Blocking the shikimate pathway amplifies the impact of carvacrol on biofilm formation in <i>Candida albicans</i>

<i>Candida albicans</i> typically thrives in a commensal relationship with humans but is also an opportunistic fungal pathogen. As an opportunistic pathogen, <i>C. albicans</i> 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 <i>C. albicans</i> growth and pathogenicity. <i>C. albicans</i> biosynthesizes aromatic amino acids and co-enzyme Q <i>de novo</i> 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, <i>ARO2</i>, and <i>ARO7</i> strains were inhibited by 150 µg/mL carvacrol, whereas the <i>ARO1</i> 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 <i>ARO1</i> 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 <i>C. albicans</i> to <i>ARO1</i> repression, with attenuated adhesion, hyphal formation, mycelial growth and biofilm formation, likely by blocking aromatic amino acid uptake.IMPORTANCEThe opportunistic pathogen <i>Candida albicans</i> 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 <i>ARO1</i> repression, and disrupting aromatic amino acid uptake by carvacrol prevent <i>C. albicans</i> biofilm formation. Thus, inhibitors of the Aro1 enzyme in combination with carvacrol are expected to shut down <i>C. albicans</i> biofilm formation and virulence.