Loss of mannosylphosphate from Candida albicans cell wall proteins results in enhanced resistance to the inhibitory effect of a cationic antimicrobial peptide via reduced peptide binding to the cell surface.
The outermost layer of the Candida albicans cell wall is enriched with mannosylated glycoproteins. We have used a range of isogenic glycosylation mutants of C. albicans, which are defective to varying degrees in cell wall protein mannosylation, to investigate the role of the outermost layer of the yeast cell wall in mediating the fungicidal action of the cationic, alpha-helical antimicrobial peptide dermaseptin S3(1-16) [DsS3(1-16)]. The degree of phosphomannan loss, and concomitant reduction in surface negative charge, from the series of glycosylation mutants correlated with reduced levels of peptide binding to the cells. In turn, the reduced peptide binding correlated with enhanced resistance to DsS3(1-16). To ascertain whether DsS3(1-16) binds to negatively charged phosphate, we studied the effect of exogenous glucosamine 6-phosphate, and glucosamine hydrochloride as a negative control, on the antifungal efficacy of DsS3(1-16). Glucosamine 6-phosphate retarded the efficacy of DsS3(1-16), and this was attributed to the presence of phosphate, because addition of identical concentrations of glucosamine hydrochloride had little detrimental effect on peptide efficacy. Fluorescence microscopy with DsS3(1-16) tagged with fluorescein revealed that the peptide binds to the outer surface of the yeast cell, supporting our previous conclusion that the presence of exterior phosphomannan is a major determinant of the antifungal potency of DsS3(1-16). The binding of the peptide to the cell surface was a transient event that was followed by apparent localization of DsS3(1-16) in the vacuole or dissemination throughout the entire cytosol. The presence of glucosamine 6-phosphate clearly reduced the proportion of cells in the population that showed complete cytosolic staining, implying that the binding and entry of the peptide into the cytosol is significantly reduced due to the exogenous phosphate sequestering the peptide and reducing the amount of peptide able to bind to the surface phosphomannan. In conclusion, we present evidence that an antimicrobial peptide, similar to those employed by cells of the human immune system, has evolved to recognize molecular patterns on the surface of pathogens in order to maximize efficacy.
Harris
Mark
M
Centre for Biomolecular Sciences, School of Biology, University of St Andrews, The North Haugh, St Andrews KY16 9ST, UK.
Mora-Montes
Héctor M
HM
School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.
Gow
Neil A R
NAR
School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.
Coote
Peter J
PJ
Centre for Biomolecular Sciences, School of Biology, University of St Andrews, The North Haugh, St Andrews KY16 9ST, UK.