A Fungal pH-Responsive Signaling Pathway Regulating Aspergillus Adaptation and Invasion into the Cornea

Fungi of the genus Aspergillus are epidemiologically important. These ascomycetes were among the first recognized etiologies of corneal infection and remain common isolates from oculomycoses.1,2 Reflecting diverse ecologic niches and risk factors, the relative prevalence of Aspergillus among patients with fungal keratitis varies widely from region to region.3–11 Aspergillus fumigatus and Aspergillus flavus are established ophthalmic pathogens, and species such as Aspergillus nidulans are emerging causes of fungal keratitis.9,12–14 The antifungal treatment of Aspergillus keratitis has been studied in animal models,15–17 but few investigations have examined the cellular and molecular pathogenesis of corneal aspergillosis.18 Recent knowledge gained from research into systemic and mucosal mycoses indicate that fungal infections involve adaptive interactions between host and pathogen.19–21 To infect tissue, fungi adjust to local conditions such as ambient pH, temperature, and nutrient supply. Aspergillus and other fungi that are pathogenic for the eye have the capabilities of colonizing and invading the injured ocular surface through mechanisms that enable growth and survival. Metabolic systems shared by several fungi include a pathway that detects and responds to extracellular pH. Known as the Pal/PacC pathway in Aspergillus sp. and the RIM101 pathway in Candida sp.,22 this process consists of cell-surface receptors and an intracellular cascade. Gene products involved in pH perception, signal transduction, and molecular processing include the fungal sensors PalI/Rim9p and PalH/Rim21p; the intracellular proteins PalF/Rim8p, PalC, and PalA/Rim20p; a calpainlike protease PalB/Rim13p; and the transcription factor PacC/Rim101p. PalB-mediated proteolytic cleavage of the PacC zinc-finger–containing transcription factor results in a functional subunit that regulates gene expression of phosphatases, proteases, and other enzymes.23–25 The Pal/PacC pathway affects fungal pathogenicity. Strains of Fusarium oxysporum, Colletotrichum acutatum, and Sclerotinia sclerotiorum that contain disruptive mutations in homologues of the pacC gene have significantly altered virulence for plants and, in the case of F. oxysporum, for mice.26–28 The Pal/PacC pathway also plays an operative part in the production of experimental aspergillosis.29 Furthermore, studies of C. albicans infections have shown the orthologous RIM101 pathway to be pivotal in candidal infection of mucous membranes and the cornea.30–32 This conserved pathway influences virulence by several fungi of clinical importance. In this study, we evaluated the role of the Pal/PacC pathway in experimental Aspergillus corneal infection. We used A. nidulans to dissect the molecular intermediates affecting fungal growth and virulence, because mutant construction and complementation is relatively easier than with other Aspergillus species.25,33,34 However, since related fungi such as Aspergillus niger and A. fumigatus encode PacC and other intermediates of the Pal/PacC pathway, studies with A. nidulans can exemplify the virulence mechanisms of Aspergillus species.35,36 We used ex vivo models of corneal wounding and infection that allowed an economical, reproducible method of producing fungal infection of porcine and human corneal tissues.37,38 Prototrophic fungal strains with palB or pacC loss-of-function mutations and their isogenic reconstituted cognates were topically applied to the epithelial surface of injured corneas. Our findings indicate that PalB-mediated signaling and PacC-regulated genetic expression affect fungal invasion and pathogenicity during the onset of Aspergillus infection of cornea.