Calcofluor Antifungal Action Depends on Chitin and a Functional High-Osmolarity Glycerol Response (HOG) Pathway: Evidence for a Physiological Role of the Saccharomyces cerevisiae HOG Pathway under Noninducing Conditions

Abstract

We have isolated several Saccharomyces cerevisiae mutants resistant to calcofluor that contain mutations in the PBS2 or HOG1 genes, which encode the mitogen-activated protein kinase (MAPK) and MAP kinases, respectively, of the high-osmolarity glycerol response (HOG) pathway. We report that blockage of either of the two activation branches of the pathway, namely, SHO1 and SLN1, leads to partial resistance to calcofluor, while simultaneous disruption significantly increases resistance. However, chitin biosynthesis is independent of the HOG pathway. Calcofluor treatment also induces an increase in salt tolerance and glycerol accumulation, although no activation of the HOG pathway is detected. Our results indicate that the antifungal effect of calcofluor depends on its binding to cell wall chitin but also on the presence of a functional HOG pathway. Characterization of one of the mutants isolated, pbs2-14, revealed that resistance to calcofluor and HOGdependent osmoadaptation are two different physiological processes. Sensitivity to calcofluor depends on the constitutive functionality of the HOG pathway; when this is altered, the cells become calcofluor resistant but also show very low levels of basal salt tolerance. Characterization of some multicopy suppressors of the calcofluor resistance phenotype indicated that constitutive HOG functionality participates in the maintenance of cell wall architecture, a conclusion supported by the antagonism observed between the protein kinase and HOG signal transduction pathways.

The Pbs2 MAP kinase kinase is essential for the oxidative-stress response in the fungal pathogen Candida albicans

“…The link between the HOG pathway and cellwall architecture has been reported in both S. cerevisiae (Alonso- Monge et al, 2001;García-Rodriguez et al, 2000;Jiang et al, 1995;Kapteyn et al, 2001) and C. albicans (Alonso-Monge et al, 1999). We describe growth differences between hog1 and pbs2 in the presence of Congo red and calcofluor white.…”

“…For example, the S. cerevisiae phr1 mutant exhibits a higher proportion of chitin and sensitivity to Nikkomycin Z and calcofluor white (Popolo & Vai, 1998), whereas mutants in chitin synthases chs2 and chs8 have less chitin synthase activity and are therefore hypersensitive to calcofluor white (Munro et al, 2003). In S. cerevisiae, mutants defective in the HOG pathway are resistant to calcofluor white, although no differences were detected in chitin synthesis during vegetative growth or during calcofluor treatment (García-Rodriguez et al, 2000). Additionally, overexpression of the PBS2 gene leads to higher b-1,3-glucan synthase activity and resistance to b-1,3-glucanases (Lai et al, 1997).…”

“…In S. cerevisiae, pbs2 and hog1 mutants have been reported to be resistant to calcofluor white (García-Rodriguez et al, 2000) and sensitive to cell wall-degrading enzymes (Zymolyase and Quantazyme) (Alonso-Monge et al, 2001; Kapteyn et al, 2001). In C. albicans, disruption of the Hog1 MAP kinase results in cells that are resistant to certain cell-wall biogenesis inhibitors (Alonso-Monge et al, 1999).…”

A PBS2 homologue from Debaryomyces hansenii shows a differential effect on calcofluor and polymyxin B sensitivity in Saccharomyces cerevisiae

“…The role of PBS2 in maintaining cell wall plasticity through its control over b-glucan synthesis has also been demonstrated (Jiang et al, 1995;Lai et al, 1997). Recently, Garcia-Rodriguez et al (2000) isolated a pbs2 mutant allele that was capable of conferring resistance to calcofluor but sustained full growth at 1.4 M NaCl, suggesting another physiological function of this gene. However, the detailed molecular mechanisms that result in these phenotypes have yet to be worked out.…”

“…Earlier studies (Boguslawski and Polazzi, 1987) have shown that S. cerevisiae cells harbouring a mutation in the PBS2 gene are sensitive to polymyxin B, an antibiotic known to damage the integrity of cell membranes (Storm et al, 1977). Recently, it has been observed that S. cerevisiae pbs2 mutants showed altered calcofluor sensitivity (Garcia-Rodriguez et al, 2000). In such a scenario, it was interesting to see whether DPBS2 could complement these phenotypes in the S. cerevisiae pbs2 mutant.…”