Identification of Proteases: Carboxypeptidase and Aminopeptidase as Putative Virulence Factors of Fusarium solani Species Complex

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Abstract
Background: Fusarium keratitis accounts for around 50% of mycotic keratitis cases. Major virulence factors produced by keratopathogenic fungi are proteases. Objective: The aim of the current study was to identify proteases contributing to corneal pathogenicity of Fusarium species. Methods: Culture filtrates from fourteen Fusarium solani species complex (FSSC) isolates and three F. delphinoides isolates were evaluated for protease activity and gelatine zymography. Mass spectroscopy was carried out using a partially purified enzyme and total extracellular extract. Protease gene expression in an in-vitro condition and an ex-vivo goat corneal infection model was measured using qRT-PCR. Specific activity was observed in a wide range and at a broad pH range; and isolates Cs1 (maximum) and Cc50 (minimum) were selected for the infection model. Results: Gene expression in in-vitro condition showed the highest fold change for proteases (C7YY94, C7Z7U2 and C7Z6W1) while in an ex-vivo infection highest fold change was seen for proteases (C7Z6W1, C7YQJ2 and C7Z7U2); in decreasing order, respectively. Expression of aminopeptidase (C7Z6W1) was 50-fold higher in the infected cornea in both isolates (Cs1 and Cc50); while expression of carboxypeptidase (C7YVF3) was 15-fold higher only in isolate Cs1. Corneal histology showed less penetration of Cc50 than Cs1 into the stroma. Mass spectrometry showed the presence of carboxypeptidase (C7YVF3) and tripeptidyl amino peptidase. Conclusion: It can be concluded that clinical isolates of FSSC produce varying amounts of proteases and differ in specific activity and gene expression in both conditions (in vitro and ex vivo). Carboxypeptidase and aminopeptidase contribute to the pathogenic potential of Fusarium solani species complex.

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