A role for the PhoP/Q regulon in inhibition of fusion between lysosomes and Salmonella-containing vacuoles in macrophages

After uptake by murine macrophages, Salmonella typhimurium is able to survi ve and replicate within specialized phagosomes called Salmonella-containing vacuoles (SCVs), which are segregated from the late endocytic pathway. The molecular basis of this process and the virulence factors required are not fully understood. In this study, we used confocal fluorescence microscopy to evaluate interactions between the endocytic pathway of the murine macrop hage cell line RAW 264.7 and different S. typhimurium strains. The analysis was carried out using the fluid-phase marker Texas red-ovalbumin and antib odies against the lysosomal enzyme cathepsin D, the late endosomal lipid ly sobisphosphatidic acid and the adaptor proteins AP-1 and AP-3. Less than 10 % of wild-type SCVs were associated with these markers at 24 h after uptake by macrophages. A similar low level of association was observed for vacuol es containing mutant strains affected in the function of the Salmonella pat hogenicity island (SPI)-2 type III secretion system or the virulence plasmi d spv operon. However, at this time point, the proportion of vacuoles conta ining phoP(-) mutant bacteria that were associated with each of the markers ranged from 25% to 50%. These results show that the regulon controlled by the PhoP/Q two-component system makes a major contribution to trafficking o f the SCV in macrophages. Segregation of SCVs from the endocytic pathway wa s also found to be dependent on bacterial proteins synthesized between 15 m in and 4 h after uptake into macrophages. However, after this time, protein synthesis was not required to maintain the segregation of SCVs from late e ndosomes and lysosomes.