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

Abstract

Many important gram-negative bacterial pathogens use highly sophisticated type III protein secretion systems (T3SSs) to establish complex host-pathogen interactions. Bacterial-host cell contact triggers the activation of the T3SS and the subsequent insertion of a translocon pore into the target cell membrane, which serves as a conduit for the passage of effector proteins. Therefore the initial interaction between T3SS-bearing bacteria and host cells is the critical step in the deployment of the protein secretion machine, yet this process remains poorly understood. Here, we use high-throughput cryo-electron tomography (cryo-ET) to visualize the T3SS-mediated Salmonella-host cell interface. Our analysis reveals the intact translocon at an unprecedented level of resolution, its deployment in the host cell membrane, and the establishment of an intimate association between the bacteria and the target cells, which is essential for effector translocation. Our studies provide critical data supporting the long postulated direct injection model for effector translocation.

Keywords: Macromolecular assembly; S. enterica; bacterial pathogenesis; host-pathogen interaction; infectious disease; membrane remodeling; microbiology; protein secretion; serovar typhie.

Figure 1.. 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.

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.

Figure 2.. 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.

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.

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.

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 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).

Figure 4.. 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.

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.