Clarissa J. Nobile

Purpose of the Review: Biofilm formation is a major adaptation of fungal pathogens that promotes persistence, immune evasion, and tolerance to antifungal therapy. Yet, despite its importance to the biofilm lifecycle, the mechanisms governing the release of cells from fungal biofilms remain poorly understood. This review examines current knowledge of fungal biofilm dispersal, with particular emphasis on the disparity between the extensive mechanistic literature in Candida albicans and the limited understanding of dispersal in other clinically important fungi. Recent Findings: In C. albicans , dispersal is a regulated process that generates predominantly yeast-form cells with transcriptional and phenotypic properties distinct from both biofilm-resident and conventional planktonic populations. These cells exhibit enhanced traits associated with adhesion, filamentation, host-cell damage, and virulence, and several molecular, cellular, and environmental factors have been implicated in regulating their production and release. In contrast, evidence for regulated dispersal in non- albicans Candida species and other fungal pathogens remains sparse. Studies of Candida auris (syn. Candidozyma auris ), Aspergillus fumigatus , and Scedosporium and Lomentospora species demonstrate that cells can be released from fungal biofilms and may exhibit distinct physiological properties, but whether these processes are mechanistically analogous to C. albicans dispersal remains unclear. Summary: Biofilm dispersal is an understudied component of the fungal biofilm lifecycle. C. albicans currently provides the strongest model for regulated fungal dispersal, but cell release alone should not be assumed to represent an equivalent developmental process across fungal species. Defining the mechanisms, cellular states, and biological consequences of biofilm cell release across diverse fungi will be essential for determining which features of dispersal are broadly shared and which reflect species-specific strategies.

Biofilms are surface-associated microbial communities that dominate natural and host-associated environments and serve as major reservoirs for infections. Current therapeutics primarily target planktonic cells and are often ineffective against biofilms, highlighting the need for biofilm-specific strategies. Here, we investigated naturally occurring L-amino acids as antibiofilm agents against Candida albicans and Staphylococcus aureus . Administration of aspartic acid, cysteine, and glutamic acid reduced biofilm formation and established biofilm biomass in both single- and dual-species settings. Moreover, composite amino acid treatment enhanced fluconazole activity during C. albicans biofilm formation. These findings indicate that targeted amino acid administration, alone or in combination with existing antimicrobials, could provide a novel strategy for preventing and treating biofilm-associated infections.

Candida parapsilosis is a major human fungal pathogen, with recent global outbreaks driven by fluconazole-resistant (FLCR-Cp) isolates that are difficult to eradicate and associated with poor clinical outcomes. However, the microbial traits enabling persistence of these outbreak lineages remain poorly defined. Here, we show that FLCR-Cp isolates responsible for prolonged, multi-country outbreaks consistently exhibit a striking low-biofilm-producing (LBP) phenotype. Contrary to the prevailing view that robust biofilm formation promotes persistence, LBP strains displayed enhanced stress tolerance, increased cell wall masking, and reduced immune recognition. These traits conferred resistance to neutrophil and macrophage killing and enhanced survival in immune cell-rich organs during systemic infection. Genome-wide transcriptomic profiling revealed extensive metabolic and regulatory rewiring in LBP strains. Whole-genome sequencing (WGS) of a global isolate collection further demonstrated that the LBP phenotype has emerged independently multiple times, supporting convergent evolution under host selection. Functional genomic analyses suggest that biofilm attenuation arises through multigenic changes, and disruption of key biofilm-associated transcriptional regulators enhanced fitness during immune interactions. Together, our findings overturn the assumption that robust biofilm formation drives outbreak persistence and instead identify biofilm attenuation as an adaptive tradeoff that promotes immune evasion and long-term survival. These results redefine our understanding of C. parapsilosis adaptation during healthcare-associated outbreaks and shift attention toward host-driven evolutionary processes than environmental persistence alone.

Candida auris (also known as Candidozyma auris) relies on extensive transporter networks to maintain cellular homeostasis and adapt to environmental stress, yet the broader regulatory roles of individual transporters remain poorly understood. Qdr3, a member of the major facilitator superfamily (MFS), contributes to multidrug resistance in several pathogenic yeasts, but its function in C. auris has not been defined. To investigate the role of Qdr3, we generated Δqdr3 mutants in C. auris strain B8441 and performed integrated phenotypic, ultrastructural, transcriptomic, and proteomic analyses. Deletion of qdr3 produced pronounced sensitivity to ionic stress without affecting basal growth or antifungal susceptibility. Transcriptomic profiling revealed extensive transcriptional remodeling characterized by reduced expression of heat-shock and cell-surface maintenance pathways alongside strong induction of mitochondrial translation and biogenesis programs. These transcriptional shifts were supported by proteomic signatures and transmission electron microscopy, which demonstrated increased mitochondrial abundance and enhanced cytoplasmic granularity. Qdr3 also influenced multicellular organization, as Δqdr3 biofilms exhibited reduced cellular aggregation and increased extracellular matrix production. Despite increased stress sensitivity, Δqdr3 mutants displayed enhanced virulence in a Galleria mellonella infection model, revealing an unexpected disconnect between stress adaptation and pathogenic potential. Together, these findings identify Qdr3 as a key regulator of cellular homeostasis that links membrane transport, mitochondrial activity, and cell-surface architecture in C. auris. These results demonstrate how disruption of a single MFS transporter can drive extensive physiological and regulatory remodeling, ultimately altering virulence in this WHO critical priority pathogen.

SUMMARY: The emerging fungal pathogen Candidozyma auris (syn. Candida auris; C. auris ) has attracted considerable attention from the scientific, clinical, and public health communities due to its multidrug resistance, environmental persistence, and high transmissibility. Since its first description in Japan in 2009, C. auris has spread rapidly worldwide, with a marked acceleration following the coronavirus disease 2019 (COVID-19) pandemic. As of December 2025, 84,941 colonization or infection cases have been reported across 82 countries spanning 6 continents. In this review, we summarize the current knowledge of the biology and global epidemiology of C. auris . We first examine its taxonomy, proposed origins, and key biological, genetic, and phenotypic characteristics, with particular emphasis on factors underlying environmental persistence, transmission dynamics, antifungal resistance, and virulence. Drawing on published literature and publicly available surveillance data from national public health authorities worldwide, we provide an updated overview of the global epidemiological landscape and evolving transmission patterns of C. auris . Finally, we discuss potential strategies to mitigate the continued and escalating global spread of this emerging multidrug-resistant fungal pathogen.

Flippases, composed of catalytic and regulatory subunits, preserve cellular integrity by maintaining the asymmetric distribution of phospholipids across the plasma membrane. Here, we provide the first functional characterization of Lem3, the regulatory subunit of the flippase complex in Candida albicans , and reveal its critical role in membrane homeostasis and pathogenesis. We demonstrate that Lem3 localizes to both the endoplasmic reticulum and plasma membrane, consistent with its role in phospholipid translocation. Phenotypic analysis of a lem3 Δ/Δ mutant showed decreased susceptibility to the alkylphosphocholine analog, miltefosine. Concurrently, the mutant was defective in the inward translocation (flip) of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) across the plasma membrane, accompanied by altered levels of ergosterol and sphingolipids, indicating the requirement of Lem3 for maintaining membrane lipid composition. Notably, LEM3 deletion suppressed the enhanced phospholipid internalization seen in an rta3 Δ/Δ mutant, suggesting that Rta3 negatively regulates Lem3 activity. The lem3 Δ/Δ strain exhibited increased susceptibility to azole antifungals, likely due to increased membrane permeability and impaired function of the major drug efflux pump Cdr1. Strikingly, the absence of LEM3 reversed drug resistance in an azole-resistant clinical isolate, reinforcing its therapeutic relevance. Finally, Lem3 was important for biofilm formation and in vivo virulence, establishing it as a key determinant of C. albicans pathogenicity. Together, these findings position Lem3 and the flippase machinery as promising targets for antifungal therapy. IMPORTANCE: Plasma membrane lipid composition, maintained by lipid translocators, is a key determinant of virulence in pathogenic fungi. Here, we demonstrate that Lem3, the regulatory subunit of the flippase complex, by regulating the asymmetric distribution of phospholipids, impacts the expression of virulence traits and modulates drug resistance in Candida albicans . These findings highlight how diverse cellular processes are interconnected through flippase-mediated lipid homeostasis. With the alarming rise in fungal infections and antifungal resistance, targeting lipid translocators such as Lem3 offers a promising avenue for the development of novel therapeutic targets.

Coccidioides is a dimorphic fungal pathogen responsible for the mammalian disease commonly known as Valley fever. While previous studies have characterized transcriptomic changes associated with its life stage transitions, the contribution of small RNAs (sRNAs) – key regulators of virulence in other pathogenic fungi – remains unexplored. Here, we profile sRNA expression across three Coccidioides posadasii morphologies – arthroconidia, mycelia, and spherules – analyzing both intracellular and extracellular RNA fractions. Utilizing RiboMarker^®️ small RNA and RNA fragment library preparation, we achieved enhanced transcriptome coverage by incorporating RNA species that are typically underrepresented or incompatible with standard sequencing workflows. We observed pronounced transcriptomic remodeling during the transition from arthroconidia to mycelia or spherules, driven primarily by changes in protein-coding transcripts, tRNA, and unannotated loci. These data identify sRNA- and fragment-producing loci that may be required for progression between the saprobic and parasitic life cycles of Coccidioides. In addition, distinct RNA fragmentation patterns were associated with each morphological state. Notably, we detected evidence of RNA export to the extracellular space, particularly snRNAs and tRNA-derived fragments, during these transitions, suggesting potential roles in cell-cell or host-pathogen communication. Together, this intra- and extracellular Coccidioides sRNA atlas provides a resource for biomarker discovery and advances our understanding of the molecular basis of fungal virulence.

Candida albicans is a common opportunistic fungal pathogen that asymptomatically colonizes most humans. Although typically a benign commensal, dysbiosis caused by antibiotic use, immune dysfunction, or epithelial barrier disruption can trigger fungal overgrowth and infection, ranging from superficial mucosal disease to life-threatening systemic candidiasis. New preclinical infection models are needed to dissect C. albicans pathogenesis in vivo across distinct infection stages and with different measurable host outcomes. The planarian Schmidtea mediterranea was previously established as an invertebrate host for studying host-pathogen interactions during C. albicans infection. S. mediterranea relies entirely on conserved innate immune mechanisms capable of overcoming infection with pathogenic microorganisms, including bacteria and fungi. Planarians' remarkable regenerative capacity and accessible stem cell populations make this organism a tractable model to analyze early immune responses, tissue repair, and pathogen clearance in vivo. This model supports simultaneous analysis of fungal virulence and host transcriptional responses, providing valuable insights into infection dynamics. Here, an updated protocol with detailed modifications, standardized procedures, and optimized steps for infecting S. mediterranea with C. albicans has been presented, designed to enhance reproducibility and enable systematic studies of fungal pathogenesis and host defense.