INTERNALIZED LISTERIA-MONOCYTOGENES MODULATES INTRACELLULAR TRAFFICKING AND DELAYS MATURATION OF THE PHAGOSOME

Previous studies have shown that early phagosome-endosome fusion event s following phagocytosis of Listeria monocytogenes are modulated by th e live organism, In the present study, we have characterized more full y the intracellular pathway of dead and live Listeria phagosomes. To e xamine access of endosomal and lysosomal markers to phagosomes contain ing live and dead Listeria, quantitative electron microscopy was carri ed out with intact cells using internalized BSA-gold as a marker to qu antify transfer of solute from endosomal and lysosomal compartments to phagosomes, To monitor the protein composition of phagosomal membrane s and to quantify transfer of HRP from endosomes and lysosomes to phag osomes, highly enriched phagosomes containing live and dead Listeria w ere isolated, Enriched phagosomal membranes were used for western blot ting experiments with endosomal and lysosomal markers, In this study, we used a listeriolysin-deficient mutant, Listeria(hly-), that is reta ined within the phagosome following phagocytosis, Western blotting exp eriments indicate that early endosomal markers (mannose receptor, tran sferrin receptor) and key fusion factors necessary for early events (N SF, alpha/beta-SNAP) but not late endosomal markers (cation dependent mannose 6-phosphate receptor) or lysosomal proteins (cathepsin D or la mp-1) accumulate on the live-Listeria phagosomal membranes, On the con trary, phagosomes containing dead-Listeria are readily accessible by b oth endocytic and lysosomal markers, Studies with radiolabeled dead- a nd live-Listeria(hly-) indicate that, following phagocytosis, degradat ion of the live microorganism is substantially delayed, These findings indicate that dead-Listeria containing phagosomes rapidly mature to a phagolysosomal stage whereas live-Listeria(hly-) prevents maturation, in part, by avoiding fusion with lysosomes, The data suggest that by delaying phagosome maturation and subsequent degradation, Listeria pro longs survival inside the phagosome/endosome assuring bacterial viabil ity as a prelude to escape into the cytoplasm.