Abstract Description

Infectious diseases typically involve a single pathogen colonising and harming its host. However, in clinical settings, co-infections also occur involving multiple pathogens. In this contribution, we studied the potential interaction between Cryptococcus (C.) neoformans and the influenza virus by investigating whether the cryptococcal yeast kexin protease (Kex2p) could activate the latent fusogenic haemagglutinin glycoprotein.HADDOCK was used to dock the haemagglutinin and Kex2p. A 6x His-tagged truncated gene encoding Kex2p was first expressed in E. coli BL 21 (DE3) cells, and the resultant recombinant protein was purified using nickel ion affinity chromatography, and the protein separation pattern was analysed using a sulphate-polyacrylamide gel electrophoresis. To confirm successful isolation, the recombinant protein was immunodetected using an anti-His tag primary antibody and mass spectrometry. The heterologously produced Kex2p was then used in a biochemical peptide cleavage assay where a fluorogenic 12-mer peptide resembling haemagglutinin at the HA1/HA2 site served as the substrate. The furin protease was used as a reference in all experiments because it is a cellular protease that activates haemagglutinin in infected humans. The in silico comparative analysis revealed that Kex2p was a suitable ligand to activate the latent haemagglutinin based on the calculated Kex2p-haemagglutinin HADDOCK and RMSD scores. When considering the peptide cleavage assay results, it was noted that the recombinant Kex2p was more biochemically efficient (p < 0.05) than recombinant furin in cleaving HA. Taken together, these findings represent the first empirical data that implicate C. neoformans Kex2p in the activation of the latent influenza virus.