Fig 1.
Insertion of the ALFA-tag into YopD does not interfere with protein function and allows for nanobody-based staining of translocons.
(A) Schematic representation of the T3SS in Yersinia enterocolitica with ALFA-tagged YopD. The T3SS connecting the bacterial and host cell membranes. The enlargement shows the translocon with ALFA-tagged YopD labeled with a fluorescently tagged nanobody (NbALFA). IM: inner bacterial membrane. PG: bacterial peptidoglycan layer. OM: outer bacterial membrane. HCM: host cell membrane. Adapted from [4,11]. (B) Model of YopD-ALFA and YopB inserted into the host cell membrane. The scheme is adapted from [23] and based on data on interactions of Pseudomonas aeruginosa PopD and PopB. The red box indicates the inserted ALFA-tag between amino acids 194 and 195 on the extracellular part of YopD. (C) Released proteins of WA-314 and WA-314 YopD-ALFA. Secreted proteins were precipitated from the culture supernatant and analyzed by Coomassie stained SDS gel (upper panel) and Western blot (lower panel) for their YopD content using specific antibodies. Black asterisks indicate the position of the YopD bands in the SDS gel. (D) Staining of YopD-ALFA in translocons. Rac1Q61L expressing HeLa cells were infected with WA-314 YopD-ALFA at an MOI of 10 for 1 h, fixed and host cell membranes were permeabilized with digitonin. Co-staining of translocon components was conducted with anti-YopB (shown in green) and anti-YopD (shown in red) antibodies and NbALFA-635 (shown in magenta). Scale bar: 2 μm. (E) Comparison of effector protein translocation by β-lactamase assay. HeLa cells pretreated with a cell permeant FRET dye (CCF4/AM) were infected for 1 h with WA-314, WA-314 pYopE-bla and WA-314 YopD-ALFA pYopE-bla at an MOI of 100 and imaged by confocal microscopy. Excitation of coumarin results in FRET to fluorescein in the uncleaved CCF4 emitting a green fluorescent signal. Cleavage of the cephalosporin core of CCF4 by the beta-lactamase tagged to a truncated YopE translocated into the host cell disrupts FRET and results in a blue fluorescent signal induced by the excitation of coumarin. Cells with incomplete CCF4 cleavage appear cyan. Scale bar: 200 μm. The percentage of green, cyan and blue cells was determined in one experiment from 354, 329 and 305 cells for WA-314, WA-314 pMK-bla and WA-314-YopD-ALFA pMK-bla, respectively. (F) Cytotoxicity assay. HeLa cells were infected for 1 h with WA-314, WA-314 YopD-ALFA and WA-314ΔYopD at an MOI of 100 and imaged by phase contrast microscopy. Depicted are phase contrast images of a representative experiment. Scale bar: 20 μm.
Fig 2.
Differential permeabilization for selective staining of YopD-ALFA in different cellular compartments.
(A) Selective nanobody staining of YopD-ALFA in different cellular compartments. The schematic (top) shows different levels of host- and bacterial cell permeabilization and according accessibility of different pools of YopD-ALFA for NbALFA staining. Rac1Q61L expressing HeLa cells were infected with WA-314 YopD-ALFA at an MOI of 10 for 1 h, fixed and stained with NbALFA-635 without prior permeabilization to specifically target translocon associated YopD-ALFA in the pre-phagosomal compartment (left, shown in red). Host cell membranes were permeabilized with digitonin and translocons located in closed phagosomes were stained with NbALFA-580 (middle, shown in magenta). Note that pre-phagosomal YopD-ALFA was already saturated with NbALFA-635 (red) during the first staining step. Finally, also the bacterial membranes were permeabilized with triton and the intrabacterial pool of YopD-ALFA was stained with NbALFA-488 (right, shown in green). Scale bar: 5 μm. (B) 2D and 3D-STED imaging of intrabacterial and translocon-associated YopD-ALFA. Rac1Q61L expressing HeLa cells were infected with WA-314 YopD-ALFA at an MOI of 10 for 1 h, fixed and stained with NbALFA-635 (shown in red) with prior permeabilization of host cell membranes using digitonin to target translocon associated YopD-ALFA. Bacterial membranes were permeabilized with triton and the intrabacterial pool of YopD-ALFA was stained with NbALFA-580 (shown in green). The images were acquired using super resolution STED microscopy. The boxed region in the left of the image is depicted as enlargements in separate channels at the side. Scale bars: 1 μm (2D-STED overview and 3D STED) and 200 nm (2D-STED enlargements).
Fig 3.
Nanobody-based live imaging of translocons: Formation and lifespan of the translocon during cell infection.
(A) Live imaging of translocons during HeLa cell infection. HeLa cells expressing myc-Rac1Q61L and GFP-LifeAct were infected with WA-314 YopD-ALFA at an MOI of 20 and incubated with NbALFA-580 diluted in the cell culture medium. Cells were imaged with a spinning disk microscope recording z-stacks every minute. Stacks for each time point were combined to one image using maximum intensity projection and one image every 5 min is shown. The left panel shows the overview image at 0 min. The boxed region in the overview image shows the area of the video depicted in still frames to the right. Dashed white lines indicate the outline of the bacteria. Scale bars: 10 μm (overview) and 2 μm (still frames). (B) Lifespan of the translocon. The lifespan of the translocons was determined using movies that recorded the YopD-signal of individual bacteria from their formation to disappearance. Experimental conditions are as in (A). n = 25 bacteria (7 independent experiments, 13 host cells) (C) Engulfment in PIP2-positive membranes precedes translocon formation in HeLa cells. HeLa cells expressing myc-Rac1Q61L and PLCδ1-PH-GFP were infected with WA-314 YopD-ALFA at an MOI of 20 and incubated with NbALFA-580 diluted in cell culture medium. Cells were imaged with a spinning disk microscope recording z-stacks every 20 s. Stacks for each time point were combined to one image using maximum intensity projection and one image every 20 s is shown. The left panel shows the overview image at 0 min. The boxed region in the overview image shows the area of the video depicted in still frames to the right. White arrows indicate the appearance of PLCδ1-PH-GFP and the first translocon signal. Scale bar: 10 μm (overview) and 2 μm (still frames). (D) Fluorescence intensities of PIP2 marker PLCδ1-PH-GFP and YopD-ALFA signals. The relative fluorescence intensities of PLCδ1-PH-GFP and NbALFA-580 signals at the bacteria in (C) were plotted to illustrate the temporal relationship of signal appearances. (E) Temporal relationship of engulfment in PIP2-positive membranes and appearance of YopD-ALFA signal. The time intervals between first occurrence of the PLCδ1-PH-GFP and first YopD-ALFA signals were measured based on live imaging experiments performed as in (C). Each dot represents one measurement. n = 43 bacteria (3 independent experiments, 13 movies). (F) Engulfment in PIP2-positive membranes precedes translocon formation in primary human macrophages. Primary human macrophages expressing PLCδ1-PH-GFP were infected with WA-314 YopD-ALFA at an MOI of 20 and incubated with NbALFA-580 diluted in cell culture medium. Cells were imaged with a spinning disk microscope recording z-stacks every minute. Stacks for each time point were combined to one image using maximum intensity projection and one image every 5 min is shown. The left panel shows the overview image at 0 min. The boxed region in the overview image shows the area of the video depicted in still frames to the right. White arrows indicate the appearance of PLCδ1-PH-GFP and the first translocon signal. Scale bars: 10 μm (overview) and 2 μm (still frames).
Fig 4.
Nanobody-based live imaging of translocons: Galectin-3 and GBP-1 recruitment upon translocon induced membrane damage.
(A) Schematic representation of galectin-3 recruitment following membrane damage during infection. Galectin-3 (shown in green) is found in the cytosol of the host cell. Translocon formation appears to induce membrane damage allowing access of galectin-3 to glycans in the lumen of vacuoles. (B) Vacuolar membrane damage by the T3SS. HeLa cells expressing myc-Rac1Q61L and GFP-galectin-3 were infected with WA-C, WA-C pTTSS and WA-314 YopD-ALFA at an MOI of 100 for 1 h, fixed and permeabilized using digitonin. Cells were stained with anti-YopD antibody, Alexa633 phalloidin and DAPI. The percentage of galectin-3 positive bacteria per cell was quantified for WA-C, WA-C pTTSS and WA-314 YopD-ALFA (n = 1680, 9 host cells; n = 508 bacteria, 7 host cells; n = 1065 bacteria, 10 host cells). Only cells harboring translocon forming bacteria were analyzed for WA-C pTTSS and WA-314 YopD-ALFA infections. (C) Fraction of galectin recruitments without and with prior YopD-ALFA signal. HeLa cells expressing myc-Rac1Q61L and GFP-galectin-3 were infected with WA-314 YopD-ALFA at an MOI of 20 and incubated with NbALFA-580 diluted in cell culture medium. Cells were imaged with a spinning disk microscope recording z-stacks every minute. Galectin-3 recruitment events were quantified with respect to whether YopD-ALFA signal is present before recruitment. n = 330 uptake events (6 independent experiments, 38 movies). (D) Temporal relationship of YopD-ALFA signal appearance and galectin-3 recruitment. HeLa cells expressing myc-Rac1Q61L and GFP-galectin-3 were infected with WA-314 YopD-ALFA at an MOI of 20 and incubated with NbALFA-580 diluted in cell culture medium. Cells were imaged with a spinning disk microscope recording z-stacks every minute. The time intervals between first occurrence of the translocon signal and first GFP-galectin-3 signals were measured. Each dot represents one measurement. n = 36 bacteria (4 independent experiments; 9 movies). (E) Galectin-3 recruitment to phagosomes containing translocon forming bacteria. Live imaging experiments were performed as in (C). The z-stacks for each time point were combined to one image using maximum intensity projection and one image every 5 min is representatively shown. Scale bar: 2 μm. The relative fluorescence intensities at the bacteria were plotted to illustrate the temporal relationship of NbALFA-580 and GFP-galectin-3 signals. (F) Loss of YopD-ALFA signal after GFP-galectin-3 recruitment. Live imaging experiments were performed as in (C). The relative fluorescence intensities of the translocon signal were measured in the last frame before and in the frames 5 min and 10 min after recruitment of GFP-galectin-3. n = 4 measurements (1 experiment, 3 host cells). (G) GBP-1 recruitment to galectin-3 positive bacteria. HeLa cells expressing myc-Rac1Q61L, mScarlet-galectin-3 (shown in green) and GFP-GBP-1 (shown in red) were infected with WA-314 YopD-ALFA at an MOI of 20 and incubated with NbALFA-580 (shown in magenta) diluted in cell culture medium. Cells were imaged with a spinning disk microscope recording z-stacks every 5 minutes. The z-stacks for each time point were combined to one image using maximum intensity projection and images are shown starting 20 min after uptake of the bacteria. Scale bar: 5 μm. (H) GBP-1 positive bacteria lack LPS antibody staining. HeLa cells expressing myc-Rac1Q61L and GFP-GBP-1 (shown in green) were infected with WA-314 YopD-ALFA at an MOI of 30, fixed and permeabilized using digitonin. Cells were stained with anti-LPS antibody (shown in magenta). The boxed regions (I, II) in the overview image are depicted as enlargements in separate channels to the right. Dashed white lines indicate the outline of the nucleus in the overview image and the bacteria in the enlargements. Scale bar: 10 μm (overview) and 2 μm (enlargement).
Table 1.
Yersinia enterocolitica strains.
Table 2.
Oligonucleotides and sequences.