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Expression of immune response genes in human corneal epithelial cells interacting with Asperrgillus flavus conidia

ABSTRACT: Human corneal epithelial cells internalize Aspergillus flavus conidia. But the biochemical response of corneal epithelial cells in response to A.flavus is not clear. In this study, NanoString analysis was used to study the immune genes expression in primary corneal epithelial cells and cell line infected with A. flavus conidia. The study revealed the differential expression of immune genes associated with TNF, Th17, NF-kB signaling and B cell receptor signaling in corneal epithelial cells. There is also difference in the response between cell line and primary cells to A. flavus implies the use of both cells to understand the host-pathogen interaction.

ORGANISM(S): Homo sapiens

PROVIDER: GSE175824 | GEO | 2021/06/01

REPOSITORIES: GEO

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Project description:A. flavus keratitis is one of the predominant fungal infections affecting the tropical parts of the world. Melanin is a virulence factor in numerous pathogenic fungi and the melanin layer maintains the cell wall structure and provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile and virulence of melanized and non-melanized conidia of A. flavus. An environmental isolate and two clinical isolates were included in this study. L-DOPA melanin pathway-specific inhibitor kojic acid (KA) as used to prepare non-melanized conidia. Conidial surface proteins were extracted using 100% formic acid at 0 ºC and the extracted proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Formic acid extracts were analyzed in an Orbitrap Velos pro-mass spectrometer. Conidial surface morphology difference was examined using scanning electron microscopy (SEM). Furthermore, lactophenol and calcofluor staining were performed to check the morphology and staining efficiency of melanized conidia (MC) and non-melanized conidia (NMC). A Galleria mellonella insect model was used to examine the virulence efficiency of MC and NMC. Kojic acid treatment inhibited melanin synthesis in A. flavus and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathway were found only in the formic acid extracts of NMC. SEM analysis showed the conidial surface morphology difference between the NMC and MC indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining of MC and NMC. Evaluation of virulence of MC and NMC in G. mellonella model showed NMC was less virulent compared to MC . Our results showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer and hence melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. G. mellonella in vivo virulence assay also confirmed that the non-melanized conidia were susceptible to host defense as in other Aspergillus pathogens.

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Expression of immune response genes in human corneal epithelial cells interacting with Asperrgillus flavus conidia

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