Disruption of the Salmonella-containing vacuole leads to increased replication of Salmonella enterica serovar typhimurium in the cytosol of epithelial cells

Abstract

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that inhabits a vacuolar compartment, called the Salmonella-containing vacuole (SCV), in infected host cells. Maintenance of the SCV is accomplished by SifA, and mutants of this Salmonella pathogenicity island 2 type III effector replicate more efficiently in epithelial cells. Here we demonstrate that enhanced replication of sifA mutants occurs in the cytosol of these cells. Increased replication of wild-type bacteria was also observed in cells treated with wortmannin or expressing Rab5 Q79L or Rab7 N125I, all of which caused a loss of SCV integrity. Our findings demonstrate the requirement of the host cell endosomal system for maintenance of the SCV and that loss of this compartment allows increased replication of serovar Typhimurium in the cytosol of epithelial cells.

FIG. 1.

Replication of serovar Typhimurium in the cytosol of HeLa epithelial cells. (A) HeLa cells were infected for 10 h with either wild-type or sifA serovar Typhimurium SL1344, as indicated, and then fixed with 2.5% paraformaldehyde. Cells were coimmunostained for LAMP-1 and Salmonella LPS, as indicated, and processed for confocal microscopy (5). Arrowheads indicate LAMP-1⁺ bacteria within Salmonella-containing vacuoles, while arrows indicate the elongated and swollen shape of LAMP-1⁻ bacteria which are present in the cytosol and undergoing replication. (B) Detection of phoP expression. HeLa cells were infected by sifA serovar Typhimurium SL1344 with a plasmid encoding the destabilized mutant of GFP driven by the phoP promoter for 10 h, and then they were processed for confocal microscopy, as above. Arrowheads indicate LAMP-1⁺ bacteria expressing high levels of GFP, while arrows indicate LAMP-1⁻ bacteria which are present in the cytosol and do not express GFP. Size bar, 10 μm.

FIG. 2.

Time lapse confocal microscopy of serovar Typhimurium replication in infected epithelial cells. HeLa cells were grown in glass chambers (Nunc) and then infected for 6 h with either the wild type or sifA serovar Typhimurium SL1344 as indicated. The medium was then replaced with HEPES-buffered growth medium, and cells were loaded with 100 nM Lysotracker DND-99 (Molecular Probes) to label acidified compartments (in red). Chambers were mounted onto an inverted Zeiss microscope with a heated incubation chamber at 35 to 37°C, and time lapse images were acquired by a Bio-Rad Radiance Plus confocal using the Lasersharp software package. Both wild-type and sifA mutant bacteria contain a plasmid (pFPV25.1) which provides constitutive expression of GFP (in green; yellow in overlap) under the rpsM promoter, as previously described (43). Rapid movement and replication of sifA mutant bacteria in the cytosol of infected cells were readily observed, in contrast to the immobile and nonreplicating bacteria in acidified vacuoles (arrows in left-hand series). Wild-type bacteria replicated in acidified vacuoles (arrows in right-hand series).

FIG. 3.

Effects of wortmannin on maintenance of the Salmonella-containing vacuole. (A) HeLa cells were infected for 6 h with wild-type serovar Typhimurium SL1344 in the presence of 100 nM wortmannin and then fixed with 2.5% paraformaldehyde. Cells were coimmunostained for LAMP-1 and LPS, as indicated, and processed for immunofluorescence microscopy. The arrow indicates LAMP-1⁻ bacteria which are present in the cytosol and undergoing replication. Sif formation was not inhibited by wortmannin treatment; a representative Sif is indicated with an arrowhead. Size bar, 10 μm. (B) Quantitation of intracellular bacteria colocalizing with LAMP-1 for experiments performed as described for panel A. The averages ± standard errors are shown for three separate experiments. ∗, P < 0.001

FIG. 4.

Expression of Rab5 Q79L blocks Sif formation and promotes the release of serovar Typhimurium from the Salmonella-containing vacuole. (A) HeLa cells were transfected with a vector encoding the Q79L mutant of Rab5 fused to GFP prior to infection for 6 h with wild-type serovar Typhimurium SL1344. Cells were then fixed with 2.5% paraformaldehyde, coimmunostained for LAMP-1 and LPS, and processed for immunofluorescence microscopy. Arrowheads indicated swollen endocytic structures that contain LAMP-1, while the arrow indicates cytosolic bacteria lacking LAMP-1 that are undergoing replication. Size bar, 10 μm. (B) HeLa cells were transfected with a vector encoding either the enhanced GFP alone or its fusion to the indicated Rab GTPase. Transfected cells were infected for 6 h with wild-type serovar Typhimurium and then fixed with 2.5% paraformaldehyde. Fixed cells were coimmunostained for LAMP-1 and LPS, and the number of transfected cells with large clusters of LAMP-1⁻ bacteria, as demonstrated for panel A, was determined. The averages ± standard errors are shown for three separate experiments. ∗, P < 0.001.