Carvacrol-Induced Vacuole Dysfunction and Morphological Consequences in Nakaseomyces glabratus and Candida albicans.

With the prevalence of systemic fungal infections caused by Candida albicans and non-albicans species and their resistance to classical antifungals, there is an urgent need to explore alternatives. Herein, we evaluate the impact of the monoterpene carvacrol, a major component of oregano and thyme oils, on clinical and laboratory strains of C. albicans and Nakaseomyces glabratus. Carvacrol induces a wide range of antifungal effects, including the inhibition of growth and hyphal and biofilm formation. Using biochemical and microscopic approaches, we elucidate carvacrol-induced hyphal inhibition. The significantly reduced survival rates following exposure to carvacrol were accompanied by dose-dependent vacuolar acidification, disrupted membrane integrity, and aberrant morphology. Germ tube assays, used to elucidate the relationship between vacuolar dysfunction and hyphal inhibition, showed that carvacrol significantly reduced hyphal formation, which was accompanied by a defective C. albicans morphology. Thus, we show a link between vacuolar acidification/disrupted vacuole membrane integrity and compromised candidal morphology/morphogenesis, demonstrating that carvacrol exerts its anti-hyphal activity by altering vacuole integrity.

Host Cell Geometry and Cytoskeletal Organization Governs Candida-Host Cell Interactions at the Nanoscale.

Candida is one of the most common opportunistic fungal pathogens in humans. Its adhesion to the host cell is required in parasitic states and is important for pathogenesis. Many studies have shown that there is an increased risk of developing candidiasis when normal tissue barriers are weakened or when immune defenses are compromised, for example, during cancer treatment that induces immunosuppression. The mechanical properties of malignant cells, such as adhesiveness and viscoelasticity, which contribute to cellular invasion and migration are different from those of noncancerous cells. To understand host invasion and its relationship with host cell health, we probed the interaction of Candida spp. with cancerous and noncancerous human cell lines using atomic force microscopy in the single-cell force spectroscopy mode. There was significant adhesion between Candida and human cells, with more adhesion to cancerous versus noncancerous cell lines. This increase in adhesion is related to the mechanobiological properties of cancer cells, which have a disorganized cytoskeleton and lower rigidity. Altered geometry and cytoskeletal disruption of the human cells impacted adhesion parameters, underscoring the role of cytoskeletal organization in Candida-human cell adhesion and implicating the manipulation of cell properties as a potential future therapeutic strategy.

Candida albicans Reactive Oxygen Species (ROS)-Dependent Lethality and ROS-Independent Hyphal and Biofilm Inhibition by Eugenol and Citral.

Candida albicans is part of the normal human flora but is most frequently isolated as the causative opportunistic pathogen of candidiasis. Plant-based essential oils and their components have been extensively studied as antimicrobials, but their antimicrobial impacts are poorly understood. Phenylpropenoids and monoterpenes, for example, eugenol from clove and citral from lemon grass, are potent antifungals against a wide range of pathogens. We report the cellular response of C. albicans to eugenol and citral, alone and combined, using biochemical and microscopic assays. The MICs of eugenol and citral were 1,000 and 256 µg/mL, respectively, with the two exhibiting additive effects based on a fractional inhibitory concentration index of 0.83 ± 0.14. High concentrations of eugenol caused membrane damage, oxidative stress, vacuole segregation, microtubule dysfunction and cell cycle arrest at the G1/S phase, and while citral had similar impacts, they were reactive oxygen species (ROS) independent. At sublethal concentrations (1/2 to 1/4 MIC), both oils disrupted microtubules and hyphal and biofilm formation in an ROS-independent manner. While both compounds disrupt the cell membrane, eugenol had a greater impact on membrane dysfunction. This study shows that eugenol and citral can induce vacuole and microtubule dysfunction, along with the inhibition of hyphal and biofilm formation. IMPORTANCE Candida albicans is a normal resident on and in the human body that can cause relatively benign infections. However, when our immune system is severely compromised (e.g., cancer chemotherapy patients) or underdeveloped (e.g., newborns), this fungus can become a deadly pathogen, infecting the bloodstream and organs. Since there are only a few effective antifungal agents that can be used to combat fungal infections, these fungi have been exposed to them over and over again, allowing the fungi to develop resistance. Instead of developing antifungal agents that kill the fungi, some of which have undesirable side effects on the human host, researchers have proposed to target the fungal traits that make the fungus more virulent. Here, we show how two components of plant-based essential oils, eugenol and citral, are effective inhibitors of C. albicans virulence traits.

From the first touch to biofilm establishment by the human pathogen Candida glabrata: a genome-wide to nanoscale view.

Candida glabrata is an opportunistic pathogen that adheres to human epithelial mucosa and forms biofilm to cause persistent infections. In this work, Single-cell Force Spectroscopy (SCFS) was used to glimpse at the adhesive properties of C. glabrata as it interacts with clinically relevant surfaces, the first step towards biofilm formation. Following a genetic screening, RNA-sequencing revealed that half of the entire transcriptome of C. glabrata is remodeled upon biofilm formation, around 40% of which under the control of the transcription factors CgEfg1 and CgTec1. Using SCFS, it was possible to observe that CgEfg1, but not CgTec1, is necessary for the initial interaction of C. glabrata cells with both abiotic surfaces and epithelial cells, while both transcription factors orchestrate biofilm maturation. Overall, this study characterizes the network of transcription factors controlling massive transcriptional remodelling occurring from the initial cell-surface interaction to mature biofilm formation.