Figure 1.
Candida albicans Gpm1 binds to human cells.
(A) Candida Gpm1 binds to HUVEC and HaCaT. Recombinant Gpm1 was added to human endothelial cells (HUVEC), keratinocytes (HaCaT) or monocytic U937 cells. After incubation and subsequent washing, bound Gpm1 was detected in flow cytometry using rabbit Gpm1 antiserum followed by Alexa Fluor 647 goat anti-rabbit IgG. Human cells without Gpm1 were used as control. Histograms are representative of three independent experiments. (B) Binding of Gpm1 to HUVEC and HaCaT was confirmed by confocal microscopy. HUVEC (top panels) or HaCaT cells (bottom panels) were incubated with Gpm1, fixed with parafolmaldehyde and after washing, bound Gpm1 was detected with rabbit Gpm1 antiserum followed by Alexa Fluor 488 goat anti-rabbit IgG (green). The cellular DNA was stained with DAPI (blue). Scale bar = 10 µm.
Figure 2.
The candida GPM1 deletion mutant bins with low intensity to human endothelial cells (HUVEC).
The candida gpm1Δ/Δ knock-out mutant bound with low intensity to HUVEC. Yeast cells of C. albicans SC5314 (wild type), gpm1Δ, gpm1Δ/Δ or gpm1Δ/Δ::GPM1 were labeled with DiD and incubated with DiO-labeled HUVEC for 120 min at 37°C at 5% CO2. After washing and detachment, HUVEC with associated (adherent and/or endocytosed) C. albicans cells were identified in flow cytometry as double-positive cells (DiO+, DiD+) and by the change in side scatter. HUVEC alone were detected as single-positive cells (DiO+, DiD−) were used as control.
Figure 3.
Gpm1 coated latex beads mediates adhesion to endothelial cells.
Gpm1 was coated to the surface of fluorescent latex beads and the coated beads were attached to HUVEC for 45°C with 5% CO2. Following incubation the fluorescent latex beads were recorded and quantified by LSM. The blue fluorescence was measured per µm2 under the oil immersion objective lens using the ZEN 2009 software. Data are mean ± SD (error bars) of three experiments. BSA coated latex beads wer used in addition.
Figure 4.
Gpm1 binds to human vitronectin.
(A) Vitronectin and fibronectin bound to immobilized Gpm1. Binding of extracellular matrix (ECM) proteins to Gpm1 was determined by ELISA. Gpm1 was immobilized on a microtiter plate and fibronectin, vitronectin, laminin, fibrinogen, collagen I, collagen III, or collagen IV were added. Following washing the bound ligands were detected with antiserum specific to each ECM protein together with HRP-conjugated specific antiserum. Binding of plasminogen to Gpm1 was used as control. (B) Vitronectin dose-dependently bound to immobilized Gpm1. Gpm1 was immobilized onto a microtiter plate, vitronectin was added at the indicated amounts, and following washing bound vitronectin was detected by rabbit vitronectin antiserum, followed by HRP-conjugated polyclonal goat anti-rabbit IgG. (C) Heparin inhibited vitronectin binding to Gpm1. Vitronectin and heparin (at the indicated amounts) were pre-incubated and then added to immobilized Gpm1. Following washing bound vitronectin was detected as in (B). (D) Interaction of vitronectin with Gpm1 is affected by ionic strength. Vitronectin was pre-incubated with NaCl (at the indicated final concentrations) and the mixture was added to immobilized Gpm1. Following washing bound vitronectin was detected as in (B). BSA and buffer were used as controls. Data represent mean values ± SD (error bars) of three independent experiments.
Figure 5.
Vitronectin blocks binding of Gpm1 to human cells.
Vitronectin when combined with Gpm1 decreased binding to human cells. Biotinylated Gpm1 (10 µg) was pre-incubated with vitronectin (10 µg) and then the mixture was added to HUVEC (A) or HaCaT (B). After washing, bound Gpm1 was detected using streptavidin-conjugated Cy5 by flow cytometry. Vitronectin when bound to Gpm1 (black dashed line) reduced Gpm1 binding to both human cell lines. Gpm1 binding in the absence of vitronectin shows prominent binding (black solid line). Human cells without Gpm1 and/or vitronectin was used as control (gray solid line). The data represent a representative experiment out of four independent experiments.
Figure 6.
Vitronectin is present on the surface of HUVEC and HaCaT cells.
Vitronectin was detected on the surface of human cells. HUVEC as well as HaCaT cells were cultivated in DMEM or RPMI, until reaching confluence. After washing, the human cells were kept in serum free medium for 24(A) Human cells were detached from the surface and vitronectin expressed on the surface of the cells was detected with specific rabbit antiserum followed by Alexa Fluor 647-conjugated goat anti-rabbit IgG as secondary antiserum by flow cytometry (solid line). The dashed line shows the same human cells that were treated with the secondary antiserum as a control. (B) Expression of vitronectin by human cells was visualized by confocal microscopy. Human cells were grown on coverslip in 24-well plate with appropriate medium until reaching confluence. After washing, the cells were maintained in serum-free medium for 24 h. Then the cells were fixed with paraformaldehyde (3%) and after extensive washing, vitronectin present on the cell surface was detected by rabbit vitronectin antiserum followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). The membrane of the human cells was visualized with Texas red-conjugated wheat germ agglutinin (red). Scale bar = 20 µm. The data represent a representative experiment out of four independent experiments.
Figure 7.
Gpm1 and vitronectin colocalize at the surface of human cells.
Vitronectin (red) and Gpm1 (green) colocalizes at the surface of human cells. Recombinant Gpm1 was attached to HUVEC or HaCaT cells that were kept in serum-free medium for 24 h. Following washing the cells were fixed with paraformaldehyde (3%). Gpm1 was detected with a monoclonal mouse Gpm1 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. Vitronectin expressed on the surface of the cells was detected by polyclonal rabbit vitronectin antiserum followed by Alexa Fluor 647-conjugated goat anti-rabbit IgG. DNA of human cells was stained with DAPI. Scale bar = 10 µm. The images show a representative experiment out of four independent experiments.