Targeting of Salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors

Abstract

Salmonella typhimurium is an intracellular bacterial pathogen that remains enclosed in vacuoles (SCV) upon entry into the host cell. In this study we have examined the intracellular trafficking route of S. typhimurium within epithelial cells. Indirect immunofluorescence analysis showed that bacteria initiated fusion with lysosomal membrane glycoprotein (lgp)-containing compartments ~15 min after bacterial internalization. This process was completed ~75 min later and did not require microtubules. Cation-independent (CI)- or cation-dependent (CD)-mannose 6-phosphate receptors (M6PRs) were not observed at detectable levels in SCV. Lysosomal enzymes showed a different distribution in SCV: lysosomal-acid phosphatase (LAP) was incorporated into these vacuoles with the same kinetics as lgps, while cathepsin D was present in a low proportion (~30%) of SCV. Uptake experiments with fluid endocytic tracers such as fluorescein-dextran sulphate (F-DX) or horseradish-peroxidase (HRP) showed that after 2 h of uptake, F-DX was present in ~75% of lgp- containing vesicles in uninfected cells, while only ~15% of SCV contained small amounts of the tracer during the same uptake period. SCV also showed only partial fusion with HRP-preloaded secondary lysosomes, with ~30% of SCV having detectable amounts of HRP at 6 h after infection. These results indicate that SCV show limited accessibility to fluid endocytic tracers and mature lysosomes, and am therefore functionally separated from the endocytic route. Moreover, the unusual intracellular trafficking route of S. typhimurium inside epithelial cells has allowed us to establish the existence of two different lgp-containing vesicles in Salmonella-infected cells: one population is separated from the endocytic route, fusogenic with incoming SCV and may arise from a secretory pathway, while the second involves the classical secondary or mature lysosomes.