A Complementation Analysis by Parasexual Recombination of Candida albicans Morphological Mutants

Abstract

Benomyl treatment (at 100 pg ml-l) of Candida albicans 1001, and other strains derived from it, determined the appearance of morphological mutants similar to those derived from UV irradiation treatment. A permanent alteration in the morphogenesis of these mutant strains determined their inability to grow by budding, to form oval yeast cells or blastospores (Yphenotype) and their growth as long filamentous forms, mostly with the appearance of pseudomycelium, giving rise to rough colonies (R phenotype). In order to carry out a genetic complementation analysis, we isolated morphological mutants that carried other genetic markers (nutritional, conditional lethal) adequate for crosses by means of protoplast fusion. Wild-type hybrids of regular mononuclear oval yeast cells and smooth colonies were obtained by crossing pairs of complementing mutants, whereas hybrids from crosses of non-complementing mutants still retained their morphological alterations. Our results define two complementation groups, which represent two genes relevant for dimorphism, whose alteration interferes with the correct transition from blastospores to mycelium. I N T R O D U C T I O NThe control of dimorphism is a key issue in research on the fungal pathogen Candida albicans. Different environmental conditions can determine a change in the budding pattern of growth of oval yeast cells, giving rise to filamentous forms of mycelial or pseudomycelial appearance. Conditions and inducers that determine mycelial growth are complex (Sevilla & Odds, 1986); a wide variety have been described and, not infrequently, data from different laboratories are contradictory (Odds, 1979). The extent to which dimorphism is controlled by specific genes whose expression would determine morphogenesis of either cellular form is controversial (Odds, 1985). As an alternative it has been suggested that metabolic regulation of permanently expressed genes could account for the formation of the major determinants of cell shape.We believe that the characterization of mutants with permanent alterations in the functions that regulate dimorphism is one of the best approaches to clarify this problem. The feasibility of isolating several types of morphological mutants of C. albicans has been demonstrated. We have taken advantage of the correlation that exists in alterations of colony and cellular morphologies, to isolate, after UV-irradiation, morphological mutants of permanent pseudomycelial growth that can be genetically analysed by means of mitotic recombination (Pomb et al., 1985). Our observations were followed by those of Slutsky et al. (1985), showing the spontaneous colonial switching that gave rise to colony phenotypes similar to that of our rough mutants, although no information was given about the cellular morphologies of these colonial variants. Hubbard et al. (1986) also described a mycelial mutant of C. albicans, and the feasibility of the genetic characterization of morphological mutants, by means of protoplast fusion has been demonstrated in our laboratory .Abbrevi...

Gene regulation during high-frequency switching in Candida albicans

“…Experiments have been performed in which switching and non-switching strains have been fused, and switching has been interpreted as being suppressed (Chu et al, 1992;Goshorn & Scherer, 1989;Gil et al, 1988) but, depending on the study, various problems in interpretation arise from the use of nonisogenic strains, the lack of distinction between suppression of the switch event and suppression of the genesis of a complex switch phenotype, the lack of quantification of switching frequencies, and analyses of switching on agar containing high levels of zinc, which suppresses the expression of many switch phenotypes, but not the switch event itself (see Soll, 1992, for a review of this literature). Another approach which was adopted involved collecting non-switching mutants, which could presumbly then be rescued by transformation with random fragments of a genomic library.…”

The Histone Deacetylase GenesHDA1andRPD3Play Distinct Roles in Regulation of High-Frequency Phenotypic Switching inCandida albicans

“…In addition, there was no effect on the phase-regulated levels of expression of the other deacetylase genes. These results demonstrate that the two deacetylase genes HDA1 and RPD3 play distinct roles in the suppression of switching, that the two play distinct and selective roles in the regulation of phase-specific genes, and that the deacetylases are in turn regulated by switching.Most strains of Candida albicans and related species switch, or can be induced to switch, between two or more general phenotypes distinguishable by colony morphology (14,41,42,44,48). Switching regulates a number of phenotypic characteristics, including putative virulence traits, and for that reason, it has been considered a higher-order virulence factor (42).…”

Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans

“…This auxotrophic mutant was used for the generation of a set of C. albicans mutants altered in their ability to undergo the dimorphic transition (Gil et al, 1988(Gil et al, ,1990. We first verified that the arginine biosynthetic defect present in strains EL2 and 1006 (arg4; Hoyer et al, 1994) hybrids when parasexual crosses between both strains were carried out.…”

“…Gene disruption of C. albicans ARG5,6 by cotransformation of pUC-ARG-U and pUC-ARG-H was optimized with respect to the concentration of both DNAs used in the assay. Protoplast fusions were performed as described previously (Gil et al, 1988). Electroporation was used for the recovery of plasmid DNA from C. albicans transformants .…”