Table 1.
Y. enterocolitica strains.
Fig 1.
Translocators YopB and YopD decorate the Yersinia cell surface after secretion by the T3SS.
(A) Analysis of YopB- and YopD expression. Lysates of Yersinia WA-314 (wild type) grown at 27°C, at 37°C (high Ca2+/non-secretion) or at 37°C in Ca2+ depleted medium (low Ca2+/secretion) were subjected to SDS-PAGE and analyzed by Western blot for expression of YopB, YopD or PepC, as loading control. (B) and (C) Immunofluorescence staining of YopB in bacterial cells under secretion and non-secretion and cell permeabilizing and non-permeabilizing conditions. Confocal immunofluorescence and corresponding differential interference contrast (DIC) images of Yersinia WA-314 subjected to indicated conditions. Diagrams depict fluorescence intensity profiles (arbitrary units, a.u.) along the arrows in the images. Scale bars: 1 μm. (C) Quantitative analysis of YopB positive bacteria. Bars represent mean ± S.D. of n = 260–800 bacteria from 2–3 independent experiments. (D) Confocal vs. STED imaging of YopB and YopD. Representative confocal and STED images (2D- or 3D-STED as indicated) and 3D reconstructions of surface localized YopB and YopD in wild type Yersinia at secretion condition. Merge of YopB (green) and YopD (red) staining and superimposition of YopB and YopD fluorescence intensity profiles along the arrow on the bacterial surface. yz projection at the level of the dashed line. Scale bars: 1 μm.
Fig 2.
STED imaging of YopB and YopD during Yersinia infection of host cells.
(A) YopB concentrates in distinct patches on host cell associated bacteria. HeLa cells (left panel) and primary human macrophages (right panel) were infected with WA-314 at a MOI of 100 for 2 h and a MOI of 10 for 20 min, respectively. Cells were stained with anti-YopB antibody, Alexa568 phalloidin (as overview; red in insets) and DAPI. White outline indicates position of the nucleus (N) and dashed lines indicate host or bacterial cell circumference. Boxed regions in the center of images are depicted as 2.5-fold enlargements at the side. Scale bars: 5 μm (overviews and insets) and 1 μm (enlargements). (B) The fraction of YopB positive, cell-associated bacteria is dependent on the target cell type. HeLa cells or primary human macrophages were infected with WA-314 at a MOI of 50 or 10, respectively, for the indicated time points and stained as in (A). Data represent mean ± S.D. of n = 4210 bacteria associated with HeLa cells from 2 experiments or n = 3047 bacteria associated with macrophages from 3 different donors (C) YopB patches by confocal microscopy can be resolved into distinct spots by STED microscopy. HeLa cells were infected with WA-314ΔYopE at a MOI of 100 for 60 min and stained for YopB using AbberiorStarRed secondary antibody. Single z-planes of parallel confocal (upper panel) and 2D-STED (lower panel) recordings of two cell-associated bacteria are depicted in overviews. Dashed lines indicate bacterial circumference. Scale bar: 1 μm. Boxed regions in overviews are depicted as 10-fold enlargements on the right. Scale bar: 0.1 μm. The number of distinct YopB signals per bacterium (WA-314YopΔE) was quantified by parallel recordings of z-stacks in confocal and 3D-STED mode. Bars represent mean ± S.D. of n = 27 YopB positive bacteria from two independent experiments; *p < 0.0001 (D) YopB spots are organized in clusters on the bacterial surface. Myc-Rac1Q61L transfected HeLa cells were infected with WA-314 at a MOI of 50 for 60 min and stained with anti-YopB antibody and Abberior635P secondary antibody. Z-stacks were recorded in 3D-STED mode and YopB spots on individual bacteria were subjected to image analysis (Methods). Identified clusters (consisting of at least 2 YopB spots) are projected in different colors and angles in a 3D image of a representative bacterium. Xyz-coordinate axes and arrows indicate position and horizontal or vertical rotation of the bacterium (see also S1 Movie). Scale bar: 1 μm (E) YopB/YopD and YopB/LcrV colocalize in STED images. WA-314ΔYopE infected HeLa cells were co-immunostained for YopB (secondary antibody AlexaFluor-594) and either YopD (Abberior-StarRed) or LcrV (Abberior-StarRed). All images show representative single planes of z-stacks recorded in 3D-STED mode. Insets represent an xz projection at the level of the dashed line. Representation of colocalizing points was generated using the “Colocalization” plugin in ImageJ. Scale bar: 1 μm (F) Merge (yellow) of green (YopD) and red (YopB) fluorescence. Superimposed intensity profiles of YopB and YopD fluorescence along the arrow in merge.
Fig 3.
Spatial coupling of translocators YopB/YopD to basal body component YscD.
(A) Schematic representation of the Yersinia injectisome. Positions of YopB/YopD, LcrV, YscF and GFP-YscD and the approximate length of basal body, needle, tip complex and translocon of a virtual injectisome are indicated [11, 19, 57, 58]. Host membrane (HM), outer membrane (OM), inner membrane (IM). (B) SIM separates YopB dots from near neighboring patches of GFP-YscD. HeLa cells were infected with Y. enterocolitica E40 GFP-YscD, stained with anti-YopB antibody and z-stacks of YopB positive bacteria were recorded with SIM. A 3D reconstruction of a representative bacterium is depicted (see also S2 Movie). Scale bar: 1 μm. Fluorescence intensity profiles along the longitudinal axis (arrow) of a GFP-YscD/YopB pair indicate a distance of 90 nm between fluorescence maxima. (C) Nearest neighbor distance analysis of YopB/D and GFP-YscD fluorescence signals. HeLa cells were infected with Y. enterocolitica E40 GFP-YscD, stained with anti-YopD or anti-YopB antibody and z-stacks of YopB/D positive bacteria were recorded with SIM. Coordinates of clearly discernible individual YopB or YopD and GFP-YscD signals were retrieved from single z-planes using ImageJ plugin TrackMate. Distances of individual YopB or YopD dots to the nearest GFP-YscD patches were analyzed by a nearest neighborhood analysis in Matlab and are depicted as histogram (mean ± S.D. = 109 ± 4 nm; 424 pairs analyzed; Methods). Corresponding YopB/YscD pairs (red and green) complying with a distance limit of 200 nm were plotted back on the original fluorescence images (white dots in GFP-YscD/YopB images) for illustration. Scale bar: 1 μm. (D) Double immunostaining of YscF and YopB in E40 GFP-YscD infected HeLa cells followed by SIM reveals conjugated fluorescence triplets displaying basal body (green), needle (white) and translocon (red). Dashed line indicates bacterial circumference. Scale bars: 100 nm. (E) Enrichment of YopD within the injectisome range. HeLa cells expressing myc-Rac1Q61L were infected with Y. enterocolitica E40 GFP-YscD and subjected to ultrathin sectioning and immunogold staining of GFP-YscD (10 nm particles) or YopD (10 nm particles). Transmission electron microscopy (TEM) images were compartmentalized into bacterial cytoplasm (yellow), periplasm and bacterial inner and outer membranes (grey), injectisome range (green, 90 nm from bacterial outer membrane) and adjacent host cell (grey). Images of representative YopD and GFP-YscD immunogold stainings are depicted. Scale bars: 100 nm. Relative labelling indices of YopD were determined for the injectisome range and the adjacent HeLa cells (n = 80 golds evaluated). Relative labelling indices of GFP-YscD were calculated for bacterial cytoplasm, bacterial inner and outer membranes (periplasm) and the host cell (n = 580 golds evaluated). *p < 0.00001 different from expected distribution (S1A–S1C Table, analysis according to [81]) (F) Double immunogold staining of YopD and GFP-YscD. Experimental conditions for HeLa cell infection as in (E). Double immunogold staining of GFP-YscD (15 nm particles) and YopD (10 nm particles). TEM images show exemplary membrane engulfed bacteria with a GFP-YscD/YopD configuration (≤ 140 nm apart; Methods) indicative of a translocon containing/fully assembled injectisome. Scale bars: 100 nm.
Fig 4.
Rac1 activity and host cell type regulate translocon formation.
(A) Lack of Rac1 inhibitor YopE and Rac1Q61L overexpression enhance translocon formation. Native HeLa cells were infected with WA-314 or WA-314ΔYopE and myc-Rac1Q61L overexpressing HeLa cells were infected with WA-314 at a MOI of 50 for 60 min. Cells were stained for YopB, myc and DAPI as indicated. Dashed lines indicate host cell circumference. Insets show strongly increased contrast settings to visualize the host cell bodies. Scale bars: 5 μm. The percentage of YopB positive bacteria of total cell associated bacteria was determined. Bars represent mean ± S.D. n = 6505 WA-314 and n = 5640 WA-314ΔYopE bacteria were investigated in 4 independent experiments (upper graph); n = 3423 bacteria (WA-314) associated to control transfected cells and n = 7882 bacteria associated to myc-Rac1Q61L transfected cells were investigated in 3 independent experiments (lower graph). (B) Digitonin extraction reveals increased incorporation of YopB and YopD into host cell membranes upon Rac1Q61L overexpression or lack of Rac1 inhibitor YopE. Experimental conditions as in (A) but with a MOI of 100. HeLa cells were lyzed with digitonin and resulting supernatants (containing membrane integrated and soluble Yops from host cells) and cell pellets (containing intact bacteria and digitonin insoluble cell components) were analyzed with Western blot for the indicated proteins. YopH serves as marker for effector translocation, myc indicates myc-Rac1QL61 expression, calnexin serves as host cell loading control and HSP60 serves as bacterial loading and lysis control. Data are representative of 3 independent experiments. (C) Quantification of YopB spots per bacterium using STED microscopy. HeLa cells and human primary macrophages (Mφ) were infected with WA-314 or WA-314ΔYopE. Cells were immunostained with anti-YopB antibody followed by AbberiorStarRed or Abberior635P secondary antibody and recorded by STED microscopy. The number of YopB spots per bacterium was analyzed using Imaris software (Methods). Bars represent mean ± S.D. of n = 20 WA-314 (HeLa), n = 27 WA-314ΔYopE (HeLa), n = 52 WA-314 (Mφ) and n = 7 WA-314ΔYopE (Mφ) bacteria investigated in 2–3 independent experiments. *p < 0.05 (D) YopB positive WA-314 and WA-314ΔYopE in macrophages at different infection times. Primary human macrophages were infected with WA-314 or WA-314ΔYopE at a MOI of 10 for the indicated time periods. The percentage of YopB positive bacteria of total cell associated bacteria was calculated. Data represent mean ± S.D. of n = 3047 WA-314 and n = 1822 WA-314ΔYopE in 2–3 independent experiments.
Fig 5.
Formation of translocons is triggered in a prevacuole, a PI(4,5)P2 enriched intermediary host cell compartment.
(A) YopB positive bacteria reside in an intermediary compartment. HeLa cells were infected with surface-biotinylated WA-314 at a MOI of 20 for 60 min and were stained with anti-LPS antibody and streptavidin-Cy5 without cell permeabilization. Cells were permeabilized and immunostained with anti-YopB antibody and DAPI. From left to right: 1. Overview of infected HeLa cell with Yersinia in different stages of internalization. Inset shows strongly increased contrast settings to visualize the host cell body. 2.–4. Enlargements of a bacterial chain showing YopB staining only in the part of the chain located in the intermediary compartment (red in merge) but not in the outside location (yellow in merge). 5. Enlargement of a YopB positive bacterium (white) in the inside compartment (no red or green). Scale bars: 5 μm (overview) and 2 μm (enlargements). Bar diagram: Distribution of YopB positive bacteria between intermediary, inside and outside compartments. Bars represent mean ± S.D. of n = 95 YopB positive bacteria recorded in 2 independent experiments. (B) Bacterial passage from the outside into the intermediary and inside compartments of macrophages. Primary human macrophages of donors 1–4 were infected with biotinylated WA-314 at a MOI of 10 for the indicated time periods and stained with anti-LPS antibody, streptavidin-Cy5 (both without cell permeabilization) and DAPI to score their localization to the outside, inside or intermediary compartments as in (A). Data represent means of at least n = 1000 bacteria investigated per donor. (C) YopB positive bacteria passage from the intermediary to the inside compartment in macrophages. Experimental conditions as in (B) but with no LPS but YopB immunostaining. Data represent mean of n = 952 YopB positive bacteria investigated from 1 donor. (D) Intermediary compartment is marked by PLCδ-PH-GFP. HeLa cells expressing PLCδ-PH-GFP and myc-Rac1Q61L were infected with surface-biotinylated WA-314 at a MOI of 10 for 60 min, stained with anti-LPS antibody and streptavidin-Cy5. Bacteria with PLCδ-PH-GFP enrichment were judged for their localization as in (A). Scale bar: 1 μm. Bars represent mean ± S.D. of n = 398 PLCδ-PH-GFP positive bacteria investigated in 1 experiment. (E) YopB spots colocalize with the PLCδ-PH-GFP marker at STED resolution. HeLa cells expressing PLCδ-PH-GFP and myc-Rac1Q61L were infected with WA-314. Cells were co-immunostained for YopB (secondary antibody Abberior-StarRed) and GFP (AbberiorStar580). Z-stacks were recorded in 3D-STED mode and z-planes (distance: 90 nm) of YopB inserted in a PLCδ-PH-GFP enriched membrane are depicted. Scale bar: 0.5 μm (F) External addition of proteinase K to infected HeLa cells degrades YopB and YopD but not YopH. HeLa cells were infected with WA-314 or WA-314ΔYopE and myc-Rac1Q61L overexpressing HeLa cells were infected with WA-314 at a MOI of 100 for 60 min as indicated. Cells were incubated with PK for 20 min. PK was inactivated with PMSF, cells were extracted with digitonin and analyzed by Western blot for YopB and YopD, YopH (translocated effector protein), myc (myc-Rac1Q61L expression) and actin (loading control). Data are representative of 3 independent experiments. (G) Rac1Q61L overexpression increases uptake of WA-314 into HeLa cells. Control or myc-Rac1Q61L expressing HeLa cells were infected with WA-314 at a MOI of 20 for 60 min, fixed and stained with anti-LPS antibody (antibody accessible bacteria in outside location) and then permeabilized and stained with the same anti-LPS antibody in combination with a different secondary antibody (antibody inaccessible bacteria in intermediary and inside compartments). Bars represent mean ± S.D. of n = 1609 bacteria in control transfected cells and n = 3482 bacteria in myc-Rac1Q61L transfected cells investigated in 3 independent experiments. (H) Rac1L61 expression significantly increases the number of YopB positive bacteria in the intermediary and inside compartments. HeLa cells expressing PLCδ-PH-GFP and either empty vector (control) or myc-Rac1Q61L were infected with WA-314 at a MOI of 50 for 60 min and stained with anti-YopB antibody. The number of YopB positive bacteria as fraction of total cell associated bacteria and their distribution between the intermediary (PLCδ-PH-GFP positive) and inside (PLCδ-PH-GFP negative) compartments was determined. Bars represent mean ± S.D. of n = 1820 bacteria in control transfected cells and n = 6020 bacteria in myc-Rac1Q61L transfected cells investigated in 3 independent experiments.