Biogenesis of Salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway

Abstract

SummaryIn epithelial cells, the intracellular pathogen Salmonella typhimurium resides and replicates within a unique cytoplasmic organelle, the Salmonella-containing vacuole (SCV). In vitro studies have shown that the SCV is a dynamic organelle that selectively acquires lysosomal glycoproteins (lgps) without fusing directly with lyosomes. Here, we have investigated early events in SCV biogenesis using immuno¯uorescence microscopy and epitope-speci®c¯ow cytometry. We show that proteins speci®c to the early endocytic pathway, EEA1 and transferrin receptor (TR), are present on early SCVs. The association of these proteins with SCVs is transient, and both proteins are undetectable at later time points when lgp and vATPase are acquired. Analysis of the fraction of SCVs containing both TR and lamp-1 showed that TR is lost from SCVs as the lgp is acquired, and that these processes occur progressively and not as the result of a single fusion/ ®ssion event. These experiments reveal a novel mechanism of SCV biogenesis, involving previously undetected initial interactions with the early endocytic pathway followed by the sequential delivery of lgp. The pathway does not involve interactions with the late endosome/prelysosome and is distinct from traditional phagocytic and endocytic pathways. Our study indicates that intracellular S. typhimurium occupies a unique niche, branching away from the traditional endocytic pathway between the early and late endosomal compartments.

Interaction of the Salmonella-containing Vacuole with the Endocytic Recycling System

“…Our data show an interaction of Rab4 with the SCV early in infection (10 min), followed by a loss from the membrane. These data coincide with maturation of the SCV as characterized by loss of early endosome antigen 1 (7). ARF6, also present on the SCV early in infection, is involved in regulating recycling to areas of membrane reorganization (35,36).…”

“…Syntaxin13-EGFP and syntaxin13⌬TM were gifts from Dr. William Trimble (University of Toronto and the Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada) (19). The Gene- Late log bacterial cultures were used for infecting HeLa cells and prepared using a method optimized for bacterial invasion (7). In brief, bacteria were grown for ϳ16 h at 37°C with shaking and then sub-cultured (1:33) in Luria-Bertani broth for 3 h. Bacterial inocula were prepared by pelleting at 10,000 ϫ g for 2 min, directly resuspended, diluted in PBS (pH 7.2), and added to cells at a multiplicity of infection of ϳ100:1 at 37°C for 10 min.…”

Coxiella burnetiiSurvival in THP-1 Monocytes Involves the Impairment of Phagosome Maturation: IFN-γ Mediates its Restoration and Bacterial Killing

“…Thus, the survival of C. burnetii in monocytic cells is associated with altered phagosomal maturation, which is reminiscent of the escape mechanism used by Salmonella enterica. Indeed, both types of pathogen-containing vacuoles acquire markers of late endosomes-early lysosomes such as Lamp-1, but are devoid of lysosomal enzymes (28,29). However, the molecular mechanisms involved in the control of phagosome maturation are likely different.…”

The Salmonella-containing vacuole—Moving with the times

“…By far the best-documented aspect of SCV biogenesis is the sequential delivery of endolysosomal membrane proteins that defines the conversion of early to intermediate SCVs. Thus the early SCV membrane is highly enriched in early endosome membrane markers, including EEA1, Rab5 and transferrin receptor, which are replaced within 20-40 min with late endosomal/lysosomal markers including Lamps and vATPase [7,8]. This change in membrane content is accompanied by a decrease in the luminal pH (pH scv ) to <4.5 [9,10] and redistribution of the SCV to a predominantly juxtanuclear position near the microtubule organizing center (MTOC) [11].…”