Sequence Variations and Protein Expression Levels of the Two Immune Evasion Proteins Gpm1 and Pra1 Influence Virulence of Clinical <i>Candida albicans</i> Isolates

<div><p><i>Candida albicans</i>, the important human fungal pathogen uses multiple evasion strategies to control, modulate and inhibit host complement and innate immune attack. Clinical <i>C. albicans</i> strains vary in pathogenicity and in serum resistance, in this work we analyzed sequence polymorphisms and variations in the expression levels of two central fungal complement evasion proteins, Gpm1 (phosphoglycerate mutase 1) and Pra1 (pH-regulated antigen 1) in thirteen clinical <i>C. albicans</i> isolates. Four nucleotide (nt) exchanges, all representing synonymous exchanges, were identified within the 747-nt long <i>GPM1</i> gene. For the 900-nt long <i>PRA1</i> gene, sixteen nucleotide exchanges were identified, which represented synonymous, as well as non-synonymous exchanges. All thirteen clinical isolates had a homozygous exchange (A to G) at position 73 of the <i>PRA1</i> gene. Surface levels of Gpm1 varied by 8.2, and Pra1 levels by 3.3 fold in thirteen tested isolates and these differences influenced fungal immune fitness. The high Gpm1/Pra1 expressing candida strains bound the three human immune regulators more efficiently, than the low expression strains. The difference was 44% for Factor H binding, 51% for C4BP binding and 23% for plasminogen binding. This higher Gpm1/Pra1 expressing strains result in enhanced survival upon challenge with complement active, Factor H depleted human serum (difference 40%). In addition adhesion to and infection of human endothelial cells was increased (difference 60%), and C3b surface deposition was less effective (difference 27%). Thus, variable expression levels of central immune evasion protein influences immune fitness of the human fungal pathogen <i>C. albicans</i> and thus contribute to fungal virulence.</p></div

Pra1 secretion and effect of secreted Pra1 on C3b/iC3b surface deposition.

<p>(<b>A</b>) Presence of Pra1 in the culture supernatant. Culture supernatant (YPD medium following overnight culture) of the selected clinical <i>C</i>. <i>albicans</i> strains (1x10⁶ cells) was separated by SDS-PAGE, transferred to a membrane and Pra1 levels were detected by rabbit Pra1 antiserum, followed by a HPR swine anti-rabbit serum as a secondary antibody. Pra1 was detected as a 60 kDa protein in culture supernatant derived from medium (lanes 3 and 4) and of high expression Gpm1/Pra1 strains (lanes 6 and 7). The additional bands of higher molecular mass and of slower mobility (arrow heads), represent Pra1 in complex with additional soluble proteins. Pra1 was not detectable in the culture supernatant derived from the two low Gpm1/Pra1 expressing strains (lanes 2 and 3). The arrow shows the 60 kDa Pra1 monomer and the position of the additional dimmer or multimeric forms and Pra1 containing complexes are likely indicated by the arrow heads. (<b>B</b>) Supernatant derived from the clinical <i>C</i>. <i>albicans</i> isolates blocked C3b/iC3b deposition on the yeast surface. The selected <i>C</i>. <i>albicans</i> clinical isolates were cultivated in YPD medium overnight, and culture supernatant derived from 5x10⁶ cells of each isolate was added to NHS (7.5%) diluted in Mg-EGTA, then heat treated C. <i>albicans</i> were challenged with this serum-supernatant mixture for 30 min at 37°C. Following washing C3b/iC3b surface deposition was analyzed by flow cytometry using goat anti C3 serum. Candida cells treated in the absence of NHS are shown as control. The histogram shown here is one representative experiment out of three performed. (<b>C</b>) The mean values of median fluorescence intensity of C3b/iC3b from three independent experiments. C3B/iC3b levels on the surface of cells incubated in NHS were set 100%. The mean values of each group, i.e. the low, medium or high Gpm1/Pra1 expressing isolates are indicated by the crossed circle in the middle of each group. The mean values of median fluorescence intensity from three independent experiments ± SD are shown.</p

Survival of the selected clinical isolates in complement active, Factor H depleted human serum.

<p>The selected clinical <i>C</i>. <i>albicans</i> isolates and the reference strain SC5314 strain were cultivated in YPD medium overnight at 30°C. Then the yeast cells (1x10⁴/ml) were incubated in complement active, Factor H depleted human serum (10%) for 60 min at 37°C. At start (time 0) and after 60 min incubation, aliquots were removed and the number of live yeast cells was determined by plating on the YPD agar plate, which then were cultivated for 2 days at 30°C. Survival yeast number at time 0 was set 100%. The mean values of each group, i.e. the low, medium or high Gpm1/Pra1 expressing isolates are indicated by the crossed circle in the middle of each group. Data shown represent the mean values ± SD of three separate independent experiments. Pra1 surface levels affect fungal adhesion and invasion to human endothelial cells.</p

Binding of the human complement regulators to selected <i>C</i>. <i>albicans</i> isolates.

<p>The selected low Gpm1- and Pra1 expressing yeast isolates (J#3 & J#11), the two medium (J#4 & J#6), as well as two high expressing strains (J#7 & J#10) and SC5314 were incubated in NHS. After washing surface bound Factor H- (<b>A</b>), C4BP- (<b>B</b>), as well as plasminogen (<b>C</b>) was detected with the corresponding anti serum in combination with secondary labeled Alexa Fluor-647 rabbit anti-goat, or goat anti-rabbit serum. Bound immune regulators were quantified by flow cytometry. The mean values of each group, i.e. the low, medium or high Gpm1/Pra1 expressing isolates are indicated by the crossed circle in the middle of each group. The data shown represent mean values ± SD of three separate experiments. Statistical significance of differences was determined using Student's <i>t</i>-test. *, <i>p</i>≤0.05; **, <i>p</i>≤0.01; ***, <i>p</i>≤0.001.Similarly, C4BP binding varied. This classical pathway regulator bound with moderate intensity to the low expressing isolates J#3 and J#11, i.e. 75% and 74%, with increased levels to the medium expressing strains, i.e. 102% and 102%, with highest intensity to the two high expressing strains, i.e. 119% and 132%. Both high expressing fungal isolates bound ca. 50% more C4BP to their surface as compared to the low expressing isolates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113192#pone.0113192.g003" target="_blank">Fig. 3B</a>).</p

Sequence variations of <i>GPM1</i> and <i>PRA1</i> in clinical <i>C</i>. <i>albicans</i> isolates.

<p>(<b>A</b>) The sequence variations identified in the thirteen clinical <i>C</i>. <i>albicans</i> isolates are indicated in the structure of the candida <i>GPM1</i> cDNA (top) and of the 747-nt Gpm1 protein (bottom). The <i>GPM1</i> gene of each clinical <i>C</i>. <i>albicans</i> isolate was amplified by PCR and the nucleotide sequences were determined. Four synonymous nucleotide exchanges were identified which appeared as homozygous or heterozygous variations and which did not affect the sequence of the Gpm1 protein (lower panel) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113192#pone.0113192.t001" target="_blank">Table 1</a>). (<b>B</b>) Sequence variations identified in the thirteen clinical <i>C</i>. <i>albicans</i> isolates in the <i>PRA1</i> gene and protein. Nucleotide exchanges are indicated for the <i>PRA1</i> cDNA (top) and for the 299 amino acid long Pra1 protein (bottom). A total of sixteen nucleotide exchanges occurred as homozygous or heterozygous variation (upper panel). Seven non synonymous nucleotide changes which affect the protein sequence are shown in <i>red characters</i>. The structure of the Pra1 protein includes the signal peptide (sp), the serine rich motive (ser), as well as the putative zinc binding region (Zn). Three amino acid exchanges, result in substitution of uncharged residues (i.e. Asn25, Gly105 or Ile111 to negatively charged residues, Asp25, Asp 105 or to a polar uncharged Ser 111 (shown with blue characters). All analyzed clinical isolates had the negatively charged Asp25, in homozygocity. In addition exchange of the non polar Gly₁₀₅ residue to negatively charged Asp residue occurs in some clinical isolates and exchange of the non polar Ile₁₁₁ to the polar, uncharged Ser residue is identified in one single isolate (<i>marked in blue</i>).</p

Gpm1 binds to human vitronectin.

<p>(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.</p

Vitronectin is present on the surface of HUVEC and HaCaT cells.

<p>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.</p

Pra1 secretion and effect of secreted Pra1 on C3b/iC3b surface deposition.

<p>(<b>A</b>) Presence of Pra1 in the culture supernatant. Culture supernatant (YPD medium following overnight culture) of the selected clinical <i>C</i>. <i>albicans</i> strains (1x10⁶ cells) was separated by SDS-PAGE, transferred to a membrane and Pra1 levels were detected by rabbit Pra1 antiserum, followed by a HPR swine anti-rabbit serum as a secondary antibody. Pra1 was detected as a 60 kDa protein in culture supernatant derived from medium (lanes 3 and 4) and of high expression Gpm1/Pra1 strains (lanes 6 and 7). The additional bands of higher molecular mass and of slower mobility (arrow heads), represent Pra1 in complex with additional soluble proteins. Pra1 was not detectable in the culture supernatant derived from the two low Gpm1/Pra1 expressing strains (lanes 2 and 3). The arrow shows the 60 kDa Pra1 monomer and the position of the additional dimmer or multimeric forms and Pra1 containing complexes are likely indicated by the arrow heads. (<b>B</b>) Supernatant derived from the clinical <i>C</i>. <i>albicans</i> isolates blocked C3b/iC3b deposition on the yeast surface. The selected <i>C</i>. <i>albicans</i> clinical isolates were cultivated in YPD medium overnight, and culture supernatant derived from 5x10⁶ cells of each isolate was added to NHS (7.5%) diluted in Mg-EGTA, then heat treated C. <i>albicans</i> were challenged with this serum-supernatant mixture for 30 min at 37°C. Following washing C3b/iC3b surface deposition was analyzed by flow cytometry using goat anti C3 serum. Candida cells treated in the absence of NHS are shown as control. The histogram shown here is one representative experiment out of three performed. (<b>C</b>) The mean values of median fluorescence intensity of C3b/iC3b from three independent experiments. C3B/iC3b levels on the surface of cells incubated in NHS were set 100%. The mean values of each group, i.e. the low, medium or high Gpm1/Pra1 expressing isolates are indicated by the crossed circle in the middle of each group. The mean values of median fluorescence intensity from three independent experiments ± SD are shown.</p

Vitronectin blocks binding of Gpm1 to human cells.

<p>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.</p

Gpm1 coated latex beads mediates adhesion to endothelial cells.

<p>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.</p