Visualization of the type III secretion mediated Salmonella–host cell interface using cryo-electron tomography
- Yale University School of Medicine, United States
- Microbial Sciences Institute, Yale University School of Medicine, United States
- McGovern Medical School, The University of Texas Health Science Center at Houston, United States
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, …
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), …
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 …
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…
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 …
Figure 3
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 …
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 …
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.
Figure 5
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.
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.39514.013Tables
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.
| Sample size | Average (nm) | Standard Deviation | Standard Error of Mean(nm) | P value campared to WT | |
|---|---|---|---|---|---|
| WT | 135 | 51.0 | 4.8 | 0.42 | |
| ∆sipB | 46 | 50.6 | 4.0 | 0.59 | 0.62 |
| ∆sipD | 61 | 46.5 | 3.9 | 0.50 | <0.0001 |
| ∆sipBCD | 46 | 45.3 | 3.0 | 0.44 | <0.0001 |
Key resources table
| Reagent type (species) or resources | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background | SB1780 (Salmonella enterica serovar Typhymurium SL1344) | PMID: 23481398 | minD::cat (wt) | Galán Laboratory (Yale University) |
| Strain, strain background | SB3542 | This study | ΔsipB minD::cat | Galán Laboratory (Yale University) |
| Strain, strain background | SB3543 | This study | ΔsipD minD::cat | Galán Laboratory (Yale University) |
| Strain, strain background | SB3141 | This study | ΔsipBCD minD::cat | Galán Laboratory (Yale University) |
| Strain, strain background | SB3046 | PMID: 28283062 | ΔspaO minD::cat | Galán Laboratory (Yale University) |
| Strain, strain background | SB3544 | This study | sipB3xFLAG minD::cat | Galán Laboratory (Yale University) |
| Strain, strain background | SB3545 | This study | sipB3xFLAG minD::cat | Galán Laboratory (Yale University) |
| Strain, strain background | SB3546 | This study | sipB3xFLAG minD::cat | Galán Laboratory (Yale University) |
| Genetic reagent | pSB3292 (Plasmid) | PMID: 28283062 | hilA in pBAD24 | Galán 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 line | HeLa | ATCC | Hela (ATCC CCL-2) | |
| Antibody | M2 | Sigma-Aldrich | F3165 | 1:1000 by volume |
| Chemical compound, drug | LB Broth | Fisher BioReagents | BP1426 | |
| Chemical compound, drug | LB Agar | Fisher BioReagents | BP1425 | |
| Chemical compound, drug | L-arabinose | VWR | 1B1473 | |
| Chemical compound, drug | Ampicillin Sodium Salt | Fisher BioReagents | BP1760-25 | |
| Other | Gold grid | Quantifoil | R 2/1 on Au 200 mesh | |
| Software, algorithm | SerialEM | PMID: 16182563 | http://bio3d.colorado.edu/SerialEM/ | Data acquisition |
| Software, algorithm | MotionCor2 | PMID: 28250466 | http://msg.ucsf.edu/em/software/motioncor2.html | Motion correction |
| Software, algorithm | Tomoauto | PMID: 26863591 | https://github.com/DustinMorado/tomoauto | Tomogram reconstruction |
| Software, algorithm | Tomo3D | PMID: 25528570 | https://sites.google.com/site/3demimageprocessing/tomo3d | Tomogram reconstruction |
| Software, algorithm | IMOD | PMID: 8742726 | http://bio3d.colorado.edu/imod/ | Tomogram reconstruction |
| Software, algorithm | I3 | PMID: 16973379 | http://www.electrontomography.org/ | Sub-tomogram averaging |
| Software, algorithm | UCSF Chimera | PMID: 15264254 | http://www.cgl.ucsf.edu/chimera/ | 3D rendering |
| Software, algorithm | UCSF ChimeraX | PMID: 28710774 | https://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 - HeLa | 458 |
| ΔsipB - HeLa | 46 |
| ΔsipD - HeLa | 52 |
| ΔsipBCD - HeLa | 86 |
| ΔspaO - HeLa | 84 |
| WT minicells | 85 |
| ΔsipB minicell | 115 |
| ΔsipD minicell | 142 |
| sipB-FLAG - HeLa | 9 |
| sipC-FLAG - HeLa | 8 |
| sipD-FLAG - HeLa | 11 |
| sipB-FLAG | 5 |
| sipB-FLAG | 7 |
| sipB-FLAG | 13 |
| Total | 1051 |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.39514.015
