Abstract Listeria monocytogenes is a Gram-positive pathogenic bacterium that causes listeriosis. We used the related non-pathogenic species Listeria innocua, which is often used as a model organism for L. monocytogenes. Bacteria grow in the form of a biofilm and resist different types of stress, and plays an important role in the survival of bacteria in the environment. Listeria biofilms are usually described as monolayers in the literature. In our work, we observed that they also form bilayer and multilayer pyramid-like structures. Fungal proteins represent a relatively unexplored group of proteins with unique properties. These include peptidase inhibitors and lectins, including cocaprin and macrocypin. Cocaprin (KKP1) is a small unexplored protein isolated from Coprinopsis cinerea that acts as an inhibitor of cysteine and aspartic peptidases. Macrocypin (Mcp1) is an inhibitor of cysteine peptidases from Macrolepiota procera. The recombinant proteins Mcp1, KKP1 and four KKP1 mutants (KKP1-N22R, KKP1-D47R, KKP1-FH32EE, KKP1-G13E) were produced in a bacterial expression system. By measuring the inhibitory activity for the enzyme papain, we confirmed the activity for macrocypin, and using the enzymes papain and pepsin, we analysed the inhibitory activity of KKP1 and its mutants. Among them, the mutant KKP1-N22R stood out, in which we successfully changed the reactive site for papain inhibition. This suggests that cocaprin has separate sites for inhibition of cysteine and aspartic peptidases. Native polyacrylamide gel electrophoresis analysis showed that KKP1 and its mutants form oligomers. We investigated the effect of isolated fungal proteins on the biofilm of L. innocua, for which we had previously successfully produced bacteria expressing different reporter proteins, of which DsRED Express proved to be the best. We also tested various markers and dyes for the detection of wild-type L. innocua. The commercial dye Mycolight Red, which binds to bacterial DNA, proved most useful. When investigating whether selected fungal proteins affect L. innocua biofilm development, we found that both cocaprin and macrocypin caused decrased adhesion of bacteria to the substrate. However, we also observed a visible effect of cocaprin on the degradation of static biofilms and the effect of macrocypin on the degradation of dynamic biofilms. Bacterial adhesion to the substrate and degradation of the mature static biofilm were more affected by the three mutants KKP1-N22R, KKP1-FH32EE, and KKP1-G13E than by KKP1. These altered amino acid residues are located on the same side of KKP1, suggesting that there is a site important for binding to L. innocua bacteria. Both cocaprin and macrocypin affect the formation of L. innocua biofilm, so it is worth analysing their effect on the pathogenic species L. monocytogenes, which is a major health problem. If the effect of selected fungal proteins on biofilm formation of pathogenic bacteria were confirmed, they could be used for further studies and development of new antimicrobial agents targeting biofilms or even drugs to treat listeriosis infections.
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