Imaging type-III secretion reveals dynamics and spatial segregation of Salmonella effectors

Abstract

The type-III secretion system (T3SS) enables gram-negative bacteria to inject effector proteins into eukaryotic host cells. Upon entry, T3SS effectors work cooperatively to reprogram host cells, enabling bacterial survival. Progress in understanding when and where effectors localize in host cells has been hindered by a dearth of tools to study these proteins in the native cellular environment. We report a method to label and track T3SS effectors during infection using a split-GFP system. We demonstrate this technique by labeling three effectors from Salmonella enterica (PipB2, SteA and SteC) and characterizing their localization in host cells. PipB2 displayed highly dynamic behavior on tubules emanating from the Salmonella-containing vacuole labeled with both endo- and exocytic markers. SteA was preferentially enriched on tubules localizing with Golgi markers. This segregation suggests that effector targeting and localization may have a functional role during infection.

Figure 1

Split-GFP can be used to detect Salmonella effectors upon translocation into host cells. (a) General strategy for detecting T3SS delivered effectors in live cells. (b) PipB2-GFP11 is detected in HeLa cells 16 hours post-infection and co-localizes with mCherry expressing Salmonella, (arrows and overlay) (MOI = 50). (c) Average distance of SCVs from infected host cell nuclei Error bars represent s.e.m. of three independent experiments (n ≥ 100 SCVs per replicate; P < 0.0001, ns = not significant) (d) Transfected PipB2-GFP_comp colocalizes with kinesin-1 by immunofluorescence in HeLa cells (arrows). Scale bars represent 10 μm.

Figure 2

Salmonella effectors SteA and SteC tagged with GFP11 are efficiently detected in host HeLa cells upon translocation by T3SS. (a) SteA-GFP_comp partially localizes with tubulated trans-Golgi membranes (GalT-mCherry). (b) SteC-GFP_comp co-localizes with coumarin-phalloidin labeled actin foci and the SCV. Both proteins were imaged at 16 hours post-infection with an MOI = 50. Scale bars represent 10 μm.

Figure 3

Time-lapse microscopy of PipB2-GFP_comp reveals highly dynamic tubules in Salmonella infected host cells. (a) HeLa cells displaying PipB2-GFP_comp (green) and Salmonella (red) overlay. Time-lapse microscopy 16 hours post-infection (MOI = 50) shows a PipB2-GFP_comp tubule (white) emanate from the SCV with multidirectional movement, bifurcation, and temporary lariat formation. (b) Highly dynamic PipB2-GFP_comp tubules are also observed in macrophage-like RAW264.7 cells (abbreviated Mϕ) at 14 hours post-infection (MOI = 20). (c) Transient PipB2-GFP_comp tubules in HeLa cells infected for 16 hours with SifA null Salmonella (MOI = 50). The overlay shows PipB2_comp (green) and LAMP1-mCherry (magenta). Scale bars represent 10 μm.

Figure 4

PipB2-GFP_comp tubules do not always co-localize with endocytic markers. (a) Infected HeLa cells (MOI = 50; 14 hours post-infection) displaying PipB2-GFP_comp and LAMP1-mCherry 48 hours post-transfection. Time-lapse microscopy reveals a nascent PipB2-GFP11 tubule which excludes the late-endosomal/lysosomal marker LAMP1-mCherry (arrow). (b) Infected HeLa cell displaying PipB2-GFP_comp and the fluid-phase endocytic marker Alexa 568-dextran. Cells were loaded with labeled dextran for 24 hrs and imaged 18 hrs post-infection. PipB2-GFP_comp labels a dynamic tubule which does not co-localize with the dextran endocytic marker (arrowheads). Scale bars represent 10 μm.

Figure 5

Tagged Salmonella effectors co-localize with the trans-Golgi and the effector SteA is selectively segregated from the endocytic system. (a) PipB2-GFP_comp co-localizes with the trans-Golgi marker GalT-mCherry (arrowheads) and highlights tubulation of GalT-mCherry⁺ membranes. (b) SteAGFP_comp co-localizes with LAMP1-mCherry on the SCV, but LAMP1-mCherry is excluded from SteAGFP_comp tubules (arrowheads). (c) Proportion of tubules labeled with PipB2- and SteA-GFP_comp co-localizing with endocytic or trans-Golgi derived membranes. Imaging experiments were performed at 12-14 hours post-infection (MOI = 50). Error bars represent s.d. from 3 independent experiments (n ≥ 20 cells per replicate; P < 0.001). Scale bars represent 10 μm.

Figure 6

Fluorescence recovery after photobleaching (FRAP) analysis of a representative PipB2-GFP_comp tubule. FRAP analysis shows directional recovery along a pre-formed filament. Similar results were obtained in 4 independent experiments. As control, LAMP1-mCherry (Magenta) shows bleached tubules remain static for the duration of the experiment. Image acquisition was performed at 16 hours post-infection (MOI = 50). Scale bar represent 10 μm.