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Live imaging of Yersinia translocon formation and immune recognition in host cells [1]

['Maren Rudolph', 'Institute Of Medical Microbiology', 'Virology', 'Hygiene', 'University Medical Center Hamburg Eppendorf', 'Hamburg', 'Alexander Carsten', 'Susanne Kulnik', 'Martin Aepfelbacher', 'Manuel Wolters']

Date: 2022-07 

Yersinia enterocolitica employs a type three secretion system (T3SS) to translocate immunosuppressive effector proteins into host cells. To this end, the T3SS assembles a translocon/pore complex composed of the translocator proteins YopB and YopD in host cell membranes serving as an entry port for the effectors. The translocon is formed in a Yersinia-containing pre-phagosomal compartment that is connected to the extracellular space. As the phagosome matures, the translocon and the membrane damage it causes are recognized by the cell-autonomous immune system. We infected cells in the presence of fluorophore-labeled ALFA-tag-binding nanobodies with a Y. enterocolitica strain expressing YopD labeled with an ALFA-tag. Thereby we could record the integration of YopD into translocons and its intracellular fate in living host cells. YopD was integrated into translocons around 2 min after uptake of the bacteria into a phosphatidylinositol-4,5-bisphosphate enriched pre-phagosomal compartment and remained there for 27 min on average. Damaging of the phagosomal membrane as visualized with recruitment of GFP-tagged galectin-3 occurred in the mean around 14 min after translocon formation. Shortly after recruitment of galectin-3, guanylate-binding protein 1 (GBP-1) was recruited to phagosomes, which was accompanied by a decrease in the signal intensity of translocons, suggesting their degradation or disassembly. In sum, we were able for the first time to film the spatiotemporal dynamics of Yersinia T3SS translocon formation and degradation and its sensing by components of the cell-autonomous immune system.

Type 3 secretion systems (T3SS) are injection machines required for virulence of various bacteria. A translocon/pore complex is formed at the tip of the injection needle, which is required for translocation of effector proteins across the host cell membrane. To date, little information is available on the composition, structure, and regulation of the translocon. Here, we present a novel method that allows us to film the spatiotemporal dynamics of the Yersinia T3SS translocon by inserting a small peptide tag into the minor translocon protein YopD that can be bound with high affinity by a corresponding fluorophore-labeled nanobody during the infection process. By this the formation and disassembly of the translocon and its sensing by components of the cell-autonomous immune system could be recorded with high temporal resolution. The described approach has the potential to provide insights into the T3SS of other bacterial species and is likely transferable also to other secretion systems.

While these approaches provided a considerable degree of spatial resolution, none of them was suitable for time resolved imaging of translocons. Live imaging of bacterium-host cell interactions using fluorescence microscopy has become a key technology for understanding bacterial infection biology [ 22 ]. However, live imaging of translocon formation and processing in host cells has not yet been accomplished. This is likely due to the elaborate und highly coordinated export of the hydrophobic translocator proteins through the T3SS needle and their interaction with the tip complex before they assemble a heteromultimeric translocon in the host cell membrane [ 16 , 23 ]. Thus, finding a label for translocon proteins that is e.g., suited for live cell imaging and super resolution and at the same time does not disturb translocon assembly has proven to be difficult. Fusion proteins of T3SS substrates with fluorescent proteins like GFP were shown to be resistant to T3SS-mediated unfolding and block the secretion path [ 12 ]. Several other tags (e.g. self-labeling enzymes Halo, CLIP, SNAP, split-GFP, 4Cys-tag/ FlAsH, iLOV) are secreted more effectively and have been used with varying degree of success for live imaging of translocated effectors [ 24 – 28 ].

In a recent cryo-electron tomography study the host cell membrane embedded translocon of Salmonella enterica minicells was found to have a total diameter of 13.5 nm [ 11 ]. In our previous work translocons of Yersinia enterocolitica were imaged by super resolution immunofluorescence techniques (STED, SIM) using antibodies against the translocator proteins YopB and YopD. Thereby, the host cellular context that promotes translocon formation could be investigated, revealing that the translocons are formed upon uptake of the bacteria into a phosphatidylinositol-4,5-bisphosphate (PIP2)—enriched pre-phagosomal compartment/prevacuole, which is still connected to the extracellular space [ 21 ].

The translocon of all investigated T3SSs consists of two hydrophobic translocator proteins, a major (YopB in Yersinia) and a minor translocator (YopD in Yersinia) harboring one and two transmembrane domains, respectively [ 16 , 17 ]. The two translocators are thought to form a heteromultimeric ring structure with an inner opening of approximately 2–4 nm in the host cell membrane [ 18 – 20 ]. Despite the central role of the translocon for effector translocation, many aspects of its regulation, assembly and composition have remained elusive. The hydrophobic nature of the translocators and the fact that the assembled translocon can only be studied when inserted into host cell membranes, up to now hindered its investigation due to a lack of suitable experimental approaches.

(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.

Injectisomes can be separated into defined substructures such as the sorting platform, the export apparatus, the needle complex, the tip complex and the translocon ( Fig 1A ). The needle complex is a multi-ring cylindrical structure embedded in the bacterial envelope connected with a 30–70 nm long needle filament, forming a narrow channel, through which the translocator and effector proteins pass in an unfolded state [ 6 , 13 ]. The needle at its distal end transitions into the tip complex, which consists of several copies of a hydrophilic translocator protein (5 copies of LcrV in Yersinia) [ 9 ]. The tip complex is involved in host cell sensing and regulates the assembly of the translocon/pore complex [ 15 ].

Type three secretion systems (T3SSs) are multi-component, syringe-like nanomachines that enable the translocation of bacterial effector proteins across the bacterial envelope into eukaryotic host cells. Numerous human pathogenic bacteria such as Yersinia, Pseudomonas, Chlamydia, Shigella and Salmonella employ T3SS-mediated effector translocation to manipulate a variety of cellular processes, ultimately determining the nature of interaction with their hosts. T3SS effector proteins are diverse in structure and biochemical activities and vary considerably between species. In contrast, the T3SS machinery—also known as the injectisome—is highly conserved across different bacterial species and has been the subject of intensive structural and functional investigation [ 1 – 14 ].

Results

Characterization of Y. enterocolitica strain WA-314 YopD-ALFA During infection with pathogenic yersiniae, a translocon/heteromultimeric pore complex composed of the translocator proteins YopB and YopD is integrated into host cell membranes, serving as an entry gate for the effector proteins (Fig 1A). In search of a method to visualize translocons in living cells using fluorescence microscopy, we inserted the ALFA-tag sequence (plus linker sequences) into the endogenous copy of yopD in the wild type strain WA-314 by CRISPR-Cas assisted recombineering, resulting in strain WA-314 YopD-ALFA (see Materials and Methods). The resulting YopD protein harbors the ALFA-tag between amino acids 194 and 195. The ALFA-tag insertion site is supposedly located in the extracellular part of YopD after it has integrated into the host cell membrane (Fig 1B) [23]. The 13 amino acid long ALFA-tag can be bound with high affinity by specific nanobodies (NbALFA) [29]. We first investigated whether WA-314 YopD-ALFA retains wild type functionalities by comparing its secretion-, translocon forming- and translocation capabilities as well as cytotoxic effect with the parental strain WA-314 (Fig 1C–1F). To analyze effector secretion, the low calcium response was exploited, which triggers secretion of translocator- and effector proteins (Yersinia outer proteins; Yops) into the supernatant, when the bacteria are placed in calcium depleted medium at 37°C [21–23]. SDS-PAGE and Western blot showed similar levels of total secreted proteins and secreted YopD-ALFA in WA-314 YopD-ALFA when compared to WA-314 (YopD-ALFA levels were compared to YopD levels), suggesting that protein secretion is unaffected in WA-314 YopD-ALFA (Fig 1C). Staining of WA-314 YopD-ALFA infected HeLa cells with fluorophore-labeled NbALFA revealed distinct fluorescence patches that were also detected by anti-YopD and anti-YopB antibodies (Fig 1D). Such patches have recently been shown to represent clusters of translocons [21]. Further, the ability of WA-314 YopD-ALFA to translocate effector proteins into host cells was examined using a well-established β-lactamase reporter system. This assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by a translocated YopE β-lactamase fusion protein. CCF4/AM-loaded HeLa cells were infected with WA-314 and WA-314 YopD-ALFA, both carrying pMK-bla encoding for YopE-β-lactamase. Both strains translocated similar amounts of a YopE β-lactamase fusion protein into host cells (Fig 1E) [30–32]. Infection of HeLa cells with Yersinia leads to rounding of the cells and this phenomenon is referred to as cytotoxicity. Cytotoxicity is mainly mediated by the action of translocated effectors YopE and YopH and is thus an indicator of a functioning T3SS machinery and translocon. Rounding of HeLa cells was induced to a similar extent by infection with WA314 and WA-314 YopD-ALFA but not with WA314ΔYopD, indicating that translocon-dependent effector translocation into host cells is unaffected in WA-314 YopD-ALFA (Fig 1F). To determine whether binding of NbALFA to translocon-associated YopD-ALFA impairs translocon function, Hela cells were infected with WA-314 YopD-ALFA in the presence of NbALFA. NbALFA had no effect on translocation of the effector YopH, as determined by Western blotting of YopH extracted by digitonin lysis from HeLa cells, or on cytotoxicity (S1 Fig). We conclude that insertion of the ALFA tag into YopD and binding of NbALFA do not affect translocon function.

Fluorescence staining of YopD-ALFA in pre-phagosomes, phagosomes and Yersinia cells In previous work we showed that translocon formation by Y. enterocolitica occurs in a specific pre-phagosomal host cell compartment, previously referred to as prevacuole [21,33]. The Yersinia-containing pre-phagosome is derived from the plasma membrane, enriched with PIP2 and characterized by a narrow connection to the extracellular space, which cannot be passed by large extracellular molecules such as antibodies (MW approx. 150 kDa), but by small molecules like streptavidin (MW 53 kDa) [21,33]. We therefore assumed that it should be feasible to stain YopD-ALFA in newly formed translocons by adding fluorophore-labeled NbALFA (MW 15 kDa) to fixed but unpermeabilized WA-314 YopD-ALFA infected cells. To test this notion and further investigate the localization of YopD-ALFA in the course of cell infection, we sequentially stained WA-314 YopD-ALFA infected HeLa cells without permeabilization (NbALFA-635), after permeabilization of the HeLa cell membranes with digitonin (NbALFA-580) and after additional permeabilization of the bacterial membranes with 0.1% Triton X-100 (NbALFA-488) (Fig 2A). In unpermeabilized HeLa cells, patchy fluorescence signals associated with bacteria could be detected (Fig 2A, left), confirming that translocon-associated YopD-ALFA can be accessed by extracellularly added NbALFA. In digitonin-permeabilized HeLa cells, additional translocon signals could be found that were not seen in unpermeabilized cells, indicating that these translocons resided in closed phagosomes (Fig 2A, middle). After additional permeabilization of the bacterial membranes with Triton X-100, the intrabacterial pool of YopD-ALFA could be visualized in all bacteria, independent of whether they displayed translocons (Fig 2A, right). The diffuse distribution of intrabacterial YopD-ALFA is in clear contrast to the patchy pattern of translocon-associated YopD-ALFA. To better resolve translocon associated from intrabacterial YopD-ALFA, we employed super resolution 2D and 3D STED microscopy (Fig 2B). While the intrabacterial distribution of YopD-ALFA remained diffuse also at this level of resolution, the extrabacterial YopD-ALFA produced distinct signals with a lateral extent of about 40 nm, which previously were identified as single translocons [21]. Overall, differential YopD-ALFA staining allows to visualize Yersinia translocons located in pre-phagosomes and phagosomes, as well as the intrabacterial YopD pool. PPT PowerPoint slide

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TIFF original image Download: 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). https://doi.org/10.1371/journal.ppat.1010251.g002

Live imaging of Yersinia translocon formation during cell infection Given the ability to stain translocons in fixed and unpermeabilized cells by external addition of fluorophore-labeled NbALFA, we hypothesized that this may also enable the recording of translocon formation in living cells. To test this possibility, live HeLa cells expressing GFP-LifeAct and myc-Rac1Q61L were infected with WA-314 YopD-ALFA in the presence of NbALFA-580 and imaged using spinning disc microscopy with one acquisition per minute. myc-Rac1Q61L was overexpressed in all Hela cell live imaging experiments because it strongly increases the percentage of bacteria forming translocons (S2 Fig) by stimulating uptake of the bacteria into the pre-phagosome [21]. GFP-LifeAct was expressed to visualize host cells and to enable the localization of the cell adhering bacteria. With this approach, appearance and disappearance of fluorescence signals corresponding to translocon-associated YopD-ALFA could be recorded over time (representative event in Fig 3A and S1 Movie). From their first visible appearance (at 5 min in Fig 3A), the number and intensity of YopD-ALFA fluorescence signals peaked after about 20 min and decreased thereafter. The mean overall lifespan of YopD-ALFA fluorescence signals, defined as their first visible appearance until their complete vanishing, was determined to be 26.6 +/- 13 min (mean +/- S.D., Fig 3B). The disappearance of the fluorescence signals was most certainly not due to photo bleaching because no decay of fluorescence was observed in recordings with considerably higher imaging frequency (e.g. acquisition rate: 3 per min in Fig 3C vs. 1 per min in Fig 3A). To further evaluate potential photobleaching, HeLa cells were infected with WA-314 YopD-ALFA and fixed with paraformaldehyde. Fixed YopD-ALFA was stained with NbALFA-580 and subjected to live imaging employing the same conditions as for NbALFA-580-stained native YopD-ALFA. The fixed YopD-ALFA fluorescence signal decreased by 18% within 105 min, as compared to a 74% decrease of the signal in a representative live imaging experiment during 40 min (S3 Fig). This indicates that only minor photobleaching occurs during the live imaging time period und that a decrease of the fluorescence signal most likely is caused by degradation or dissolution of YopD-ALFA. Taken together, for the first time we filmed assembly and disassembly or degradation of T3SS translocons in living host cells, thus providing new insights into the spatiotemporal dynamics of this central T3SS activity. PPT PowerPoint slide

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TIFF original image Download: 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). https://doi.org/10.1371/journal.ppat.1010251.g003

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010251

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