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

Abstract

After uptake by murine macrophages, Salmonella typhimurium is able to survive 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 macrophage cell line RAW 264.7 and different S. typhimurium strains. The analysis was carried out using the fluid-phase marker Texas red-ovalbumin and antibodies against the lysosomal enzyme cathepsin D, the late endosomal lipid lysobisphosphatidic 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 vacuoles containing mutant strains affected in the function of the Salmonella pathogenicity island (SPI)-2 type III secretion system of the virulence plasmid spv operon. However, at this time point, the proportion of vacuoles containing 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 of the SCV in macrophages. Segregation of SCVs from the endocytic pathway was also found to be dependent on bacterial proteins synthesized between 15 min and 4 h after uptake into macrophages. However, after this time, protein synthesis was not required to maintain the segregation of SCVs from late endosomes and lysosomes.