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Phagolysosomal
Fusion In Striped Bass (Morone saxatilis) Macrophages Infected With Mycobacterium
marinumDavid 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.
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