Ecm33p is a widely distributed fungal protein with functional relevance, clearly demonstrated by Δ mutant phenotypes, mainly related to the cell wall. Homology searches with genes identified Ecm33p, as well as the two other proteins of its family: Pst1p and the product of . Ecm33p is a 423 aa protein which has the typical features of cell-surface GPI proteins and is able to complement Δ cell wall defects. Heterozygous (RML1) and homozygous (RML2) mutants of were obtained, as well as a single and a double reintegrant (RML3 and RML4, respectively). mutant strains displayed an aberrant morphology, being more rounded and bigger than the wild-type, suggesting morphogenetic defects. They also exhibited cell wall defects, with enhanced sensitivity to different compounds that interfere in polymerization of cell wall components (Calcofluor white, Congo red and hygromycin B) and a marked tendency to flocculate extensively. In addition, CaEcm33p is required for normal yeast-to-hyphae transition . In liquid medium (5 % serum), the transition was delayed in mutants, and after 24 h the culture contained very abnormal large and rounded cells. On solid medium (10 % serum, Spider or SLADH) RML2 failed to produce hyphae and media invasiveness. showed a gene dosage effect, demonstrated by the intermediate phenotype of the heterozygous mutants RML1 and confirmed by Northern blot analysis. Furthermore, CaEcm33p is also involved in virulence. In a murine systemic model of infection, 100 % mouse survival and no kidney or brain colonization were obtained 30 days after infection with 10 cells of any homozygous or heterozygous Δ mutant tested. In contrast, all mice infected with parental or RML4 (two Ca copies reintegrated) strains died in a few days, showing that, in these conditions, two copies were required for virulence.


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