Abstract Description

Cryptococcus neoformans is one of the few pathogens that can breach the neuroprotective blood-brain barrier (BBB) to reach the brain. Thus, this paper aimed to elucidate the role of the cryptococcal proteolytic enzyme, yeast kexin protease (Kex2p), in disrupting the integrity of the brain endothelial cells that line the BBB by cleaving the tight junction protein, occludin. First, the cryptococcal recombinant Kex2p was isolated from the lysate of E. coli BL 21 (DE 3) cells. Following this, functional studies were then performed using the heterologously produced Kex2p to test if: 1) Kex2p could cleave a 20-mer mimetic peptide based on the occludin, and 2) overlying an artificial BBB made of b.End5 endothelial cells with Kex2p compromise the BBB by assessing sodium fluorescein (NaFl) permeability and TEER measurements. The HADDOCK programme was also used to predict the binding modes and energies of the Kex2p and occludin complex. For this DOCKing experiment, furin was also used as a control serine protease. It was determined that Kex2p could cleave the occludin with biochemical efficiency similar to that of furin (p > 0.05), and lead to increased NaFl penetration and a decrease in TEER, suggesting exposure of b.End5 cells to Kex2p could have resulted in occludin hydrolysis. This was supported by the HADDOCK results, wherein it was determined that the Kex2p-occludin had a more favourable score of -103.4 +/- 10.9 and RMSD of 7.3 +/- 0.3 than the furin-occludin complex, which had a score of -92.3 +/- 1.1 and RMSD of 1.1 +/- 0.7. These DOCKing results confirm that Kex2p may be a suitable ligand to interact with occludin. Taken together, these findings highlight Kex2p as an important microbial factor that may be targeted to limit cells from reaching the brain. A future study should now consider generating a cryptococcal kex2Δ mutant strain and a corresponding reconstituted strain, then assess the efficiency of these two strains to traverse an artificial BBB relative to the wildtype strain.