Genomic Insights into Candidozyma auris Clade II: Comparative Phylogenomics and Structural Validation of Fluconazole Resistance Mechanisms

<i>Candidozyma auris</i> (formerly <i>Candida auris</i>) is an emerging multidrug-resistant fungal pathogen with confirmed cases in over 30 countries. Although whole-genome sequencing (WGS) analysis defined distinct clades during characterization of underlying genetic mechanism behind multidrug resistance, Clade II remains under-evaluated. In this study, a three-level comparative genomic strategy (Global, Clade, Phenotype) was employed by integration of unbiased genome-wide comparative SNP screening (GATK v4.1.9.0), targeted BLAST profiling (BLAST+ v2.17.0), and in silico protein analysis (ColabFold v1.5.5; DynaMut2 v2.0) for systematic evaluation of mechanisms of antifungal resistance in thirty-nine Clade II <i>C. auris</i> clinical isolates and fourteen reference strains. Global and clade-level analyses confirmed that all the clinical isolates belong to Clade II, according to phylogenetic clustering and mating type locus (<i>MTL</i>) conservation. At the phenotype level, a distinct subclade of fluconazole-resistant mutants was identified to have a heterogenous network of mutations in seven key enzymes associated with cell membrane dynamics and the metabolic stress response. Among these, four core mutations (<i>TAC1B</i>, <i>CAN2</i>, <i>NIC96</i>, <i>PMA1</i>) were confirmed as functional drivers based on strict criteria during multitier in silico protein analysis: cross-species conservation, surface exposure, active site proximity, thermodynamic stability, and protein interface interaction. On the other hand, three high-level fluconazole-resistant clinical isolates (≥128 μg/mL) that lacked these functional drivers were subjected to comprehensive subtractive genomic profiling analysis. The absence of coding mutations in validated resistance drivers, yeast orthologs, and convergent variants suggests that there is an alternative novel non-coding or regulatory mechanism behind fluconazole resistance. These findings highlight Clade II's evolutionary divergence into two distinct trajectories towards the development of a high level of fluconazole resistance: canonical protein alteration versus regulatory modulation.