those of C. albicans suggesting an important role of this particular virulence attribute in the pathogenesis of infections caused by C. tropicalis and C. parapsilosis.

Conclusions: Our results reveal new insight into host-pathogen interactions during infections by two important, recently emerging, fungal pathogens.

No MeSH data available.


Related in: MedlinePlus

Interactions of Candida spp. cell wall-associated proteins with fibronectin (FN), vitronectin (VTN) and laminin (LAM). In panels a and b, the plots are presented for saturable binding of biotinylated cell wall-associated proteins extracted from filamentous forms of C. albicans, C. parapsilosis and C. tropicalis to immobilized FN (a) and of cell wall-associated proteins of C. parapsilosis and C. tropicalis to immobilized VTN and LAM (b) (5 pmoles of FN, VTN or LAM adsorbed into wells of MaxiSorp microplates with the unoccupied surfaces blocked with BSA). Panels c and d show the respective plots for the displacement of biotinylated cell wall-associated proteins (40 μg/ml for FN binding or 15 μg/ml for VTN and LAM binding) from microplate-immobilized ECMPs (3 pmoles of FN or 1.25 pmoles of VTN or LAM per well) by soluble FN, VTN and LAM added at increasing concentrations. Wells without immobilized ECMPs but coated with BSA served as controls and the values obtained for those wells were subtracted from the total binding values. Results from representative experiments are presented, in which data points represent mean values from three determinations (three wells) ± standard deviation
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Fig3: Interactions of Candida spp. cell wall-associated proteins with fibronectin (FN), vitronectin (VTN) and laminin (LAM). In panels a and b, the plots are presented for saturable binding of biotinylated cell wall-associated proteins extracted from filamentous forms of C. albicans, C. parapsilosis and C. tropicalis to immobilized FN (a) and of cell wall-associated proteins of C. parapsilosis and C. tropicalis to immobilized VTN and LAM (b) (5 pmoles of FN, VTN or LAM adsorbed into wells of MaxiSorp microplates with the unoccupied surfaces blocked with BSA). Panels c and d show the respective plots for the displacement of biotinylated cell wall-associated proteins (40 μg/ml for FN binding or 15 μg/ml for VTN and LAM binding) from microplate-immobilized ECMPs (3 pmoles of FN or 1.25 pmoles of VTN or LAM per well) by soluble FN, VTN and LAM added at increasing concentrations. Wells without immobilized ECMPs but coated with BSA served as controls and the values obtained for those wells were subtracted from the total binding values. Results from representative experiments are presented, in which data points represent mean values from three determinations (three wells) ± standard deviation

Mentions: Further experiments were thus focused on the proteinaceous components of the fungal cell wall. These were extracted from pseudohyphal forms of the two investigated NAC species and, for comparative purposes, from C. albicans hyphae, by a procedure that is postulated to protect the native binding activities toward biological ligands. Therefore, strong denaturants or otherwise harsh conditions could not be applied, which inevitably limited the yield of the protein extraction process. Of the two glucanases used, β-1,3-glucanase was employed to hydrolyze the entire β-1,3-glucan network to release all proteins connected with or embedded in this structure while β-1,6-glucanase was used to cleave the anchors through which the main adhesins are known to be covalently bound. After these treatments, more than 95 % of the cells remained viable, excluding the possibility of a significant contamination of the obtained protein mixtures with cytoplasmic proteins. The obtained cell wall-associated proteins possessed binding activity toward fibronectin, vitronectin and laminin, as determined using two kinds of binding tests: (1) saturable binding of biotinylated ECMPs (Fig. 3a, b), a test similar to that used for the whole cells and presented in Fig. 1 above; and (2) a competitive test based on the displacement of the biotinylated cell wall-associated proteins from microplate-immobilized fibronectin, vitronectin or laminin by soluble ECMPs (Fig. 3c, d). Notably, the highest displacement (in a range of 50–60 % at 10-fold molar excess of soluble protein) was observed for fibronectin. Thus, the binding of vitronectin and laminin might be considered to be less “specific”, particularly for C. parapsilosis.Fig. 3


Fibronectin-, vitronectin- and laminin-binding proteins at the cell walls of Candida parapsilosis and Candida tropicalis pathogenic yeasts.

Kozik A, Karkowska-Kuleta J, Zajac D, Bochenska O, Kedracka-Krok S, Jankowska U, Rapala-Kozik M - BMC Microbiol. (2015)

Interactions of Candida spp. cell wall-associated proteins with fibronectin (FN), vitronectin (VTN) and laminin (LAM). In panels a and b, the plots are presented for saturable binding of biotinylated cell wall-associated proteins extracted from filamentous forms of C. albicans, C. parapsilosis and C. tropicalis to immobilized FN (a) and of cell wall-associated proteins of C. parapsilosis and C. tropicalis to immobilized VTN and LAM (b) (5 pmoles of FN, VTN or LAM adsorbed into wells of MaxiSorp microplates with the unoccupied surfaces blocked with BSA). Panels c and d show the respective plots for the displacement of biotinylated cell wall-associated proteins (40 μg/ml for FN binding or 15 μg/ml for VTN and LAM binding) from microplate-immobilized ECMPs (3 pmoles of FN or 1.25 pmoles of VTN or LAM per well) by soluble FN, VTN and LAM added at increasing concentrations. Wells without immobilized ECMPs but coated with BSA served as controls and the values obtained for those wells were subtracted from the total binding values. Results from representative experiments are presented, in which data points represent mean values from three determinations (three wells) ± standard deviation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4595241&req=5

Fig3: Interactions of Candida spp. cell wall-associated proteins with fibronectin (FN), vitronectin (VTN) and laminin (LAM). In panels a and b, the plots are presented for saturable binding of biotinylated cell wall-associated proteins extracted from filamentous forms of C. albicans, C. parapsilosis and C. tropicalis to immobilized FN (a) and of cell wall-associated proteins of C. parapsilosis and C. tropicalis to immobilized VTN and LAM (b) (5 pmoles of FN, VTN or LAM adsorbed into wells of MaxiSorp microplates with the unoccupied surfaces blocked with BSA). Panels c and d show the respective plots for the displacement of biotinylated cell wall-associated proteins (40 μg/ml for FN binding or 15 μg/ml for VTN and LAM binding) from microplate-immobilized ECMPs (3 pmoles of FN or 1.25 pmoles of VTN or LAM per well) by soluble FN, VTN and LAM added at increasing concentrations. Wells without immobilized ECMPs but coated with BSA served as controls and the values obtained for those wells were subtracted from the total binding values. Results from representative experiments are presented, in which data points represent mean values from three determinations (three wells) ± standard deviation
Mentions: Further experiments were thus focused on the proteinaceous components of the fungal cell wall. These were extracted from pseudohyphal forms of the two investigated NAC species and, for comparative purposes, from C. albicans hyphae, by a procedure that is postulated to protect the native binding activities toward biological ligands. Therefore, strong denaturants or otherwise harsh conditions could not be applied, which inevitably limited the yield of the protein extraction process. Of the two glucanases used, β-1,3-glucanase was employed to hydrolyze the entire β-1,3-glucan network to release all proteins connected with or embedded in this structure while β-1,6-glucanase was used to cleave the anchors through which the main adhesins are known to be covalently bound. After these treatments, more than 95 % of the cells remained viable, excluding the possibility of a significant contamination of the obtained protein mixtures with cytoplasmic proteins. The obtained cell wall-associated proteins possessed binding activity toward fibronectin, vitronectin and laminin, as determined using two kinds of binding tests: (1) saturable binding of biotinylated ECMPs (Fig. 3a, b), a test similar to that used for the whole cells and presented in Fig. 1 above; and (2) a competitive test based on the displacement of the biotinylated cell wall-associated proteins from microplate-immobilized fibronectin, vitronectin or laminin by soluble ECMPs (Fig. 3c, d). Notably, the highest displacement (in a range of 50–60 % at 10-fold molar excess of soluble protein) was observed for fibronectin. Thus, the binding of vitronectin and laminin might be considered to be less “specific”, particularly for C. parapsilosis.Fig. 3

Bottom Line: The major individual compounds of the fungal cell wall that bound fibronectin, vitronectin and laminin were found to comprise two groups: (1) true cell wall components similar to C. albicans adhesins from the Als, Hwp and Iff/Hyr families; and (2) atypical (cytoplasm-derived) surface-exposed proteins, including malate synthase, glucose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, enolase, fructose-1,6-bisphosphatase, transketolase, transaldolase and elongation factor 2.The adhesive abilities of two investigated non-albicans Candida species toward extracellular matrix proteins were comparable to those of C. albicans suggesting an important role of this particular virulence attribute in the pathogenesis of infections caused by C. tropicalis and C. parapsilosis.Our results reveal new insight into host-pathogen interactions during infections by two important, recently emerging, fungal pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, Gronostajowa 7, 30-387, Krakow, Poland. andrzej.kozik@uj.edu.pl.

ABSTRACT

Background: Candida parapsilosis and C. tropicalis increasingly compete with C. albicans-the most common fungal pathogen in humans-as causative agents of severe candidiasis in immunocompromised patients. In contrast to C. albicans, the pathogenic mechanisms of these two non-albicans Candida species are poorly understood. Adhesion of Candida yeast to host cells and the extracellular matrix is critical for fungal invasion of hosts.

Methods: The fungal proteins involved in interactions with extracellular matrix proteins were isolated from mixtures of β-1,3-glucanase- or β-1,6-glucanase-extractable cell wall-associated proteins by use of affinity chromatography and chemical cross-linking methods, and were further identified by liquid chromatography-coupled tandem mass spectrometry.

Results: In the present study, we characterized the binding of three major extracellular matrix proteins-fibronectin, vitronectin and laminin-to C. parapsilosis and C. tropicalis pseudohyphae. The major individual compounds of the fungal cell wall that bound fibronectin, vitronectin and laminin were found to comprise two groups: (1) true cell wall components similar to C. albicans adhesins from the Als, Hwp and Iff/Hyr families; and (2) atypical (cytoplasm-derived) surface-exposed proteins, including malate synthase, glucose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, enolase, fructose-1,6-bisphosphatase, transketolase, transaldolase and elongation factor 2.

Discussion: The adhesive abilities of two investigated non-albicans Candida species toward extracellular matrix proteins were comparable to those of C. albicans suggesting an important role of this particular virulence attribute in the pathogenesis of infections caused by C. tropicalis and C. parapsilosis.

Conclusions: Our results reveal new insight into host-pathogen interactions during infections by two important, recently emerging, fungal pathogens.

No MeSH data available.


Related in: MedlinePlus