The combinatorial action of hyphal growth and candidalysin is critical for promoting <i>Candida albicans</i> oropharyngeal infection

<i>Candida albicans</i> is one of the most common fungal pathogens, yet much remains unknown about how its virulence factors cooperate to promote pathogenicity. To investigate this, CRISPR-Cas9 technology was used to create a panel of 19 single, double, triple, and quadruple deletion mutant strains targeting four established virulence factors: <i>ALS3</i> (adhesin/invasin), <i>ECE1</i> (candidalysin toxin), <i>HGC1</i> (hypha formation regulator), and <i>SAP2</i> (protease). <i>In vitro</i>, the deletion of each gene had differing impacts across multiple characterization assays. The <i>hgc1</i>∆/∆ mutant was unable to form hyphae under inducing conditions, leading to downstream impairment of epithelial invasion. The <i>als3</i>∆/∆ mutant exhibited significantly reduced adhesion and invasion into epithelial cells, resulting in attenuated cellular damage. The <i>ece1</i>∆/∆ mutant displayed significantly reduced epithelial damage, cell signaling, and immune activation. The phenotype of the <i>sap2</i>∆/∆ mutant resembled that of wild type but was unable to degrade protein. In an immunocompromised murine model of oropharyngeal infection, hyphal growth and candidalysin production were the dominant drivers of elevated fungal burden, innate immune responses, and mortality. Following a 5-day infection with <i>hgc1</i>∆/∆ and <i>ece1</i>∆/∆ single gene deletion strains, mice had survival rates of 100% and 80%, respectively, compared to 15% in wild-type infected mice. Notably, 100% survival was also observed following challenge with all <i>hgc1</i>∆/∆ and <i>ece1</i>∆/∆ combination mutants. This study demonstrates that specific <i>C. albicans</i> virulence attributes act in combination to promote mucosal infection, with hyphal growth and candidalysin production being a critical driver of oropharyngeal infection.IMPORTANCE<i>Candida albicans</i> has been classified by the WHO as a "critical priority" pathogen, highlighting the urgent need for a greater understanding of the mechanisms that enable it to cause disease. <i>C. albicans</i> possesses numerous virulence attributes, but how they synergize during infection is not well understood. Here, using reverse genetics, we dissect the individual and combinatorial roles of four <i>C. albicans</i> virulence factors (Als3p, candidalysin, hyphal growth, and Sap2p) <i>in vitro</i> and in an <i>in vivo</i> murine model of oropharyngeal candidiasis. Increasing the number of <i>C. albicans</i> gene deletions correlated with reduced oral fungal burden, with hyphal growth and candidalysin together being critical for infection, inflammation, and mortality during oropharyngeal infection. These findings demonstrate that virulence attributes act cooperatively as a collective network to promote pathogenicity, a finding also observed in plant fungal pathogens. Our approach has identified specific fungal virulence factors that can be targeted for new treatment strategies against <i>C. albicans</i> infections.