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

Nature Methods volume 7, pages 325–330 (2010)Cite this article

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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.

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Figure 1: Split-GFP can be used to detect Salmonella effectors upon translocation into host cells.
Figure 2: Salmonella effectors SteA and SteC tagged with GFP11 efficiently detected in host HeLa cells upon translocation by T3SS.
Figure 3: Time-lapse microscopy of PipB2 GFPcomp signal revealed highly dynamic tubules in Salmonella-infected host cells.
Figure 4: PipB2 GFPcomp tubules did not always localize with endocytic markers.
Figure 5: Tagged Salmonella effectors localize with the trans-Golgi and the effector SteA is selectively segregated from the endocytic system.
Figure 6: FRAP analysis of a representative PipB2 GFPcomp tubule shows directional recovery along a preformed tubule.

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Acknowledgements

We thank C. Detweiler (University of Colorado) for careful review of the manuscript and for providing Salmonella strains. Plasmids pKD46 pKD3, and pKD4 were obtained from B. Wanner (Purdue University) by means of C. Detweiler (University of Colorado). We acknowledge the Creative Training in Molecular Biology grant (US National Institutes of Health 5 T32GM07135-33) and the University of Colorado for financial support.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, USA

    Schuyler B Van Engelenburg & Amy E Palmer

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  1. Schuyler B Van Engelenburg
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  2. Amy E Palmer
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Contributions

S.B.V. and A.E.P. designed research; S.B.V. performed research; S.B.V. and A.E.P. analyzed data; S.B.V. and A.E.P. wrote the manuscript.

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Correspondence to Amy E Palmer.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 1213 kb)

Supplementary Movie 1

Ectopically expressed PipB2 GFPGFPcomp in the absence of all Salmonella effectors shows distinct dynamics within HeLa cells when compared to T3SS introduced PipB2 GFPcomp. HeLa cells were transiently transfected with PipB2-GFP11 and GFP1–10. PipB2 GFPcomp shows a steady state distribution at the cell periphery and perinuclear region. Image sequence was acquired 48 h after transfection. Images were acquired every 15 s and movie displayed at 10 FPS. Scale bar, 10 μm. (MOV 177 kb)

Supplementary Movie 2

PipB2 GFPcomp protein displays dynamic behavior in the context of the entire effector subset. PipB2-GFP11 SL1344 Salmonella-infected HeLa cell transiently expressing GFP1–10. PipB2 GFPcomp localizes with the SCV (center) and labels highly dynamic tubules protruding from this compartment. Images were acquired at 16 h after infection (MOI = 50). Images were acquired every 10 s and movie displayed at 10 FPS. Scale bar, 10 μm. (MOV 1090 kb)

Supplementary Movie 3

PipB2 GFPcomp dynamics are recapitulated in Salmonella-infected RAW264.7 macrophage-like cells. RAW264.7 cells were transduced with GFP1–10 and infected with PipB2-GFP11 expressing Salmonella. PipB2 GFPcomp (green) localized with intracellular Salmonella (red) and labels dynamic tubules. Images were acquired at 14 h after infection and 48 h after transduction (MOI = 20). Images were acquired every 10 s and movie displayed at 12.5 FPS. Scale bar, 10 μm. (MOV 111 kb)

Supplementary Movie 4

PipB2 GFPcomp highlights transient and destabilized tubules in the absence of the T3SS effector SifA. GFP1–10 expressing HeLa cells were infected with PipB2-GFP11 SL1344 Salmonella null for sifA. Image sequence was acquired at 16 h after infection (MOI = 50). Images were acquired every 10 s and movie displayed at 7 FPS. Scale bar, 10 μm. (MOV 902 kb)

Supplementary Movie 5

PipB2 GFPcomp displays dynamic behavior that is not always recapitulated by the fluid-phase endocytic tracer Alexa Fluor 595–dextran. GFP1–10 expressing HeLa cells infected with PipB2-GFP11 SL1344 Salmonella (18 h after infection; MOI = 50) were pulsed with Alexa Fluor 595–extran for 24 h to label all endocytic compartments. Notice that there is very little movement of the dextran-positive vesicles in the infected cell whereas there is rapid movement in the non-infected cells. We believe this is a consequence of the fact that Salmonella seizes membrane/endocytic cargo as an infection proceeds, which gives rise to a reduction in the number of endocytic vesicles and a reduction in the motility of Salmonella-induced tubules/endocytic membranes as has been previously reported8,22. Alexa Fluor 595–dextran tracer (top, red) and PipB2 GFPcomp (bottom, green). Images were acquired every 10 s and movie displayed at 30 FPS. (MOV 1142 kb)

Supplementary Movie 6

Tubulated PipB2 GFPcomp tubules are observed which do not localize with the late-endosomal/lysosomal marker LAMP1-mCherry. PipB2-GFP11 SL1344 infected HeLa cells transiently expressing GFP1–10 and LAMP1-mCherry were imaged 16 h after infection. PipB2 GFPcomp (top, green) and LAMP1-mCherry (bottom, magenta). Images were acquired every 15 s and movie displayed at 30 FPS. (MOV 1798 kb)

Supplementary Movie 7

SteA GFPcomp tubules exclude the late-endosomal/lysosomal marker LAMP1-mCherry. GFP1–10 and LAMP1-mCherry expressing HeLa cells infected with SteA-GFP11 SL1344 were imaged at 14 h after infection (MOI = 50). LAMP1-mCherry (magenta), SteA GFPcomp (green), and overlay (white). Images were acquired every 10 s and movie displayed at 10 FPS. Scale bar, 10 μm. (MOV 155 kb)

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Van Engelenburg, S., Palmer, A. Imaging type-III secretion reveals dynamics and spatial segregation of Salmonella effectors. Nat Methods 7, 325–330 (2010). https://doi.org/10.1038/nmeth.1437

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