The SPI-2 type III secretion system restricts motility of Salmonella-containing vacuoles - PubMed (original) (raw)

The SPI-2 type III secretion system restricts motility of Salmonella-containing vacuoles

Amy E Ramsden et al. Cell Microbiol. 2007 Oct.

Abstract

Intracellular replication of Salmonella enterica occurs in membrane-bound compartments, called Salmonella-containing vacuoles (SCVs). Following invasion of epithelial cells, most SCVs migrate to a perinuclear region and replicate in close association with the Golgi network. The association of SCVs with the Golgi is dependent on the Salmonella-pathogenicity island-2 (SPI-2) type III secretion system (T3SS) effectors SseG, SseF and SifA. However, little is known about the dynamics of SCV movement. Here, we show that in epithelial cells, 2 h were required for migration of the majority of SCVs to within 5 microm from the microtubule organizing centre (MTOC), which is located in the same subcellular region as the Golgi network. This initial SCV migration was saltatory, bidirectional and microtubule-dependent. An intact Golgi, SseG and SPI-2 T3SS were dispensable for SCV migration to the MTOC, but were essential for maintenance of SCVs in that region. Live-cell imaging between 4 and 8 h post invasion revealed that the majority of wild-type SCVs displaced less than 2 microm in 20 min from their initial starting positions. In contrast, between 6 and 8 h post invasion the majority of vacuoles containing sseG, sseF or ssaV mutant bacteria displaced more than 2 microm in 20 min from their initial starting positions, with some undergoing large and dramatic movements. Further analysis of the movement of SCVs revealed that large displacements were a result of increased SCV speed rather than a change in their directionality, and that SseG influences SCV motility by restricting vacuole speed within the MTOC/Golgi region. SseG might function by tethering SCVs to Golgi-associated molecules, or by controlling microtubule motors, for example by inhibiting kinesin recruitment or promoting dynein recruitment.

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Figures

Fig. 1

Migration of SCVs to the MTOC/Golgi region of HeLa cells. A. Representative images of HeLa cells infected with wt S. Typhimurium for 2 h, in the presence or absence of nocodazole or BFA. Cells were fixed in ice-cold methanol and coimmunolabelled for giantin (blue in merged images), γ-tubulin (red in merged images) and S. Typhimurium (green in merged images). Images were acquired by confocal microscopy and represent combined projections of multiple Z-sections. Higher magnification of cells in boxed region. Scale bars, 5 μm. B. Percentage of SCVs within 5 μm of the MTOC in BFA-, nocodazole-treated or untreated HeLa cells. _P_-values are indicated above bars and were obtained by comparing nocodazole- or BFA-treated cells to untreated cells at the same time point.

Fig. 2

Time-lapse video microscopy analysis of the movement of SCVs towards the Golgi in HeLa cells. To allow visualization of the Golgi, HeLa cells were transiently transfected with a vector encoding MannII-EGFP. Transfected cells were infected with wt S. Typhimurium expressing the red fluorescent protein (DsRed). A. Graphical representation of the distance between red fluorescence centroid (Salmonella) and green fluorescence centroid (Golgi) in every frame of six time-lapse sequences; images were taken at 1 min intervals. Each colour represents a single SCV. B. In the upper left panel an SCV trajectory (corresponding to the blue curve in Fig. 2A), is superimposed on the fluorescent and DIC image of the infected HeLa cell. The boxed region is magnified in subsequent panels, which show merged fluorescent images at 30 min intervals (see Supplementary Movie S1). Scale bar, 5 μm. C. Change of speed over time is shown for two distinct SCVs. Data are from time-lapse sequences where images were acquired at 5 s intervals. One SCV (blue line), had an average speed of 0.045 ± 0.004 μm; the other (red line) had an average speed of 0.017 ± 0.001 μm s−1.

Fig. 3

Requirement of the SPI-2 T3SS and effector SseG for SCV association with the MTOC. A. Representative images of HeLa cells infected for 2 h and 8 h with sseG mutant S. Typhimurium. Infected cells were fixed in ice-cold methanol and triple labelled for Salmonella (green in merged images), giantin (blue in merged images) and γ-tubulin (red in merged images). Cells were analysed by confocal microscopy and images represent combined projections of multiple z-sections. Scale bars, 5 μm. B. Percentage of ssaV mutant, sseG mutant and wt S. Typhimurium bacteria within 5 μm from the MTOC over an 8 h time-course. Error bars not apparent if less than 0.1; _P_-values are indicated above bars, compared with corresponding wt values.

Fig. 4

Effect of BFA on SCV association with the MTOC. A. HeLa cells were infected with wt S. Typhimurium and then incubated in the presence or absence of BFA. Cells were then fixed at 8 h post inoculation and coimmunolabelled for γ-tubulin (red in merged images), Salmonella (green in merged images) and giantin (blue in merged images) and analysed by confocal microscopy. Images represent combined projections of multiple z-slices. Scale bars, 5 μm. B. Percentage of bacteria within 5 μm of the MTOC at 4 h, 6 h and 8 h p.i. _P_-values are indicated above bars and are derived from comparisons with corresponding untreated cells.

Fig. 5

Effect of nocodazole on the redistribution of vacuoles containing sseG mutant bacteria. A. Representative images of HeLa cells infected with wt S. Typhimurium, either left untreated or exposed to nocodazole at 2 h p.i., and fixed 6 h later. Cells were labelled for Salmonella (green in merged images), giantin (blue in merged images) and γ-tubulin (red in merged images) and analysed by confocal microscopy. Images represent combined projections of multiple z-slices; scale bars correspond to 5 μm. B. Percentage of sseG mutant bacteria within 5 μm of the MTOC following addition of nocodazole; _P_-value is indicated above bars.

Fig. 6

Live imaging analysis of wt, ssaV and sseG mutant SCVs. A. Percentages of small (less than 2 μm) and large (greater than 2 μm) displacing wt, ssaV and sseG mutant SCVs between 4 h and 6 h p.i., and between 6 h and 8 h p.i. The number of SCVs for each category is indicated above the bar, as is the _P_-value from comparing sseG and ssaV mutant SCVs with wt SCVs within the same time period. B. Trajectories of all vacuoles sampled, containing wt, ssaV and sseG mutant bacteria. SCV trajectories were reoriented to start at x,y = 0 in a standardized grid; small displacing SCVs are in red and large displacing SCVs are in black. C. Images from Supplementary material Movies S2–4. ManII-EGFP-expressing HeLa cells, infected with wt (upper panel), sseG mutant (middle panel) or ssaV mutant (lower panel) DsRed-expressing S. Typhimurium. The first image shows full SCV trajectories superimposed on the merged image at 0 min; the consecutive images are 5 min apart. Scale bar, 5 μm. Images were acquired every min for 20 min.

Fig. 7

Analysis of wt, ssaV and sseG vacuole directionality and speed between 6 h and 8 h p.i. SCVs were imaged every 5 s for 5 min. A. Trajectories of small and large displacing vacuoles containing wt, ssaV and sseG mutant bacteria. Trajectories were reoriented to start at x,y = 0 with the first displacement vector pointing in the same direction (+x, +y) in a standardized grid. The number of SCVs examined and the average cosine are indicated for each category. B. Mean speeds of small and large displacing vacuoles containing wt, ssaV and sseG bacteria. C. Scatter plot in which the speeds of individual 5 s movements of vacuoles containing wt, sseG and ssaV bacteria undergoing large displacements were plotted against the distance of the vacuole from the Golgi centroid at the end of its 5 s movement. Spearman correlation _R_-value and _P_-values are indicated.

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The SPI-2 type III secretion system restricts motility of Salmonella-containing vacuoles - PubMed (original) (raw)