Abstract
Perforin-2 is vital for successful containment and elimination of bacterial infection regardless of infection route or pathogen cell wall composition. Lacking Perforin-2 or having a mutant allele for this pore forming bactericidal protein prevents bacterial clearance in animal models and humans.
Using a pharmacological approach, we confirmed the importance of cytosolic tail ubiquitination as an activator of Perforin-2 trafficking. We demonstrate that treatment of macrophages with MLN4924 disrupts intracellular trafficking of Perforin-2 as evident by the loss of LPS induced aggregation of Perforin-2 in endosomes. Disruption of trafficking also leads to the inhibition of Perforin-2 dependent killing of the intracellular pathogens and mirrors the effect of Cif, a bacterial effector which also disrupts NEDD8 modification.
Following localization to the phagosome, recognition and binding to target membranes is necessary to facilitate pore formation. A region within the P2 domain; the β-hairpin is necessary for target membrane binding. It is hypothesized that the overall positive charge of this fold along with its hydrophobic tip make it ideal for target membrane binding. Here we demonstrate that deletion of the β-hairpin or elimination of its hydrophobic tip greatly diminish bactericidal activity.
Finally, we functionally characterize a clinically relevant human variant responsible for persistent polymicrobial infections and advance our knowledge of a candidate therapy, IFN-γ. Patient-derived phagocytes exhibited impaired bactericidal activity against S. Typhimurium and Y. pseudotuberculosis, a phenotype that likely underpins the patient’s recurrent and non-resolving infections. Structural investigation of the variant revealed the patient’s mutation (Y430*) is a nonsense mutation. Y430* causes the loss of the P2 domain and transmembrane domain. Thus, we hypothesize that the variant polypeptide is secreted to the extracellular milieu, leaving the patient with a haploinsufficiency phenotype.