1. Microbiology and Infectious Disease
Download icon

Visualization of the type III secretion mediated Salmonella–host cell interface using cryo-electron tomography

  1. Donghyun Park
  2. Maria Lara-Tejero
  3. M Neal Waxham
  4. Wenwei Li
  5. Bo Hu
  6. Jorge E Galán
  7. Jun Liu  Is a corresponding author
  1. Yale University School of Medicine, United States
  2. Microbial Sciences Institute, Yale University School of Medicine, United States
  3. McGovern Medical School, The University of Texas Health Science Center at Houston, United States
Research Article
Cite this article as: eLife 2018;7:e39514 doi: 10.7554/eLife.39514
5 figures, 2 videos, 3 tables and 1 additional file

Figures

Figure 1 with 1 supplement
In situ structures of host-free S. Typhimurium T3SS injectisome in wild-type (WT), ΔsipB, and ΔsipD minicells.

(A) A central section of a tomogram showing S. Typhimurium minicell containing multiple injectisomes. (B–D) Central sections of sub-tomogram averages showing injectisomes of WT, ΔsipB, and ΔsipD, respectively. (E) A schematic of the injectisome. Outer membrane (OM), peptidoglycan (PG), sorting platform, and inner membrane (IM) of S. Typhimurium are annotated. (F–H) Central sections of tomograms showing injectisomes from strains expressing epitope-tagged (FLAG) SipB, SipC, and SipD, respectively. Yellow arrow indicates antibody bound to the epitope-tag.

https://doi.org/10.7554/eLife.39514.002
Figure 1—figure supplement 1
Detection of FLAG-epitope-tagged SipB, SipC, and SipD Central slices from representative tomograms showing.

(A) sipB-FLAG, (B) sipC-FLAG, (C) antibody-free sipD-FLAG, and (D) antibody-bound sipD-FLAG needles. (E-H) Sub-tomogram averages of FLAG-epitope-tagged S. Typhimurium strains shown in panels (A-D), respectively. Yellow arrows indicate anti-FLAG antibodies bound to the epitope-tag. (I) Quantification of anti-FLAG antibody bound needles.

https://doi.org/10.7554/eLife.39514.003
Figure 2 with 2 supplements
Visualization of the T3SS mediated Salmonella-Host interactions.

(A) A central slice showing a S. Typhimurium minicell interacting with a host. Plasma membrane (PM) of HeLa cell, outer membrane (OM) and inner membrane (IM) of S. Typhimurium are annotated. (B) 3D rendering of the tomogram shown in (A). (C) Tomographic slices showing injectisomes interacting with the host PM. Blue arrows indicate needles attached to the host PM. Direction of the arrow represents the angle of needle perpendicular to the host PM.

https://doi.org/10.7554/eLife.39514.005
Figure 2—figure supplement 1
Cultivation of mammalian cells (HeLa) on EM grid for cryo-ET.

(A) Phase contrast microscopy image of HeLa cells grown on a gold Quantifoil grid. (B) A zoom-in view of the boxed area in panel a. (C) A snapshot of HeLa cell edge in a low-magnification montage. (D) Tomographic slice of the boxed area in panel C showing cellular features such as actin filaments and microtubules.

https://doi.org/10.7554/eLife.39514.006
Figure 2—figure supplement 2
Inter-membrane spacing between outer membrane and plasma membrane.

(A-C) Central slices of tomograms showing different Salmonella - host cell contacts in the presence of T3SS injectisomes. (D-F) The zoom-in views of the boxed regions in the tomographic slices from panels (A-C) respectively. (G-I) Central slices of tomograms showing different Salmonella - host cell contacts without the presence of T3SS injectisomes. (J-L) The zoom-in views of the boxed regions in the tomographic slices from panels (G-I), respectively. Plasma membrane (PM) of HeLa cell, outer membrane (OM) and cytoplasmic membrane (CM) of S. Typhimurium are annotated. (M) Average membrane spacing between S. Typhimurium minicells and HeLa cells at different positions as indicated across the bottom of the bar graph. Error bars indicate s.e.m. Data were compared using an unpaired t test. (N) A summary of statistical measures including average, standard deviation, and standard error of mean.

https://doi.org/10.7554/eLife.39514.007
In situ structural analysis of the interface between the T3SS needle and the host membrane reveals a novel structure of the intact translocon.

(A) A schematic representation of the S. Typhimurium injectisome with a box highlighting the area used for alignment and classification. (B–E) Central sections and (F–I) 3-D surface views of class averages showing different spacings between the needle and the plasma membrane (PM). (J–L) Central sections of the sub-tomogram averages of the interface between the host PM and the needle of WT, ΔsipB, and ΔsipD, respectively. (M) Cross-section and (N) diagonal view of the surface rendering of the translocon in panel (J).

https://doi.org/10.7554/eLife.39514.009
Figure 4 with 1 supplement
Deletion of the protein translocases disrupts the T3SS-dependent intimate attachment to the host PM, and the formation of the translocon.

(A) Percentage of minicells attached to the host membrane via needle-membrane contact. Data were compared using a chi-squared test. (B, C) Central slices from tomograms showing the ΔsipBCD injectisomes interacting with the host PM. (E, F) Central slices from tomograms showing the ΔsipB injectisomes interacting with the host PM. (H, I) Central slices from tomograms showing the ΔsipD injectisomes interacting with the host PM. Blue arrows indicate needles attached to the host PM. Red arrows indicate unattached needles. (D, G, J) Schematic models depicting needle-attachment patterns from three mutants, respectively.

https://doi.org/10.7554/eLife.39514.010
Figure 4—figure supplement 1
Gallery of snapshots from host-free and host-interacting S. Typhimurium minicells.

Central slices from representative tomograms showing (A) host-free and (B) host-interacting S. Typhimurium minicells from WT, ΔsipB, ΔsipD, ΔsipBCD, and ΔspaO strain.

https://doi.org/10.7554/eLife.39514.011
Model of the S. Typhimurium injectisome interacting with the host cell membrane.

(A) A schematic diagram of S. Typhimurium interacting with the host cell. (B) Molecular model of the T3SS injectisome at the Salmonella-host cell interface.

https://doi.org/10.7554/eLife.39514.012

Videos

Video 1
A typical reconstruction shows the detailed interaction between the T3SS machines and the target cell and the membrane remodeling.
https://doi.org/10.7554/eLife.39514.008
Video 2
An animation shows the T3SS mediated Salmonella-host interaction and a plausible pathway of effector translocation.
https://doi.org/10.7554/eLife.39514.013

Tables

Table 1
Needle lengths of S. Typhimurium WT, ΔsipB, ΔsipD and ΔsipBCD cells.

A summary of statistical measures including needle length average, standard deviation, and standard error of mean. Data were compared using an unpaired t test.

https://doi.org/10.7554/eLife.39514.004
Sample sizeAverage
(nm)
Standard DeviationStandard Error of Mean(nm)P value campared to WT
WT13551.04.80.42
sipB4650.64.00.590.62
sipD6146.53.90.50<0.0001
sipBCD4645.33.00.44<0.0001
Key resources table
Reagent type
(species) or
resources
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain
background
SB1780
(Salmonella enterica
serovar Typhymurium
SL1344)
PMID: 23481398minD::cat (wt)Galán Laboratory
(Yale University)
Strain, strain
background
SB3542This studyΔsipB minD::catGalán Laboratory
(Yale University)
Strain, strain
background
SB3543This studyΔsipD minD::catGalán Laboratory
(Yale University)
Strain, strain
background
SB3141This studyΔsipBCD minD::catGalán Laboratory
(Yale University)
Strain, strain
background
SB3046PMID: 28283062ΔspaO minD::catGalán Laboratory
(Yale University)
Strain, strain
background
SB3544This studysipB3xFLAG minD::catGalán Laboratory
(Yale University)
Strain, strain
background
SB3545This studysipB3xFLAG minD::catGalán Laboratory
(Yale University)
Strain, strain
background
SB3546This studysipB3xFLAG minD::catGalán Laboratory
(Yale University)
Genetic
reagent
pSB3292
(Plasmid)
PMID: 28283062hilA in pBAD24Galán Laboratory
(Yale University)
Genetic
reagent
minD::cat P22
(P22 bacteriophage lysate)
Galán Laboratory
(Yale University)
P22 lysate from
SB1780 S. Typhimurium strain
Source of minD::
cat allele
Cell lineHeLaATCCHela (ATCC CCL-2)
AntibodyM2Sigma-AldrichF31651:1000 by volume
Chemical
compound, drug
LB BrothFisher BioReagentsBP1426
Chemical
compound, drug
LB AgarFisher BioReagentsBP1425
Chemical
compound, drug
L-arabinoseVWR1B1473
Chemical
compound, drug
Ampicillin Sodium SaltFisher BioReagentsBP1760-25
OtherGold gridQuantifoilR 2/1 on Au 200 mesh
Software,
algorithm
SerialEMPMID: 16182563http://bio3d.colorado.edu/SerialEM/Data acquisition
Software,
algorithm
MotionCor2PMID: 28250466http://msg.ucsf.edu/em/software/motioncor2.htmlMotion correction
Software,
algorithm
TomoautoPMID: 26863591https://github.com/DustinMorado/tomoautoTomogram
reconstruction
Software,
algorithm
Tomo3DPMID: 25528570https://sites.google.com/site/3demimageprocessing/tomo3dTomogram
reconstruction
Software,
algorithm
IMODPMID: 8742726http://bio3d.colorado.edu/imod/Tomogram
reconstruction
Software,
algorithm
I3PMID: 16973379http://www.electrontomography.org/Sub-tomogram
averaging
Software,
algorithm
UCSF ChimeraPMID: 15264254http://www.cgl.ucsf.edu/chimera/3D rendering
Software,
algorithm
UCSF ChimeraXPMID: 28710774https://www.rbvi.ucsf.edu/chimerax/3D rendering
Table 2
Number of tomograms collected and analyzed.
https://doi.org/10.7554/eLife.39514.014
Number of tomograms collected
WT - HeLa458
ΔsipB - HeLa46
ΔsipD - HeLa52
ΔsipBCD - HeLa86
ΔspaO - HeLa84
WT minicells85
ΔsipB minicell115
ΔsipD minicell142
sipB-FLAG - HeLa9
sipC-FLAG - HeLa8
sipD-FLAG - HeLa11
sipB-FLAG5
sipB-FLAG7
sipB-FLAG13
Total1051

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)