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Abstract
The human pathogenic yeast Candida albicans utilizes host complement regulators for immune evasion. Here we identify the first fungal protein that binds Factor H and FHL-1. By screening a protein array of 4088 proteins of Saccharomyces cerevisiae, phosphoglycerate mutase (ScGpm1p) was identified as a Factor H- and FHL-1-binding protein. The homologous C. albicans Gpm1p (CaGpm1p) was cloned and recombinantly expressed as a 36-kDa His-tagged protein. Purified CaGpm1p binds the host complement regulators Factor H and FHL-1, but not C4BP. The CaGpm1p binding regions in the host proteins were localized; FHL-1 binds via short consensus repeats (SCRs) 6 and 7, and Factor H utilizes two contact regions that are located in SCRs 6 and 7 and in SCRs 19 and 20. In addition, recombinant CaGpm1p binds plasminogen via lysine residues. CaGpm1p is a surface protein as demonstrated by immunostaining and flow cytometry. A C. albicans gpm1(-/-) mutant strain was generated that did not grow on glucose-supplemented but on ethanol- and glycerol-supplemented medium. Reduced binding of Factor H and plasminogen to the null mutant strain is in agreement with the presence of additional binding proteins. Attached to CaGpm1p, each of the three host plasma proteins is functionally active. Factor H and FHL-1 show cofactor activity for cleavage of C3b, and bound plasminogen is converted by urokinase-type plasminogen activator to proteolytically active plasmin. Thus, the surface-expressed CaGpm1p is a virulence factor that utilizes the host Factor H, FHL-1, and plasminogen for immune evasion and degradation of extracellular matrices.
Highlights
Candida albicans is the most important human pathogenic yeast and causes disseminated infections [1, 2]
Identification of ScGpm1p as a Factor H- and FHL-1-binding Protein—We have previously shown that host complement regulators Factor H and FHL-1 bind to the pathogenic yeast C. albicans [15]
The C. albicans homologue C. albicans Gpm1p (CaGpm1p) showed the highest homology to the S. cerevisiae protein (78%)
Summary
After blocking of nonspecific binding sites, the cells were incubated at 4 °C overnight with rabbit anti-CaGpm1p antiserum or with preimmune serum (1:50 dilution). After incubation at 4 °C for 30 min with CaGpm1p antiserum (1:100 dilution in 1% BSA-PBS), C. albicans yeast cells were washed with PBS supplemented with 1% BSA (1% BSA-PBS). CaGPM1 was reintroduced into the null mutant strain CAP4 by transformation with the 5.6-kilobase SacI/PstI insert of pG3 that harbors the CaGPM1 gene, upstream as well as downstream regions for homologous recombination, and the URA3 gene as a selectable marker, and yielded CAP5. Candida ELISA—For Candida ELISAs, C. albicans yeast cells from an overnight culture were washed with PBS, diluted into carbonate-bicarbonate buffer (Sigma) to 1 ϫ 107 cells/ml and immobilized onto a microtiter plate (MaxiSorb, Nunc) at 4 °C overnight. Plasmin activity was measured at 37 °C in intervals of 30 min by recording the absorbance at 405 nm (SpektraMax 190, Molecular Devices)
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