Plant-based essential oils are promising anti-virulence agents against the multidrug-resistant opportunistic pathogen Candida albicans. Gas chromatography–mass spectrometry of Cinnamomum zeylanicum (cinnamon) leaf and Eugenia caryophyllus (clove) flower bud essential oils revealed eugenol (73 and 75%, respectively) as their major component, with β-caryophyllene, eugenyl acetate, and α-humulene as common minor components. Cinnamon leaf and clove essential oils had minimum inhibitory concentrations of 600 and 500 µg/mL, respectively against the C. albicans RSY150 reference strain and 1000 and 750 µg/mL, respectively for the clinical reference strain ATCC 10231. The combined oils are additive (FICI = 0.72 ± 0.16) and synergistic (0.5 ± 0.0) against RSY150 and the clinical reference strain, respectively. Mycelial growth was inhibited by sublethal concentrations of either essential oil, which abolished colony growth. At half of the lowest combined lethal concentration for the two oils, the yeast-to-hyphal transition and mycelial growth was potently inhibited. Mutant strains als1Δ/Δ, als3Δ/Δ, hwp1Δ/HWP1+, and efg1Δ/Δ were sensitive to either or both oils, especially efg1Δ/Δ. In conclusion, oils of cinnamon leaf and clove and their combination significantly impact C. albicans virulence by inhibiting hyphal and mycelial growth.

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.

Candida albicans typically thrives in a commensal relationship with humans but is also an opportunistic fungal pathogen. As an opportunistic pathogen, C. albicans relies heavily on its ability to assimilate nutrients, for which it must compete with the host and other microorganisms. Amino acid biosynthesis, sensing, and uptake play pivotal roles in C. albicans growth and pathogenicity. C. albicans biosynthesizes aromatic amino acids and co-enzyme Q de novo through the shikimate pathway, including the Aro1, Aro2, and Aro7 enzymes, but also has amino acid transporters for uptake from the environment. Thus, antifungal approaches targeting aromatic amino acid biosynthesis must simultaneously inhibit amino acid biosynthesis and uptake. Herein, we investigate the plant-based antifungal, carvacrol, in conjunction with aromatic amino acid biosynthetic mutants, as a potential anti-candidal strategy. Growth of the WT, ARO2 , and ARO7 strains were inhibited by 150 µg/mL carvacrol, whereas the ARO1 mutant was slightly more sensitive (with MIC 125 µg/mL). All repressed mutants exposed to carvacrol are partially rescued in the presence of para-aminobenzoic acid (PABA) (CoQ precursor), indicating that blocking the shikimate pathway impacts both aromatic amino acid and CoQ biosynthesis. Moreover, carvacrol at sublethal concentrations significantly inhibits ARO1 adhesion and hyphal formation, along with pre-attached and pre-formed hyphae, ultimately impacting biofilm metabolic activity and biomass accumulation and significantly reducing biofilm growth. In summary, carvacrol increases the sensitivity of C. albicans to ARO1 repression, with attenuated adhesion, hyphal formation, mycelial growth and biofilm formation, likely by blocking aromatic amino acid uptake. IMPORTANCE: The opportunistic pathogen Candida albicans remains the leading cause of candidemia and invasive candidiasis (IC), causing significant morbidity and mortality in immunocompromised patients. Our current arsenal of effective antifungal drugs is limited in number, mechanistic diversity, and efficacy, are cytotoxic and associated with antifungal resistance, necessitating the development of novel antifungals and combination therapies. Here, we show how simultaneously blocking the shikimate pathway, through ARO1 repression, and disrupting aromatic amino acid uptake by carvacrol prevent C. albicans biofilm formation. Thus, inhibitors of the Aro1 enzyme in combination with carvacrol are expected to shut down C. albicans biofilm formation and virulence.