Phenotypic switching of Candida tropicalis is associated with cell damage in epithelial cells and virulence in Galleria mellonella model

Candida species are the most common causes of human fungal infection worldwide. The pathogenic status of Candida spp. is associated with modulation of virulence determinants in response to environmental changes, and impairment of host defenses. Among Candida putative virulence factors, phenotypic switching is associated with fungal adaptability to environmental changes during invasion of host organism. Switching is a biological event associated with generation of phenotypic heterogeneity that occurs in a small fraction of the population, is random, reversible and represents an epigenetic state. In yeast, phenotypic switching drives variable changes, leading to the emergence of colonies with altered morphologies. Our group has previously described a high-frequency switching of colony morphology in several clinical isolates of Candida tropicalis. The isolates could switch spontaneously, heritably and reversibly between at least six different phenotypes, not including the white-opaque transition. One of the identified switching system that include three colony phenotypes: smooth (parental phenotype) and two related switch variants (crepe and rough) was associated with in vitro changes in virulence traits, including biofilm formation, morphogenesis and hemolysis, as well as switching from itraconazole susceptibility to resistance. Soll et al. were the first to assess switch phenotypes in C. tropicalis during the course of a prolonged Candida infection in a compromised host. Further, a white-opaque phenotypic switch was described for C. tropicalis that shows some similarities to that in Candida albicans. Although these studies have begun to explore the mechanism of phenotypic switching in C. tropicalis, the role of switching in this species remains an open question. Porman et al. demonstrated that a white-opaque phenotypic switching in C. tropicalis regulates a cryptic program of sexual mating, potentially giving rise to strains with increased virulence. The white-opaque transition also is associated with sexual biofilm formation in C. tropicalis, where biofilms are formed exclusively by opaque cells. C. tropicalis together with the majority of the medically relevant Candida species belongs to the CTG clade and is genetically close to C. albicans. The frequency of infections caused by C. tropicalis has increased over the last years, particularly in tropical regions; in some settings, bloodstream infections due to C. tropicalis have been associated with higher mortality than other Candida species. In the present study, we address for the first time in the literature, if phenotypic switching affects virulence in C. tropicalis. To this end, we employed all three colony phenotypes (smooth, crepe and rough) described previously and revertants (strains that switched back from variant to parental phenotype) for the evaluation of the capacity of damaging epithelial cells and virulence in Galleria mellonella. The switched strains (crepe and rough variants) were obtained as individual subclones from a clinical isolate exhibiting a smooth colony dome. The crepe and rough variants exhibited an irregular and structured dome surface and were obtained at frequencies of 5£10¡3 and 3£10¡2, respectively. In the present study, we initially determined the reversibility of crepe and rough variants phenotypes to the smooth phenotype (parental phenotype) by visually scoring of four days old growth colonies on YPD agar medium (Gibco) at 28 C. To calculate the reversibility frequencies, a total of 6,000 colonies from each variant