Targeting Exogenous β-Defensin to the Endolysosomal Compartment via a Biotic Guided Missile System

CARVELLI L; MORALES CR; LIBIN Y

San Francisco, California

Congreso; 2016 ASCB Annual Meeting; 2016

American Society for Cell Biology

Viral, bacterial and protozoal infections are a preoccupation with respect to the evolution of virulence and iatrogenic enhancement due to cross-species transmission and antibiotic resistance. A number of bacteria, viruses and protozoa take advantage of receptor-mediated endocytosis, a critical function of eukaryotes, by binding to surface proteins and receptors and by using their endocytic pathway to infect cells. Once in the endosomes and/or phagosomes, the pathogens complete their life cycle by overriding normal lysosomal functions. Most notably, pathogens evade lysosomal degradation by the selective retention of Rab GTPases in the membranes of the vacuoles that they occupy in host cells during infection, inhibiting the fusogenic function and acidification of these organelles and/or the activity of the lysosomal hydrolases. Recently, our laboratory identified the lysosomal targeting signal of prosaposin, which is recognized by the sorting receptor ?sortilin?. Based on this evidence, we tested whether the antimicrobial peptide β-Defensin linked to the targeting sequence of prosaposin (β-Defensin-prosaposin-V5) could be redirected from its secretory pathway to the endolysosomal compartment. To this end, β-Defensin-prosaposin-V5 was transfected into COS-7 cells. The subcellular distribution of β-Defensin-prosaposin-V5 was analyzed by confocal microscopy and differential centrifugation. Confocal microscopy demonstrated that β-Defensin-prosaposin-V5 overlaid with the lysosomal marker LAMP1, indicating that the construct reached endosomes and lysosomes. Differential centrifugation also showed that β-Defensin-prosaposin-V5 was in the lysosomal fractions. In addition, a binding inhibition assay demonstrated that β-Defensin-prosaposin-V5 bound specifically to sortilin. Similarly, the delivery of β-Defensin-prosaposin-V5 was abolished after overexpressing a truncated sortilin. These results indicate that the prosaposin C-terminus and D/C-domain (prosaposin targeting sequence) was an effective biotic ?guidance system? to redirect β-Defensin to the endolysosomal compartment. In the future, this and other fusion proteins with antimicrobial properties will be assembled to our ?biotic missile? to target pathogens growing within these compartments.