Human corneal epithelial cells internalize Aspergillus flavus spores by actin mediated endocytosis

Human corneal epithelial cells (HCE) play a significant role in the innate immune response by secreting cytokines and antimicrobial peptides when they encounter fungal pathogens. But the detailed mechanism of attachment and engulfment of the fungal conidia by HCE cells is not well understood. Here, we show the phagocytosis of A. flavus conidia by the RCB2280 cells and primary HCE cultures using confocal microscopy and proteomic analysis of conidia containing phagosomes. Phalloidin staining showed actin polymerization, leading to an actin ring around engulfed conidia. Cytochalasin D, inhibited the actin mediated endocytosis of the conidia. Immunolabeling of early endosomal markers CD71 and EEA1, and late endosomal markers LAMP1, Rab7, and Cathepsin G showed that endosomal proteins were recruited to the site of conidia, and maturation of the conidia containing phagosomes. Lysotracker red-DND 99 labelling showed the acidification of the phagosomes containing conidia. Phagosome specific proteome analysis confirmed the recruitment of various phagosomal and endosomal proteins to the conidia containing phagosomes. These results show that the ocular surface epithelium contributes actively to anti-fungal defense by phagocytosis of invading fungal conidia.