Interaction with amoeba drives virulence-associated phenotypes in the Candida haemulonii complex

Predation by amoebae can induce adaptive traits in microorganisms, enhancing their resilience and pathogenic potential. This study investigated whether interaction with Acanthamoeba castellanii modulates the virulence of environmental fungi Candida haemulonii complex species. Our findings reveal that adaptation to an amoebic host induces significant phenotypic changes in C. duobushaemulonii isolated from a beetle's gut, leading to four distinct phenotypes: smooth-pink, rough-pink, intense-pink, and hyphae-white. These variants exhibited increased aggregation and filamentation. Amoeba-adapted isolates produced over 15-fold more biofilm biomass and more than 2.2-fold greater extracellular matrix than non-adapted isolates. Transcriptional analysis revealed upregulation of virulence-associated genes, including dimorphism regulators WOR1 ( > 2.4-fold), CPH1 (4.8-fold), and HGC1 ( > 1.9-fold) as well as biofilm-related genes ALS1 ( > 3.6-fold), BCR1 ( > 14.5-fold), and EGF1 ( > 1.6-fold), demonstrating amoeba-induced enhancement of pathogenic traits. Furthermore, adaptation enhanced fungal tolerance to environmental stressors and resulted in more than a 1.3-fold increase in the secretion of hydrolytic enzymes, including esterase, phospholipase, and secreted aspartyl proteinase. Pathogenicity assays using Galleria mellonella revealed significantly increased virulence of amoeba-adapted isolates, with a reduction in the lethal dose (LD₅₀) by 71.5-97.1% compared to non-adapted controls (NA: 2.52 ± 0.22 × 10⁶ CFU/mL; P10:HW: 7.17 ± 2.14 × 10⁴ CFU/mL; P10:RP: 1.25 ± 0.15 × 10⁵ CFU/mL). These findings suggest that environmental interaction with amoebae can promote the emergence of virulence-associated phenotypes in C. duobushaemulonii, providing a model for understanding how environmental pressures may facilitate fungal pathogenesis.