Mechanism of action of litopenin PvHCt, a strictly antifungal peptide processed from hemocyanin in a penaeid shrimp

A histidin-rich antifungal peptide, litopenin PvHCt, which corresponds to the 23 amino acid C-terminal sequence of the shrimp respiratory protein hemocyanin, has been previously identified in the plasma of the penaeid shrimp Litopenaeus vannamei. It is generated by proteolytic cleavage in response to a microbial challenge. Similarly, a C-terminal fragment of hemocyanin, astacidin-1, displaying antimicrobial activity had been isolated previously from crayfish plasma. These peptides are believed to contribute to crustacean defence. The phenomenon of in vitro antimicrobial peptide generation from a respiratory pigment, already observed with hemoglobin, thus appears not to be restricted to mammals. Litopenin PvHCt displays a broad spectrum of antifungal activity with minimum inhibitory concentrations (MICs) in the range 3-50 µM (12.5 µM against the shrimp pathogen Fusarium oxysporum). Its activity would be based on the inhibition of spore germination. To contribute to the elucidation of the mechanism of PvHCt antifungal activity, we determined its three-dimensional structure by circular dichroism (CD), NMR and molecular modelling and examined its effects on the F. oxysporum spore ultrastructure by transmission electron microscopy (TEM). We also investigated the PvHCt-induced damages of F. oxysporum plasma membrane, using Sytox Green uptake and subsequent detection by fluorescence microscopy and a liposome permeabilization assay.