| Summary: | The intracellular fate of microorganisms ingested by phagocytes may be determined
by the specific receptors mediating their uptake. To test this hypothesis, the subcellular
location of Pseudomonas aeruginosa in human monocyte-derived macrophages upon
phagocytosis via opsonic versus non-opsonic receptors was investigated by indirect double
immunofluorescence. Opsonic ingestion of P. aeruginosa is glucose-independent while non
opsonic phagocytosis of this bacterium by macrophages requires the presence of glucose.
Therefore, opsonic and non-opsonic phagocytosis of P. aeruginosa were differentiated by
presenting immunoglobulin-coated bacteria to macrophages in glucose-free medium and
unopsonized bacteria to the phagocytes in the presence of glucose. Compartments of the
opsonic and non-opsonic phagocytic pathways of P. aeruginosa in human monocyte-derived
macrophages were defined by double-labelling infected macrophages with antibodies specific
for different endocytic compartments (lysosomes, endosomes, etc.) and with polyclonal
antibodies to P. aeruginosa. Both opsonized and unopsonized P. aeruginosa colocalized with
the lysosomal-associated membrane glycoprotein, LAMP-i, which is found predominantly in
lysosomes. Ingested opsonized P. aeruginosa appeared to colocalize with this LAMP-i +
compartment at a faster rate than unopsonized P. aeruginosa. When cells were preloaded with
rhodamine-ovalbumin to label secondary lysosomes, a small fraction of ingested bacteria that
were phagocytosed via the two routes entered these labelled compartments. Brefeldin A, a
drug which inhibits transport of newly synthesized membrane proteins and secretory proteins,
and monensin, an ionophore which inhibits endosome acidification , did not influence the
ingestion and intracellular fate of either opsonized or unopsonized P. aeruginosa. Mannose 6-phosphate receptor (MPR), an antigenic marker enriched in the late endosome, showed little
colocalization with either opsonized or unopsonized P. aeruginosa. These studies suggest that
both opsonized and unopsonized P. aeruginosa enter functionally similar pathways after
phagocytosis by macrophages and that the phagosomes ultimately fuse with LAMP-1
compartments regardless of the receptor mediating the ingestion. Although it is not possible
to determine definitively the stage at which the phagocytosed P. aeruginosa converged with
the endocytic pathway, they appeared to do so at a stage that is distal to the late endosome,
probably with prelysosomes. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate
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