Analyses of hyphal diversity in Trichosporonales yeasts based on fluorescent microscopic observations

In dimorphic yeasts, hyphal growth is primarily associated with infection and mycosis progression, with Trichosporon asahii causing deep-seated mycosis and summer-type hypersensitivity pneumonitis. Magnesium accelerates hyphal growth in T. asahii, leading to multi-septation, vacuolar expansion, and decreased lipid droplet size. However, the commonality of these phenotypes has not been studied in Trichosporonales yeasts. Therefore, to explore whether similar magnesium-induced phenotypes occur across Trichosporonales yeasts, we examined hyphal growth, multi-septation, vacuolar extension, and lipid droplet size and number in 30 species. Cell length increased with magnesium treatment in 13 yeasts: 5 Trichosporon (T. asahii, Trichosporon aquatile, Trichosporon asteroides, Trichosporon coremiiforme, and Trichosporon ovoides), three Apiotrichum (Apiotrichum brassicae, Apiotrichum montevideense, and Apiotrichum veenhuisii), three Cutaneotrichosporon (Cutaneotrichosporon cavernicola, Cutaneotrichosporon cutaneum, and Cutaneotrichosporon dermatis), Pascua guehoae, and Takashimella koratensis. C. dermatis also underwent pseudo-hyphal growth. Multi-septation increased in seven dimorphic yeasts, including five Trichosporon spp., Trichosporon faecale, and C. dermatis. The vacuolar area was significantly extended in T. asahii, T. aquatile, T. ovoides, and C. dermatis. Lipid droplet size increased only in Trichosporon inkin; however, it decreased in T. asahii, T. coremiiforme, and T. faecale. Additionally, lipid droplet number was preferentially altered in Apiotrichum and Cutaneotrichosporon. These results suggested that magnesium-induced multi-septation and vacuolar area expansion phenotypically distinguish Trichosporon hyphae from Apiotrichum and Cutaneotrichosporon hyphae and distinguish C. dermatis pseudo-hyphae from Cutaneotrichosporon spp. Moreover, differences in lipid droplet size can discriminate species within Trichosporon. Our results suggest that phenotypic alteration via magnesium treatment can contribute to the characterization of Trichosporonales yeasts. These findings provide insights into fungal pathogenesis and may support new treatment strategies.IMPORTANCEMagnesium sulfate considerably affects hyphal growth and cellular organization in Trichosporon asahii. To examine the commonality of this phenotype in Trichosporonales, we treated 30 Trichosporonales yeasts with magnesium sulfate and observed genus-level phenotypic alterations. Using cell length measurement, lipid droplet staining, septum staining, and vacuole staining, considerable hyphal diversity was observed among Trichosporonales. Notably, differences in the multi-septation phenotype and vacuolar size distinguished Trichosporon hyphae from Apiotrichum and Cutaneotrichosporon hyphae and distinguished Cutaneotrichosporon dermatis from other Cutaneotrichosporon spp. Moreover, differences in lipid droplet phenotype divided Trichosporon hyphae into two groups. Our study revealed the relationship between hyphal morphology and phylogenetic systematics in Trichosporonales.