Interaction with amoeba drives virulence-associated phenotypes in the <i>Candida haemulonii</i> complex

Predation by amoebae can induce adaptive traits in microorganisms, enhancing their resilience and pathogenic potential. This study investigated whether interaction with <i>Acanthamoeba castellanii</i> modulates the virulence of environmental fungi <i>Candida haemulonii</i> complex species. Our findings reveal that adaptation to an amoebic host induces significant phenotypic changes in <i>C. duobushaemulonii</i> 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 <i>WOR1</i> ( > 2.4-fold), <i>CPH1</i> (4.8-fold), and <i>HGC1</i> ( > 1.9-fold) as well as biofilm-related genes <i>ALS1</i> ( > 3.6-fold), <i>BCR1</i> ( > 14.5-fold), and <i>EGF1</i> ( > 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 <i>Galleria mellonella</i> 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 <i>C. duobushaemulonii</i>, providing a model for understanding how environmental pressures may facilitate fungal pathogenesis.