Influence of phagosomal contents on the apparent inhibition of phagosome-lysosome fusion mediated by polyanionic substances in mouse peritoneal macrophages

Abstract

The study of fusion of phagosomes with secondary lysosomes in macrophages is facilitated by assessing transfer of fluorescent or electron-opaque markers (or both) from the lysosomes to the phagosomes. When certain virulent viable pathogens are phagocytosed by mouse peritoneal macrophages, phagosome-lysosome fusion (P-LF) is inhibited. Nonviable counterparts ordinarily cannot impose this block. A similar, but spurious, block to P-LF seems to be mediated from the lysosomal domain following sequestration of certain polyanionic substances. This block has been judged to be relieved by, for example, heat-killed yeasts and various viable bacteria designated as fusion-inducing microorganisms, acting from the phagosome. In this study we tested this concept and believed it to be unfounded. Macrophages labeled with Thorotrast and incubated with dextran sulfate were offered a variety of viable and heat-killed microorganisms for phagocytosis: Saccharomyces cerevisiae, Mycobacterium lepraemurium, Streptococcus faecalis, and Escherichia coli. By electron microscopy, a transfer of Thorotrast to phagosomes up to 18 h was seen to be highly suppressed as compared with controls, but was not notably different for any of the targets, whether viable or not. Instead, inert 0.45-micron carboxylated polystyrene beads (the smallest target) showed the most delivery of marker. If polyanionic agents truly inhibited fusion, then "fusiogenic" microorganisms should free the marker for delivery. If polyanions do not inhibit P-LF and only trap the marker, the behavior of the various targets would correspond to what we found.