The virulence of Candida species depends on many environmental conditions, including extracellular pH and concentration of alkali metal cations. Tests of the tolerance/sensitivity of four pathogenic Candida species (C. albicans, C. dubliniensis, C. glabrata, and C. parapsilosis) to alkali metal cations under various growth conditions revealed significant differences among these species. Though all of them can be classified as rather osmotolerant yeast species, they exhibit different levels of tolerance to different salts. C. parapsilosis and C. albicans are the most salt-tolerant in general; C. dubliniensis is the least tolerant on rich YPD media and C. glabrata on acidic (pH 3.5) minimal YNB medium. C. dubliniensis is relatively salt-sensitive in spite of its ability to maintain as high intracellular K(+)/Na(+) ratio as its highly salt-tolerant relative C. albicans. On the other hand, C. parapsilosis can grow in the presence of very high external NaCl concentrations in spite of its high intracellular Na(+) concentrations (and thus lower K(+)/Na(+) ratio) and thus resembles salt-tolerant (halophilic) Debaryomyces hansenii.

Yeasts tightly regulate their intracellular concentrations of alkali metal cations. In Saccharomyces cerevisiae, the Nha1 Na(+) /H(+) -antiporter and Ena1 Na(+) -ATPase, mediate the efflux of toxic sodium and surplus potassium. We report the characterization of Candida glabrata CgCnh1 and CgEna1 homologues. Their substrate specificity and transport properties were compared upon expression in S. cerevisiae, and their function characterized directly in C. glabrata. The CgCnh1 antiporter and the CgEna1 ATPase transport both potassium and sodium when expressed in S. cerevisiae. CgEna1p fully complements the lack of S. cerevisiae own Na(+) -ATPases but the activity of the CgCnh1 antiporter is lower than that of ScNha1p. Candida glabrata deletion mutants and analyses of their phenotypes revealed that though both transporters have a broad substrate specificity, their function in C. glabrata cells is not the same. Their differing physiological roles are also reflected in their regulation of expression, CgENA1 is highly upregulated by an increased osmotic pressure or sodium concentration, whereas CgCNH1 is expressed constitutively. The Cnh1 antiporter is involved in the regulation of potassium content and the Ena1 ATPase in sodium detoxification of C. glabrata cells. This situation differs from S. cerevisiae, where the Nha1 antiporter and Ena ATPases both participate together in Na(+) detoxification and in the regulation of K(+) homeostasis.

Fluconazole-sensitive (SC 5314) and -resistant (clinical isolate 1173) Candida albicans strains were compared in terms of their osmotolerance and tolerance to toxic sodium cations. The two strains did not differ in their tolerance to high osmotic pressure in general but exhibited distinct sensitivities to sodium cations. Although the fluconazole-resistant 1173 strain contained, under all conditions tested, significantly lower intracellular amounts of Na+, it was much more sodium sensitive than the SC 5314 strain. The addition of subinhibitory concentrations of fluconazole to media supplemented with NaCl significantly influenced the growth of both strains and resulted in substantially elevated intracellular sodium concentrations compared with growth in medium containing NaCl but no fluconazole.

Background The virulence of Candida species depends on many environmental conditions. Extracellular pH and concentration of alkali metal cations belong among important factors. Nevertheless, the contribution of transporters mediating the exchange of alkali metal cations for protons across the plasma membrane to the cell salt tolerance and other physiological properties of various Candida species has not been studied so far. Results The tolerance/sensitivity of four pathogenic Candida species to alkali metal cations was tested and the role of one of the cation transporters in that tolerance (presumed to be the plasma-membrane Na + /H + antiporter) was studied. The genes encoding these antiporters in the most and least salt sensitive species, C. dubliniensis and C. parapsilosis respectively, were identified, cloned and functionally expressed in the plasma membranes of Saccharomyces cerevisiae cells lacking their own cation exporters. Both Cp Cnh1 and Cd Cnh1 antiporters had broad substrate specificity and transported Na + , K + , Li + , and Rb + . Their activity in S. cerevisiae cells differed; Cp Cnh1p provided cells with a much higher salt tolerance than the Cd Cnh1 antiporter. The observed difference in activity was confirmed by direct measurements of sodium and potassium efflux mediated by these antiporters. Conclusion We have cloned two genes encoding putative Na + /H + antiporters in C. parapsilosis and C. dubliniensis , and characterized the transport properties of encoded proteins. Our results show that the activity of plasma-membrane Na + /H + antiporters is one of the factors determining the tolerance of pathogenic Candida species to high external concentrations of alkali metal cations.

Background Kexin-like proteinases are a subfamily of the subtilisin-like serine proteinases with multiple regulatory functions in eukaryotes. In the yeast Saccharomyces cerevisiae the Kex2 protein is biochemically well investigated, however, with the exception of a few well known proteins such as the α-pheromone precursors, killer toxin precursors and aspartic proteinase propeptides, very few substrates are known. Fungal kex2 deletion mutants display pleiotropic phenotypes that are thought to result from the failure to proteolytically activate such substrates. Results In this study we have aimed at providing an improved assembly of Kex2 target proteins to explain the phenotypes observed in fungal kex2 deletion mutants by in vitro digestion of recombinant substrates from Candida albicans and C. glabrata . We identified CaEce1, CA0365, one member of the Pry protein family and CaOps4-homolog proteins as novel Kex2 substrates. Conclusion Statistical analysis of the cleavage sites revealed extended subsite recognition of negatively charged residues in the P1', P2' and P4' positions, which is also reflected in construction of the respective binding pockets in the ScKex2 enzyme. Additionally, we provide evidence for the existence of structural constrains in potential substrates prohibiting proteolysis. Furthermore, by using purified Kex2 proteinases from S. cerevisiae , P. pastoris , C. albicans and C. glabrata , we show that while the substrate specificity is generally conserved between organisms, the proteinases are still distinct from each other and are likely to have additional unique substrate recognition.