28th ANNUAL EASTERN FISH HEALTH WORKSHOP April 21-25, 2003 Phagolysosomal Fusion In Striped Bass (Morone saxatilis) Macrophages Infected With Mycobacterium marinum David T. Gauthier and Wolfgang K. Vogelbein College of William & Mary, Virginia Institute of Marine Science, 1208 Greate Road, Gloucester Point, VA 23062 Macrophages form the first line of defense against mycobacterial infection in both endotherms and poikilotherms.  Pathogenic mycobacteria are readily phagocytosed by these cells but are not degraded, thus becoming intracellular parasites.  It is widely thought that mycobacteria of mammalian clinical significance, such as M. tuberculosis and M. avium, survive in macrophages by suppressing phagosomal acidification and fusion with lysosomes.  Mycobacterium marinum, a commonly reported pathogen of fish, is also capable of survival within fish macrophages.  In the course of studying in vitro and in vivo interactions between M. marinum and striped bass (Morone saxatilis) macrophages, however, we have frequently observed M. marinum phagosomes that appear to have fused with lysosomes, based on the presence of electron-opaque material within the phagosome.  To more clearly demonstrate phagolysosomal fusion, lysosomes of cultured striped bass macrophages were labeled with both BSA-coated gold beads (Au-BSA) and horseradish peroxidase (HRP).  Macrophages were then pulsed with either live or heat-killed M. marinum and fixed for transmission electron microscopy at 2 to 72 hours post-infection.  Internalized peroxidase was localized ultrastructurally via the diaminobenzidine (DAB) reaction.  Discrete, membrane-limited compartments within macrophages were labeled with HRP and/or Au-BSA.  These compartments were shown to fuse frequently with M. marinum-containing phagosomes.   Between 60% and 90% of M. marinum phagosomes were fused with HRP-containing lysosomes, while between 20% and 50% of mycobacterial phagosomes contained gold beads.  Fusion rates for either HRP or Au-BSA did not appear to differ between phagosomes containing live or heat-killed bacilli.  Both live and heat-killed mycobacteria appeared to be refractory to degradation within fused phagolysosomes for up to three days.  Return to 28th Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page