Multidimensional assessment of in-host fitness costs of echinocandin resistance in the opportunistic fungal pathogen <i>Candida glabrata</i> reveals the niche-specific requirement for <i>FKS1</i> and <i>FKS2</i> during infection and gut colonization

Host stresses are often considered a major barrier against the emergence of echinocandin resistance (ECR) in prominent infecting organisms like <i>Candida albicans</i> due to fitness defects. Yet, ECR strains of <i>C. glabrata</i> carrying diverse amino acid changes in Fks1 and Fks2 are increasingly reported as breakthrough infections. Nonetheless, the impact of equivalent mutations in different <i>FKS</i> alleles on fitness has not been systematically studied. Herein, we employed a diverse array of <i>ex vivo</i> and <i>in vivo</i> models to address these questions among clinically relevant ECR mutants. All ECR mutants retained fitness during interaction with THP1 macrophages and neutrophils. Whereas a strain with a Fks2^F659del or <i>fks2Δ</i> showed fitness defects during interaction with macrophages and neutrophils. Fks2^F659del showed a unique susceptibility to numerous stresses, especially the combination of alternative carbon sources, low pH, and H₂O₂. Consistent with failure in mounting adaptive oxidative stress response during exposure to H₂O₂, transcriptomic analysis of intracellular Fks2^F659del highlighted the dysregulation of oxidative stress response genes, whereas intracellular <i>fks2Δ</i> showed hallmarks of metabolic dysregulation. Intriguingly, the Fks2^F659del mutant was outcompeted by wild type and Fks2^F659V in <i>in vivo</i> gut colonization and systemic infection models. Importantly, whereas both <i>FKS1</i> and <i>FKS2</i> were required to establish gut colonization, only <i>FKS2</i> was required for systemic infection. Therefore, our study supports the notion that the prevalence of ECR mutants among <i>C. glabrata</i> strains is likely driven by its ability to retain fitness across diverse niches. Furthermore, we identified that the essentiality of <i>FKS1</i> and <i>FKS2</i> is similarly dictated by niche-specific requirements.