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Intracellular trafficking of opsonized and unopsonized Pseudomonas aeruginosa in human monocyte-derived macrophages

University of British Columbia

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.

Thesis/Dissertation

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2009-03-03

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http://hdl.handle.net/2429/5379

Microbiology and Immunology

University of British Columbia

UBCV

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