Candida albicans Hgt1p, a multifunctional evasion molecule: complement inhibitor, CR3 analogue, and human immunodeficiency virus-binding molecule

Abstract

Background: The complement system is tightly controlled by several regulators. Two of these, factor H (FH) and C4b-binding protein (C4BP), can be acquired by pathogens conveying resistance to complement attack. The aim of the study was to characterize the FH binding molecule of Candida albicans, a potentially life-threatening yeast.

Methods: The gene coding for this molecule was identified by probing an expression library and homozygous deletion mutants of the respective gene were constructed. Binding and functional assays were undertaken to compare wild-type and knockout strains.

Results: The high-affinity glucose transporter 1 (CaHgt1p) was identified as an FH-binding molecule. Homozygous hgt1Δ/Δ deletion mutants, but not the restored strain in which HGT1 was reintegrated, showed a decreased binding of FH and even of C4BP, demonstrating its function as an FH- and C4BP-binding protein. This led to an enhanced terminal complement complex deposition after incubation with human serum; CaHgt1p thus functions as complement inhibitor. hgt1Δ/Δ mutants failed to form rosettes with complement-coated sheep erythrocytes, and show reduced binding to HIV-gp160, implying that a complement receptor 3 (CR3) moiety, known as fungal HIV binding molecule is lacking.

Conclusions: CaHgt1p is a multifunctional evasion molecule, as complement inhibitor, CR3 analogue and HIV receptor.

Figure 1

Morphology of Candida albicans high-affinity glucose transporter 1 (hgt1)Δ/Δ. Microscopic analyses of cellular morphology, in part via immunofluorescence using anti-Candida immunoglobulin G (IgG) (A–C), of C. albicans hgt1Δ/Δ (B, E) compared with SN152 parental (A, D) and hgt1Δ/Δ::HGT1 restored (C, F) strains after 24 hours incubation in RPMI at 30°C (A–C) or at 37°C (D–F). Representative examples of quadruplicate experiments are shown.

Figure 2

Binding of factor H (FH) by Candida albicans high-affinity glucose transporter 1 (hgt1)Δ/Δ-qualitative assessment. Immunofluorescence analyses of binding of FH from ethylenediaminetetraacetic acid (EDTA)–plasma (A–C) or in its purified form (D–F) by hgt1Δ/Δ (B, E) compared with SN152 parental (A, D) and hgt1Δ/Δ::HGT1 restored (C, F) strains via goat anti-FH IgG. Representative examples of quadruplicates experiments are shown.

Figure 3

Binding of factor H (FH) and C4b- binding protein (C4BP) by Candida albicans high-affinity glucose transporter 1 (hgt1)Δ/Δ–quantitative assessment. Binding of FH (A) and C4BP (B) to SN152 parental (or wild type [WT], black columns), hgt1Δ/Δ (chequered columns), and hgt1Δ/Δ::HGT1 restored (striped columns) strains was assessed by determining the percentage of binding cells within the entire population. Results from quadruplicate experiments performed on 4 different days are shown; **, P <.01.

Figure 4

Deposition of terminal complement complex (TCC) on Candida albicans high-affinity glucose transporter 1 (hgt1)Δ/Δ as a measure of complement activation on the yeast cell surface. Deposited TCC was detected by flow cytometry using mouse antineoC9 antibodies followed by fluorescein isothiocyanate (FITC)–labelled antimouse antibodies on SN152 parental (black columns), hgt1Δ/Δ (checkered columns), and hgt1Δ/Δ::HGT1 restored (striped columns) strains after incubation with normal human serum (NHS) (A) or after preincubation with factor H (50 μg/mL) followed by incubation with NHS (B). Percentages of deposited TCC on the yeast cell surface are shown. Cells incubated in buffer only were used as 100% control. Results from quadruplicate experiments performed on 4 different days are shown; *, P <.05, **, P <.01.

Figure 5

Rosette formation on Candida albicans hgt1Δ/Δ as a measure of CR3 analogue expression on the yeast cell surface. Rosettes (indicated by arrows) of C. albicans SN152 parental and high-affinity glucose transporter 1 (hgt1)Δ/Δ::HGT1 restored (C) strains after incubation with complement-coated sheep erythrocytes for 1 hour. No rosettes were observed for hgt1Δ/Δ at 1 hour (not shown) and not even after extended periods of 12 hours (B) and 24 hours. Results from quadruplicate experiments performed on 4 different days are shown.

Figure 6

Binding of recombinant human immunodeficiency virus (HIV) gp160 to Candida albicans. Binding of recombinant HIV gp160 to C. albicans SN152 parental (black column), high-affinity glucose transporter 1 (hgt1)Δ/Δ (chequered column) and hgt1Δ/Δ::HGT1 restored (striped column) strains was determined by yeast cell enzyme-linked immunosorbent assay (ELISA). Human HIV antibody-positive serum (1:500) and antihuman-AP IgG served as detecting reagents. Results from quadruplicate experiments performed on 4 different d are shown; * P <.05.