Virotrap Reveals Salmonella SopB as A Ubiquitinated Cargo for Host ESCRT-0

Virotrap Reveals Salmonella SopB as A Ubiquitinated Cargo for Host ESCRT-0 | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var 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Steene , View ORCID Profile Igor Fijalkowski , Veronique Jonckheere , View ORCID Profile Leander Meuris , View ORCID Profile Mathieu JM Bertrand , View ORCID Profile Petra Van Damme , View ORCID Profile Virginie Stévenin , View ORCID Profile Sven Eyckerman doi: https://doi.org/10.1101/2025.08.19.669813 Margaux De Meyer 1 VIB-UGent Center for Medical Biotechnology, VIB , Ghent, Belgium 2 Department of Biomolecular Medicine, Faculty of Medicine and Health Sciences, Ghent University , Ghent, Belgium 3 RIP Unit, Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University , Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Margaux De Meyer Annick Verhee 1 VIB-UGent Center for Medical Biotechnology, VIB , Ghent, Belgium 2 Department of Biomolecular Medicine, Faculty of Medicine and Health Sciences, Ghent University , Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hanna Grzesik 1 VIB-UGent Center for Medical Biotechnology, VIB , Ghent, Belgium 2 Department of Biomolecular Medicine, Faculty of Medicine and Health Sciences, Ghent University , Ghent, Belgium 4 Department of Cellular and Molecular Medicine, Faculty of Medicine, KU Leuven , Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hanna Grzesik Delphine De Sutter 1 VIB-UGent Center for Medical Biotechnology, VIB , Ghent, Belgium 2 Department of Biomolecular Medicine, Faculty of Medicine and Health Sciences, Ghent University , Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jon Huyghe 5 Department of Biomedical Molecular Biology, Faculty of Sciences, Ghent University , Ghent, Belgium 6 VIB-UGent Center for Inflammation Research, VIB , Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jon Huyghe Louis Delhaye 1 VIB-UGent Center for Medical Biotechnology, VIB , Ghent, Belgium 2 Department of Biomolecular Medicine, Faculty of Medicine and Health Sciences, Ghent University , Ghent, Belgium 7 Cancer Research Institute Ghent (CRIG), Ghent University , Ghent, Belgium 8 OncoRNALab, Center for Medical Genetics Ghent (CMGG), Ghent University , Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Louis Delhaye Tessa Van de Steene 1 VIB-UGent Center for Medical Biotechnology, VIB , Ghent, Belgium 2 Department of Biomolecular Medicine, Faculty of Medicine and Health Sciences, Ghent University , Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Igor Fijalkowski 3 RIP Unit, Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University , Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Igor Fijalkowski Veronique Jonckheere 3 RIP Unit, Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University , Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Leander Meuris 1 VIB-UGent Center for Medical Biotechnology, VIB , Ghent, Belgium 3 RIP Unit, Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University , Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Leander Meuris Mathieu JM Bertrand 5 Department of Biomedical Molecular Biology, Faculty of Sciences, Ghent University , Ghent, Belgium 6 VIB-UGent Center for Inflammation Research, VIB , Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mathieu JM Bertrand Petra Van Damme 3 RIP Unit, Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University , Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Petra Van Damme For correspondence: sven.eyckerman{at}vib-ugent.be Virginie Stévenin 9 Department of Cell and Chemical Biology, Oncode Institute, Leiden University Medical Center (LUMC) , Leiden, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Virginie Stévenin For correspondence: sven.eyckerman{at}vib-ugent.be Sven Eyckerman 1 VIB-UGent Center for Medical Biotechnology, VIB , Ghent, Belgium 2 Department of Biomolecular Medicine, Faculty of Medicine and Health Sciences, Ghent University , Ghent, Belgium 7 Cancer Research Institute Ghent (CRIG), Ghent University , Ghent, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sven Eyckerman For correspondence: sven.eyckerman{at}vib-ugent.be Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The pathogenic bacterium Salmonella survives and replicates in host cells within a Salmonella -containing vacuole (SCV). To build this niche, Salmonella uses secreted effectors, such as SopB, a phosphoinositide phosphotransferase that modifies host phospholipid fluxes at the plasma membrane and SCV. These lipid modifications impact host protein recruitment and activity, implicating SopB in bacterial internalization, SCV biogenesis, and inflammatory signaling. Yet, interactions of SopB with host proteins have remained ill-defined. Here, we employ the unique Virotrap mass spectrometry-based technology to identify a new set of SopB-associated host proteins. We demonstrate the direct interaction of SopB with the ubiquitin-binding domains of the ESCRT-0 subunit HGS, and we show that SopB promotes ESCRT-0 recruitment at the SCV where they colocalize. As the ESCRT machinery plays a central role in cargo sorting and membrane remodeling, we propose a new SopB-dependent mechanism by which Salmonella controls host membrane dynamics. Introduction Salmonella is one of the primary agents responsible for foodborne illnesses and diarrheal diseases globally [ 1 ] and is classified as a high-priority pathogen by the WHO [ 2 ]. Salmonella enterica subspecies enterica serovar Typhimurium ( S . Typhimurium) typically induces self-limiting gastroenteritis in humans but can elicit life-threatening systemic infections, particularly in immunocompromised individuals [ 3 ]. Infection is set off with the active invasion of non-phagocytic intestinal epithelial cells through the induction of ruffles and bacterial uptake. Within enterocytes, the pathogen creates an intracellular niche known as the Salmonella -containing vacuole (SCV), providing an environment that enables bacterial residence and replication [ 4 ]. Next to its intravacuolar residence, other intracellular lifestyles are adopted by S . Typhimurium, comprising approximately 10% bacteria escaping the vacuole, which results in hyper-replication within the cytosol [ 5 , 6 ], or dormant salmonellae inside a divergent vacuole in human enterocytes [ 7 ]. The orchestration of invasion, SCV maturation, and intracellular pathogenesis is dictated by a repertoire of over 40 virulence factors, called effectors, which are secreted through the type-3 secretion systems (T3SS)-1 and -2, encoded on Salmonella pathogenicity islands SPI-1 and -2, respectively [ 8 , 9 ]. T3SS-1 effector SopB ( Salmonella outer protein B), recently characterized as a phosphoinositide phosphotransferase [ 10 , 11 ], has been implicated in a range of functions throughout the infection process [ 12 , 13 ]. First, SopB contributes to efficient bacterial internalization by modulating phosphoinositide fluxes at the plasma membrane. These lipidic changes results in the activation of SH3-containing guanine nucleotide exchange factor (SGEF) that stimulate CDC42-dependent actin rearrangements, and promote membrane ruffling necessary for SCV and infection-associated macropinosome (IAM) formation [ 12 , 14 , 15 ]. Notably, ectopically expressing SopB in eukaryotic cells results in the formation of macropinosomes [ 16 ]. Next, SopB promotes the acute synthesis of phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2) [ 10 ] and thereby activates the host pro-survival kinase AKT that supports intracellular bacterial growth [ 17 ]. Shortly after cellular entry of the pathogen, SopB is mono-ubiquitinated at multiple lysine residues, which favors its translocation to the SCV and downregulates its activity at the plasma membrane [ 18 , 19 ]. At the SCV, SopB orchestrates the early design of the intracellular niche by generating PI(3)P through recruitment of RAB5 and VPS34 [ 20 ]. This alteration in membrane phospholipid composition enhances recruitment of sortin nexins SNX1 and SNX3, leading to spacious vacuole-associated tubules (SVAT) formation, SCV shrinkage and subsequent maturation [ 21 – 23 ]. Despite in-depth investigations into SopB’s phospholipid-modifying properties, ubiquitination-dependent localization [ 18 , 24 ], and modulation of various host cell signaling pathways [ 25 ], the links between these distinct – yet interdependent – biological aspects remain largely unresolved. While the identification of protein interaction partners is commonly used for such mechanistic investigations, mapping of the complete SopB interactome using classical approaches is hindered by SopB-specific challenges, including the high cytotoxicity resulting from SopB intracellular accumulation upon forced expression and the potential role of a lipid local environment [ 11 ]. An innovative interactomics strategy, called Virotrap, involves fusing a protein of interest to HIV-1 Gag to trap interacting protein complexes and identify them using mass spectrometry (MS) [ 26 , 27 ]. By avoiding cell lysis, Virotrap allows a comprehensive description of a protein’s micro-environment, thus potentially revealing novel interaction partners. In this study, we leverage the unique properties associated with the Virotrap technology to characterize the SopB micro-environment and reveal a previously unidentified interplay of SopB with the host ESCRT machinery, typically involved in sorting of plasma membrane receptors [ 28 ]. Especially, our findings demonstrate a direct and ubiquitin-dependent interaction between Salmonella SopB and HGS at IAMs and at the SCV. This newfound interaction sheds light on how the extensively studied SopB effector manipulates the host cell through targeting of a central endocytic host complex. Results Virotrap Charts the SopB Endocytic Membrane Environment To provide new insight into the biology of the Salmonella effector SopB, we examined the host interactome of SopB using the Virotrap technology ( Fig. 1a ). This approach is based on the fusion of a protein of interest (so-called ‘Bait’) with the human immunodeficiency virus-1 (HIV-1) Gag protein. Upon expression, Gag-Bait forms virus-like particles (VLPs) that comprise co-sorted interaction partners. After purification of the released VLPs from the cell supernatant, mass spectrometry (MS)-based proteomics reveals the content of the particles, including interaction partners bound to the bait protein. This approach supports the characterization of weak and valency-dependent interactions within the bait micro-environment [ 29 ], including modification-dependent interactions [ 30 , 31 ], and allows the analysis of proteins whose intracellular accumulation would have triggered cytotoxicity [ 31 ]. Based on these properties, we generated a Gag-SopB fusion construct to enable Virotrap analysis. Western blot confirmed that SopB fusion with Gag and expression in HEK293T cells results in SopB detection in VLPs collected in the culture medium ( Suppl. Fig. 1 ). Next, we analyzed the proteomic composition of Gag-SopB VLPs in comparison with a negative control VLP by MS analysis, as previously described [ 30 , 32 ]. In total, 478 human proteins showed differential abundance in VLPs containing SopB versus control ( Fig. 1b ). To investigate this extensive SopB interactome, we first identified cellular compartments associated with the significant hits using gene ontology enrichment analysis ( Fig. 1c ). Consistent with SopB’s documented endosomal localization [ 33 ], Gag-SopB VLPs demonstrated significant enrichment of 47 endosomal proteins ( Fig. 1b, c ), involving multiple endocytic host complexes ( Fig. 1c ). Intriguingly, all three subunits of the ESCRT-0 complex, namely HGS, STAM and STAM2, as well as the accessory ESCRT proteins HD-PTP and Endofin exhibited significant enrichment with the SopB bait protein ( Fig. 1b ). Together, HGS, STAM, STAM2, HD-PTP and Endofin have been described to comprise the machinery needed for sorting of ubiquitinated cargoes, in particular membrane receptors, into intraluminal vesicles (ILVs) within multivesicular bodies [ 34 ]. Download figure Open in new tab Figure 1. Virotrap maps the host environment of Salmonella SopB. (a) Overview of Virotrap. A fusion of the bait, e.g., effector protein of interest, to the C-terminus of the Gag polyprotein (myristoylated; pink zigzag line) of human immunodeficiency virus type 1 (HIV-1) is transiently expressed in HEK293T cells. Along with the Gag-bait fusion, FLAG-tagged vesicular stomatitis virus glycoprotein (VSV-G) is expressed and becomes embedded in the plasma membrane. Gag facilitates multimerization and localization of the fusion protein at the plasma membrane, giving rise to virus-like particle (VLP) budding. Consequently, host preys are trapped inside VLPs that can be captured in the cell medium by anti-FLAG immunoprecipitation. LC-MS/MS-based analysis of the VLP content subsequently allows identification of significant bait co-enriched host proteins. (b) Volcano plot displaying the differential enrichment between Gag-SopB wild-type (WT) and Gag-eDHFR in Virotrap (limma, p-value < 0.05). (c) Gene Ontology (Cellular Component) enrichment analysis for enriched proteins (p-value < 0.05) with SopB in Virotrap. STRING v12.0 was used for enrichment analysis. 15 significant terms are shown (FDR < 0.05). (d) Volcano plot displaying the differential enrichment between SopB-C460S catalytic mutant and SopB-WT in Virotrap (limma, p-value < 0.05). (e) Volcano plot displaying the differential enrichment between SopB-K527A catalytic mutant and SopB-WT in Virotrap (limma, p-value < 0.05). (f) Gene Ontology (Molecular Function) enrichment analysis for enriched proteins (p-value < 0.05) with SopB-WT compared to C460S in Virotrap. STRING v12.0 was used for enrichment analysis. 10 significant terms are shown (FDR < 0.05). (g) Gene Ontology (Molecular Function) enrichment analysis for enriched proteins (p-value < 0.05) with SopB-WT compared to C460S in Virotrap. STRING v12.0 was used for enrichment analysis. 10 significant terms are shown (FDR < 0.05). (h) Interaction network representation of significant endosomal protein complexes identified in Virotrap for SopB-WT. Connections (edges) depict high-confidence physical interactions with a STRING score of at least 0.7. (i) VENN diagram showing overlap of significant hits among all three SopB variant Virotrap screens upon comparison with Gag-eDHFR. (j) Heatmap representing LFQ intensities for ESCRT-0 components over all SopB variant Virotrap comparisons. For a more in-depth functional characterization of the SopB interactome, we performed comparative Virotrap analyses with either the catalytically inactive mutant SopB-C460S [ 35 ], or with the catalytically reduced mutant SopB-K527A. We confirmed that both mutants were effectively detected in VLPs collected in the culture medium ( Suppl. Fig 1 ). This comparison revealed 86 significantly enriched hits for the WT effector form, including multiple sorting nexins (SNX2, SNX3, SNX9, SNX18, and SNX27), components of the cargo-selective retromer complex (VPS26A, VPS29, VPS35), components of the exocyst complex (EXOC1/SEC3, EXOC2/SEC5, EXOC3/SEC6, and EXOC4/SEC8), clathrin chains (CLTA, CLTB, and CLTC) and ESCRT-associated proteins (HD-PTP and Endofin) ( Fig. 1d, e ). Highly similar results were obtained upon comparison of the WT and K527A forms ( Fig. 1g ), indicating that reduced enzymatic activity is sufficient to significantly impact the SopB-host interactome. Gene ontology enrichment of the differential SopB interactome aligned with molecular functions and biological processes associated with SopB’s enzymatic activity, including “phosphatidylinositol binding” [ 11 ], “vesicle tethering in exocytosis” [ 36 , 37 ], “retrograde transport, endosome to Golgi” [ 21 ], and “endocytic recycling” [ 38 ] among others ( Fig. 1f, g ). Overall, the SopB-WT, C460S, and K527A interaction profiles shared a total of 131 significant proteins, while 333 hits are specifically enriched in the SopB-WT profile ( Fig. 1i ), which underscores the importance of SopB catalytic activity in defining its interactome. Remarkably, accessory ESCRT-0 proteins HD-PTP and Endofin, highly enriched hits in the SopB-WT interaction profile, were not enriched with the catalytic mutants ( Fig. 1d, e, h, j ). In contrast, HGS, STAM and STAM2 were enriched independently of SopB catalytic activity, suggesting a two-step interaction of SopB with the ESCRT-0 complex and accessory proteins. Taken together, these Virotrap results capture the SopB host environment and its dependence on SopB’s enzymatic activity: notably pondering a novel interaction between the host ESCRT-0 complex and SopB. SopB Interacts with the Host ESCRT-0 Machinery The detection of ESCRT-0 components HGS, STAM and STAM2 in both SopB WT and catalytic mutant interactomes suggests a catalytic activity–independent interaction. Therefore, we tested for potential direct interactions between these host factors and SopB occurs, using a split-luciferase NanoBiT protein–protein interaction assay in HEK293T cells ( Fig. 2a ). This assay offers high sensitivity, making it well-suited for detecting transient or weak interactions in a cellular context [ 39 ]. HGS, STAM and STAM2 were genetically fused in both orientations to the NanoLuc C-terminal small fragment (SmBiT), whereas SopB was fused to the larger N-terminal fragment (LgBiT) ( Fig. 2b ). All possible fusion combinations were evaluated for luciferase complementation, measuring luminescence relative to the HaloTag negative control setup ( Fig. 2c , Suppl. Fig. 2 ). We observed high relative luminescence for all ESCRT-0 proteins with SopB demonstrating direct interaction between these host and Salmonella proteins ( Fig. 2c ). Download figure Open in new tab Figure 2. SopB interacts with host ESCRT-0 components. (a) Schematic representation of the NanoBiT protein-protein interaction assay. SopB (bait) and ESCRT-0 subunits (HGS, STAM, STAM2; prey) are genetically fused to either LgBiT- or SmBiT-encoding portions of the NanoLuc luciferase. Upon expression of the fusion proteins, the luciferase reconstitutes when an interaction between bait and prey occurs. After administration of the luciferase substrate furimazine, luminescence can be measured to detect a potential interaction. (b) Constructs used for NanoBit: LgBiT-tagged SopB with SmBiT-tagged HGS, STAM and STAM2 at either the N- or C-terminus. (c) NanoBiT protein-protein interaction assay evaluating the interaction between SopB C-terminally tagged with LgBiT and ESCRT-0 proteins HGS, STAM, and STAM2. The results represent three technical replicates. The experiment represents the results of three independent repeats. Statistical significance was determined using one-way ANOVA (***p < 0.001, ****p-value < 0.0001). (d) Confocal imaging of stable inducible HeLa-HGS-mScarlet cells. Top row: SopE2-EGFP or SopB-EGFP transiently expressed without doxycycline. Middle and bottom rows: the same conditions with doxycycline induction (50 ng/mL); showing HGS-mScarlet localization relative to SopE2 or SopB). The scale bars indicate 10 µm (overview) and 2 µm (insets). Accessory ESCRT-0 proteins are indicated in non-transparent red. (e) Quantification of SopB and SopE2 co-localization with HGS. Data were collected from 26 cells from three independent experiments. Statistical significance was determined using an unpaired t-test (****p-value < 0.0001). Accessory ESCRT-0 proteins are indicated in non-transparent red. (f) Fluorescence intensity profiles of EGFP and mScarlet channels across the circumference of three representative macropinosomes. Arrowheads mark coincident peaks indicating SopB-HGS co-localization. Next, we used live microscopy to assess the intracellular localization of SopB and HGS upon co-expression in epithelial cells. Heterologous expression of EGFP-SopB in HeLa cells showed its localization on small intracellular vesicles, as well as macropinosomes formed upon SopB expression, in alignment with previous studies ( Fig. 2d ) [ 33 ]. To follow HGS localization with minimal overexpression side-effects, we engineered inducible HGS-mScarlet expressing HeLa cells allowing a controlled low HGS-mScarlet expression using an optimized low doxycycline concentration. We observed HGS-mScarlet localizing at small intracellular vesicles ( Fig. 2d ), which corresponds to its described endo- and lysosomal localization (Human Protein Atlas [ 40 ]). Upon SopB expression, HGS was also observed at the SopB-induced macropinosome, where it co-localizes with SopB. Further, to determine the role of SopB in HGS localization on macropinosomes, we monitored HGS localization upon SopB-independent macropinocytosis, by expressing the Salmonella effector SopE2 ( Fig. 2d ). Similar to SopB, SopE2 induced the formation of macropinosomes where HGS is recruited, showing that HGS recruitment at macropinosomes, per se, does not require SopB. SopE2 displayed dispersed localization all over the cytoplasm while the punctate SopB pattern localized to small intracellular vesicles and macropinosomes positive for HGS ( Fig. 2e ). Strikingly, SopB was exclusively observed in HGS-positive areas. Especially, macropinosome membrane microdomains with high SopB fluorescent intensity co-localized with microdomains with high HGS fluorescent intensity ( Fig. 2f ). In summary, SopB co-localizes with HGS on macropinosome microdomains but HGS localization on these structures is independent of SopB. HGS Localizes at Salmonella Infection Sites and Displays SopB-Dependent Recruitment at the SCV Following ESCRT-0 and SopB colocalization at the host endomembranes upon SopB heterologous expression, we aimed to characterize ESCRT-0 subcellular SopB-dependent positioning during infection. To this end, we performed time-lapse microscopy of stable inducible HeLa-HGS-mScarlet cells infected with fluorescent WT Salmonella (SL1344-WT) or Salmonella lacking SopB (SL1344-Δ sopB ). Within the first five minutes after cell entry, we observed HGS recruitment to large vesicles and SCVs ( Fig. 3a ). Using fluorescent dextran, we confirmed that the large vesicles positive for HGS are newly formed IAMs ( Fig. 3b ). Overall, HGS accumulation was observed on almost all SCVs during the course of the time-lapse ( Fig. 3c ) and about 75% of IAMs ( Fig. 3d ). Interestingly, infection with SL1344-Δ sopB resulted in approximately a fourth reduction in HGS recruitment to SCVs ( Fig. 3c ), but did not significantly impacted HGS recruitment at IAMs ( Fig. 3d ). The latter observation is in line with the partial HGS-positive character of the macropinosomes visible with SopE2 overexpression ( Fig. 2d ). Download figure Open in new tab Figure 3: HGS is recruited at Salmonella infection sites. (a) Time-lapse imaging of HeLa cells expressing mScarlet-HGS upon doxycycline induction (white) and infected with GFP-expressing Salmonella SL1344 WT (in green). Bacteria are added to the cells just before the beginning of the acquisition to visualize entry and early events. See also Suppl. Video 1 (cropped view). Yellow arrow-head: HGS-positive SCV, Blue arrow-head: HGS-positive IAMs. Scale bars: 10 µm (overview) and 5 µm (insets). (b) Time-lapse microscopy of HeLa cells expressing mScarlet-HGS upon doxycycline induction (in white) and infected with GFP-expressing Salmonella SL1344 WT (in green) in the presence of fluorescent Dextran (in yellow). Dextran and Bacteria are added to the cells for 30 min and washed (W) before the beginning of the acquisition to visualize IAM dynamics. See also Suppl. Video 2 (cropped view). Scale bars: 10 µm (overview) and 5 µm (inset). (c) Quantification of SCVs that become HGS-positive over the course of the time-lapse in HeLa cells. Over 180 SCVs were scored across three independent experiments (mean ± SEM). Statistical significance was determined using an unpaired t-test ***p < 0.001). Arrowheads in representative images indicate HGS-positive (green) and HGS-negative (red) SCVs. The scale bars indicates 10 µm. (d) Quantification of IAMs that become HGS-positive over the course of the time-lapse in HeLa cells. Over 180 IAMs were scored across three independent experiments (mean ± SEM). Statistical significance was determined using an unpaired t-test (ns, not significant). Arrowheads in representative images indicate HGS-positive (green) and HGS-negative (red) IAMs. Scale bars indicates 5 µm. (e) SopB localization during infection using SL1344(SopB-Flag)-dsRed upon depletion of HGS. HeLa cells were infected and fixed at 30 minutes post-infection. SopB-Flag was visualized using mouse anti-Flag antibodies and donkey anti-mouse DyLight-488. Cells were stained using Deep Red cell mask (white). Scale bar indicates 1 µm. Next, we examined SopB recruitment at the SCV with and without depletion of HGS with siRNAs. First, we monitored SCV and IAM formation upon HGS silencing by time-lapse microscopy using the PI(3)P biosensor 2xFYVE [ 41 ] and did not observe significant changes in the formation of the SCV or IAMs compared to the control condition ( Suppl. Fig. 2 ). Subsequently, we infected the HGS silenced HeLa cells with a Salmonella strain expressing a FLAG-tag SopB followed by an anti-FLAG immunodetection. We did not observe any alterations of SopB localization to the SCV upon HGS depletion ( Fig. 3e ). Together, we found that ESCRT-0 localizes at IAMs and SCVs – compartments known to associate with SopB during infection [ 42 ] – and our results suggest that SopB promotes HGS recruitment to the SCV, rather than the reverse. SopB is Recognized as a Ubiquitinated Cargo for ESCRT-0 Host ESCRT-0 (HGS, STAM and STAM2) directly binds ubiquitin moieties of ubiquitinated cargo targeted for incorporation into ILVs on multivesicular bodies [ 43 ]. Taking advantage of the Virotrap MS data, we searched for diGly modifications indicative of ubiquitination sites and confirmed previously reported ubiquitination of SopB at multiple lysines: K19, K41, K93 and K541 [ 24 , 44 , 45 ] ( Fig. 4a ). Notably, these ubiquitinations appeared independent of SopB catalytic activity in our Virotrap assay. Considering the relevance of ubiquitination of the effector in vivo , this finding confirms the validity of using SopB heterologous expression to study interactions dependent on this post-translational modification. Given the remarkable mono-ubiquitination pattern of SopB, we explored whether its interaction with ESCRT-0 is dependent on this modification. To study SopB’s mode of binding to ESCRT-0, four HGS domain deletion mutants were constructed including removal of the VHS (Vps-27, Hrs and STAM) ubiquitin-binding domain, ubiquitin-interacting motif (UIM) domain, and a proline-rich region (PR) known to facilitate cargo binding independent of ubiquitination ( Fig. 4b-c ). We then conducted a NanoBiT protein-protein interactions assay with these constructs and observed a drastic drop of relative luminescence upon SopB binding to HGS deleted of both ubiquitin-binding domains ( Fig. 4d ). VHS deletion alone did not lead to a noticeable effect on the luminescent readout, whereas ΔUIM markedly reduced the signal, indicating that the HGS-UIM is the primary determinant for SopB binding. Thus, SopB interaction with the ESCRT-0 machinery is dependent on HGS ubiquitin-binding properties. In addition, removal of the PR domain did not abolish SopB–HGS interaction, consistent with an auxiliary ubiquitin-independent contact previously described for interleukin (IL)-2 receptor recognition [ 46 ]. As SopB ubiquitination was previously linked to its prolonged retention at the SCV [ 44 ], and given our observation that SopB promotes HGS recruitment at the SCV ( Fig. 3e ), we propose that SopB mono-ubiquitination may act as a molecular mimicry for the recruitment of HGS at the SCV ( Fig. 4e ). Following ESCRT-0 recruitment, the catalytic activity of SopB likely promotes subsequent recruitment of the complex accessory proteins, as suggested by the comparative SopB-WT and catalytic mutant Virotrap assay ( Fig. 1h, j ). Download figure Open in new tab Figure 4. SopB is recognized as a ubiquitinated cargo for ESCRT-0 (a) Heat map of identified SopB peptides bearing diGly (K-ε-GG) remnants indicative of Lys ubiquitination across Virotrap datasets (WT and mutants). Intensities were normalized by median subtraction; crossed boxes indicate not detected. (b) HGS deletion constructs used in NanoBiT: VHS, Vps-27, Hrs and STAM; UIM, ubiquitin-interacting motif; CBD, clathrin-binding domain; PR, proline-rich; CC, coiled-coil; FYVE, Fab1p, YOTB, Vac1p, and EEA1 zinc finger. (c) Immunoblot analysis of SmBiT-V5-tagged HGS constructs expressed in HEK293T cells. Tubulin was used as a loading control. (d) NanoBiT assay measuring the interaction between SopB and HGS mutant variants. Statistical significance was determined using one-way ANOVA (*****p-value < 0.0001). (e) Model displaying ubiquitin-dependent binding of SopB with ESCRT-0 at the SCV and the differential recruitment of accessory host factors in comparison with a catalytic mutant SopB ( Fig. 1 ). Discussion Salmonella deploys a diverse arsenal of effectors to manipulate host cell signaling. Among these, the inositol phosphatase SopB, discovered over two decades ago [ 51 ], remains incompletely understood despite extensive study, with emerging functions continuing to reveal its multifunctionality [ 21 , 35 , 38 , 42 , 52 , 53 ]. Dissecting the specific contribution of SopB requires precise mapping of its host interaction network. Using the ultra-sensitive Virotrap interactomics platform [ 26 ] ( Fig. 1a ), well-suited for proteins that are cytotoxic upon (over)expression [ 31 ], as is the case for SopB [ 11 ], we generated the most comprehensive SopB-host interactome to date that includes both novel and described host interactions ( Fig. 1b, f-g ). Virotrap revealed known SopB-associated host proteins, including the retromer subunit VPS35 [ 47 ] and multiple sorting nexins (SNX3, SNX9, SNX18) [ 13 , 38 , 52 ] ( Fig. 1b, h ). The host cargo-selective retromer complex, composed of VPS26, VPS29, and VPS35, acts together with specific SNX dimers (SNX1, SNX2, SNX3, SNX5, SNX6, or SNX27) to mediate endosomal protein sorting and trafficking on and from the endosome via tubular vesicles [ 54 ]. In addition to VPS35, we detected VPS26A and VPS29, indicating recruitment of the complete cargo-selective retromer trimer. We also identified new SopB associations SNX2 and SNX27, both previously localized to the SCV [ 55 ]. SNX2 supports SNX3-dependent tubule formation for mannose-6-phosphate receptor (M6PR) cycling [ 56 , 57 ], a process known to be subverted by SopB [ 52 ], while SNX27 functions as a cargo adaptor in recycling to the plasma membrane, opposed to SNX2 and SNX3, which route cargo to the trans-Golgi network (TGN) [ 54 ]. In addition, SNX9 and SNX18 that remodel membranes at the plasma membrane independent of retromer [ 58 ], were also identified as potential SopB interactors ( Fig. 1b, h ). The diversity of SNX subfamilies recovered suggests that Virotrap captures the SopB interactome at both the plasma membrane and endosomal compartments, reflecting its multi-compartment localization at the invasion sites and the SCV [ 18 , 19 ]. Moreover, our interactomics data position SopB within a broad host membrane–remodeling network, directly linking its phosphoinositide phosphatase activity to retromer, SNAP receptor (SNARE), and exocyst complexes ( Fig. 1f, g ). The enrichment of STX3, STX7, VAMP3, and VAMP7 with wild-type SopB compared to the C460S and K527A mutant suggests selective fusion events between host endo-/lysosomal compartments, while enrichment of EXOC1, 2, 3 and -4 implies involvement of the complete heterotetrameric exocyst subcomplex. This aligns with recent evidence for SopB-dependent assembly of EXOC2 and EXOC3 at Samonella invasion sites that required the C460 residue to enhance bacterial uptake [ 37 ], and our results extends it to additional subunits. Based on our data, SopB also engages SNX3, SNX9, and SNX18 through phosphoinositide remodeling, which is consistent with prior work [ 13 , 38 , 52 ]. Remarkably, the endosomal complexes enriched with SopB serve different modes of membrane remodeling: protein removal via internalization (ESCRT), tubular extrusion (retromer), fusion (SNARE), and membrane microdomain organization (clathrin and flotillin) that coordinates these events [ 59 , 60 ]. Together, these results indicate that SopB mobilizes multiple endosomal machineries to modulate the membrane composition and dynamics of the SCV. Among the novel insights revealed by the SopB Virotrap interactome, we identified a significant association between the effector and the entire host ESCRT-0 complex, comprising HGS, STAM and STAM2 ( Fig. 1a ), notably independent of SopB’s enzymatic activity ( Fig. 1h, j ). The ESCRT-0 machinery is a critical regulator of endosomal sorting, responsible for directing ubiquitinated receptors into the lumen of multivesicular bodies (MVBs) via the formation of ILVs [ 61 ]. Upon activation, many cell surface receptors undergo ubiquitination and are internalized into early endosomes, where they are either recycled back to the cell surface or targeted for lysosomal degradation. Through the sequential action of the ESCRT complexes ( i.e ., ESCRT-0, -I, -II, and -III) ubiquitinated cargo is sorted into ILVs after which the MVB fuses with the lysosome for receptor turnover or to the plasma membrane for exocytosis [ 62 ]. Next to receptor trafficking, ESCRT functions in micro-autophagy, autophagosome closure, membrane repair and cytokinesis among others [ 63 ], and is exceptionally conserved across eukaryotes [ 64 ]. The phosphatase-independent enrichment of ESCRT-0 with SopB, together with the pivotal roles of both SopB and ESCRT in endomembrane dynamics, led us to further explore this intriguing interaction. Using NanoBiT assays, we confirmed direct binding between SopB and ESCRT-0 subunits HGS, STAM, and STAM2 ( Fig. 2c ). Additionally, confocal microscopy revealed co-localization of HGS with heterologously expressed SopB at host endomembranes ( Fig. 2d-f ), consistent with both SopB and ESCRT-0’s localization to PI(3)P-enriched membranes [ 65 , 66 ]. Notably, HGS localization to macropinosomes appeared to be independent of SopB, as similar localization was observed with the expression of SopE2 ( Fig. 2d ), another Salmonella T3SS-1 effector involved in ruffle formation, bacterial entry and macropinosome formation [ 12 , 67 ]. Considering ESCRT-0’s established role in cargo sorting within specialized membrane subregions [ 60 ], it is possible that SopB and HGS are co-sorted into ILVs at these sites, potentially together with other host proteins. This hypothesis is supported by the reported presence of SopB on exosomes derived from Salmonella -infected cells [ 68 ]. To explore whether our observations are recapitulated during infection, we examined ESCRT-0 dynamics in infected cells. HGS, the core ESCRT-0 subunit, was found to accumulate at IAMs shortly after bacterial entry and at SCVs around 30 minutes post-infection ( Fig. 3a-d ) , paralleling the established spatiotemporal distribution of SopB [ 42 ]. Notably, SopB enhances the recruitment of HGS to the SCV ( Fig. 3c ), suggesting that it modulates ESCRT-0 localization during infection. SopB is mono-ubiquitinated on multiple lysine residues in host cells [ 24 , 44 ]. Mining our Virotrap data for diGly modifications corroborated reported ubiquitination of SopB at multiple lysines— K19, K41, K93, and K541 ( Fig. 4a ). Ubiquitination has been associated with SopB relocation from the plasma membrane to the SCV in Caco-2 cells [ 24 ] and with prolonged SopB retention at the SCV in HeLa cells [ 44 ]. Interestingly, these lysines appear to be highly conserved across diverse Salmonella serovars [ 44 ], underscoring their importance in Salmonella pathogenesis. Indeed, our NanoBiT assay support an essential role for ubiquitination in SopB binding to ESCRT-0 ( Fig. 4d ). Deletion of both ubiquitin-binding domains of HGS (VHS and UIM) abolished the interaction with SopB in NanoBiT. Recent research suggests that multimeric ESCRT-0 assembles in response to localized increases of PI3P at the membrane and engages multiple low-affinity ubiquitin interactions with high overall avidity upon condensation of ubiquitinated cargo [ 69 , 70 ]. Consequently, it is likely that the loss of both ubiquitin-binding domains of HGS is required to disrupt SopB-ESCRT-0 binding. The evolutionary stability and central role of the ESCRT machinery make it an attractive target for intracellular pathogens, yet direct exploitation by Salmonella effectors had not been documented. Our findings identify SopB as a bacterial effector that binds and recruits the host ESCRT-0 complex. The distinctive mono-ubiquitination pattern of SopB, together with its ubiquitin-dependent interaction with HGS, supports a model in which SopB functions as a molecular mimic of ubiquitinated host cargo, possibly modulating SCV membrane composition ( Fig. 4e ). Whether such sophisticated mimicry extends to other pathogens remains an open and compelling avenue for future research. Funding This work was supported by the Research Foundation Flanders (FWO) (project G042918N to S.E.; project G045921N to P.V.D.; projects 3G046420, G0A7L24N and EOS grant G0I5722N to M.J.M.B.; post-doctoral fellowship 1270825N to J.H.); the Dutch Research Council (NWO) (Veni grant VI.Veni.222.124 to V.S.); the European Research Council (ERC) under the European Union’s research and innovation programme (Starting Grant PROPHECY, grant agreement 803972, to P.V.D.); the Special Research Fund (BOF) – Concerted Research Actions (GOA) (projects BOF-GOA-2022-0003-03, BOF16-GOA-023 to S.E.; project BOF23-GOA-001 to P.V.D. and M.J.M.B.); Ghent University grant (project BOF-BAF-4Y-2024-01-462 to S.E.; project iBOF ATLANTIS 01IB3920 to M.J.M.B., post-doctoral mandate BOF22-PDO-024 to L.D.). Materials and Methods Cell Culture HEK293T cells originate from [ 73 ] (Rufer lab, CHUV, Lausanne) and were maintained in high-glucose Dulbecco’s Modified Eagle Medium containing GlutaMAX (DMEM; Gibco, cat no. 10566016), supplemented with 10% fetal bovine serum (FBS, Gibco, cat no. 10270106) and 100 U/mL penicillin/streptomycin (Gibco, cat no. 15070063). HeLa cells were cultured in Minimum Essential Medium (MEM; Gibco, cat. no. 11534496), supplemented with non-essential amino acids (Gibco, cat. no. 11140035) and sodium pyruvate (Gibco, cat. no. 11360070). Cells were kept in a humidified incubator at 37 °C and 5% CO2. Cloning and Plasmids Cloning was performed using Escherichia coli strain DH10B with standard chemical transformation. Salmonella Typhimurium SL1344 genomic DNA was extracted as described in [ 32 ] and served as a template for sopB coding sequence amplification. Human coding sequences were PCR-amplified from cDNA synthesized using the SuperScript IV first-strand synthesis system (Thermo Fisher, cat. no. 18091050). The cDNA was derived from RNA isolated from HEK293T or HeLa cells using TRIzol reagent (Thermo Fisher, cat. no. 15596026). Amplicons were purified by means of a Nucleospin® Gel and PCR clean-up kit (Machinery-Nagel) or E.Z.N.A.® Plasmid DNA Mini Kit II. (Omega Biotek) according to the manufacturers’ instruction. Coding sequences were cloned into the Gateway® pDONR221 and pMET7-GAG-SP1 [ 30 ] using BP Clonase II Enzyme (Invitrogen) and LR Clonase II Plus Enzyme (Invitrogen), respectively, for Virotrap. The pMD2.G (VSV-G envelope-expressing plasmid; Addgene, plasmid no. 12259) was retrieved from Addgene, the pcDNA3-FLAG-VSV-G (Addgene, plasmid no. 80606) plasmid was provided by the Eyckerman lab and the pSVsport vector was obtained from Life Technologies. Other constructs were created using the BsaI-based Golden Gate assembly cloning system as described in [ 74 ] for genetic fusions with EGFP, mScarlet, LgBiT-FLAG and SmBiT-V5. Mutant SopB constructs were generated from the wild-type SopB coding sequence using the Quickchange II site-directed mutagenesis kit (Agilent, cat. no. 200523). Stable Cell Line Generation To generate HeLa cell lines stably expressing HGS-mScarlet, lentiviral constructs were prepared encoding HGS-mScarlet along with a puromycin resistance cassette for antibiotic selection. Subsequently, these constructs were used to produce lentiviruses following the procedure outlined in [ 74 ]. HeLa cells equipped with the doxycycline-inducible rtTA/tTS system, as detailed [ 75 ], were transduced with HGS-mScarlet lentivirus at an MOI of 1. The medium was replaced the next day. Two days after transduction, 1 µg/mL of puromycin was introduced into the cell culture medium, based on previously established kill-curves in the lab. Selection using puromycin continued for at least two days, or until no cells from the parental lines survived under the same conditions. Virotrap Virotrap was performed as described previously [ 76 , 77 ]. In brief, ten million HEK293T cells were seeded per T75 flask in complete DMEM and transfected the next day using polyethylenimine (PEI) reagent (linear 25 kDa, Polysciences, Inc.). The transfected DNA/PEI mixture consisted of 0.71 μg pcDNA3-FLAG-VSV-G, 0.36 μg pMD2.G, 6.43 μg pMET7-GAG-SP1-sopB (mutant) or 3.75 µg pMET7-GAG-SP1-eDHFR and 2.67 µg pSVsport (control samples) and 37.5 μL PEI (1 mg/mL solution in MQ, pH 7.0) per T75. The cellular supernatant was harvested 48 h after transfection, spun at 1500xg for 3 minutes at room temperature and filtered through a 0.45 μm Millex® filter (Millipore). Per sample (equivalent of T75), 20 μL MyOne Streptavidin T1 beads (10 mg/mL; Invitrogen), washed in 20 mM TRIS HCl pH 7.5 and 150 mM NaCl, was loaded with 2 μL anti-FLAG BioM2-biotin antibodies (1 mg/mL; ANTI-FLAG® BioM2, cat no. F9291, Sigma Aldrich) in 200 μL washing buffer by end-over-end rotation and incubation for 2 h. VLPs were allowed to bind the anti-FLAG-coated beads for 2 h by end-over-end rotation at room temperature. Bead-bound VLP complexes were washed once with washing buffer and eluted using 20 µL elution buffer (20 mM TRIS HCl pH 7.5, 150 mM NaCl, 200 µg/ml FLAG-peptide) and incubate for 30 minutes at 37°C. Subsequently, VLPs were lysed by addition of 2.2 µL amphipathic polymer solution (Amphipol A8-35, Anatrace; final concentration of 1 mg/mL) and incubation for 10 minutes. For protein concentration, proteins were pelleted from the lysates by acidification (0.2% final concentration formic acid) [ 78 ]. After acidification, protein pellets were dissolved in 20 μL 50 mM triethylammonium bicarbonate (TEAB) buffer (pH 8.5), boiled and digested overnight using 0.5 μg of sequence-grade modified trypsin (Promega). After a final acidification step (0.4% formic acid, final concentration), samples were separated on an UltiMate™ 3000 RSLCnano (Thermo Scientific) and analyzed on a Q Exactive HF instrument (Thermo Scientific; 7.5 μL injected, 1.5 h long run) as described previously [ 79 , 80 ]. Mass spectrometry Data Analysis Searches were performed using MaxQuant (Version 1.6.6.0) [ 81 ] against the human SwissProt Proteome Database (Release 2022-02) complemented with eDHFR, FLAG-VSV-G, VSV-G, Gag and SL1344 SopB protein sequences. In MaxQuant, multiplicity was set to one, indicating that no labels were used. Furthermore, we performed label-free quantification (LFQ) using MaxQuant’s standard settings with a minimum of two ratio counts and considering unique and razor peptides for protein quantification. A decoy database of reversed protein sequences was used to estimate FDR, and 1% FDR threshold was applied. Di-glycine, methionine oxidation and N-terminal protein acetylation were set as variable modifications and trypsin/P was set as the digestion enzyme allowing two missed cleavages. N-terminal acetylation was included in protein quantification. The MaxQuant ProteinGroups data file was processed using R Studio (R Foundation for Statistical Computing, V1.3.959) and custom R scripts. The dataset was further filtered based on reversed hits, potential contaminants and proteins only identified by site. For identifications with LFQ values calculated for all bait replicates ( i.e ., 4 valid values), LFQ intensities were log2 transformed and missing values were imputed using the QRILC function with default parameters from the imputeLCMD package in R [ 82 ]. Significance was assessed using limma in R [ 83 ] as previously demonstrated [ 32 ]. Replicate samples were grouped and compared to the control samples (Gag-eDHFR or Gag-SopB-WT) in a pairwise analysis. Basic data handling and Pearson correlation calculations were performed in Perseus V1.6.6.0 [ 84 ]. Network analysis was done using the STRING database [ 85 ] and Cytoscape [ 86 ] for additional visualization and ClueGo [ 87 ] enrichment analyses. Infection Salmonella infections were conducted following the procedures described in [ 88 ]. Bacteria were cultured overnight in 3 mL of LB containing 0.3 M NaCl and 50 μg/mL of either ampicillin or kanamycin at 37°C shaking at 220 r.p.m. On the day of infection, the bacteria were further cultured at a 1:21 dilution in the same medium and conditions for 3 hours until the late exponential growth phase. Following this, the bacteria were washed and resuspended in EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgCl 2 , 5 mM glucose, 25 mM HEPES, pH 7.3). The bacterial density was assessed using optical density at 600 nm and adjusted to the required multiplicity of infection (MOI) in warm EM buffer. All infections were performed at an MOI of 20. Cells were rinsed with EM buffer before bacterial addition and incubated for 30 minutes at 37°C in 5% CO 2 . Subsequent to this, cells were washed three times with sterile EM buffer to remove any extracellular bacteria. Depending on the specific experimental timeline, cells were either immediately fixed or continued to be incubated in EM buffer containing 10% FBS and 100 μg/mL gentamicin for one hour. For extended time points, the medium was replaced after one hour with EM buffer containing 10% FBS and gentamicin at 10 μg/mL. Confocal Microscopy HeLa cells were seeded into each well of an 8-well imaging μ-Slide (Ibidi GmbH, cat. no. 80826). For transient expression studies, cells in each well were transfected with 100 ng of DNA using a 1:3.5 ratio of Fugene HD (Promega, cat. no. E2311) for 18 hours before imaging. Transfection mixtures were prepared in reduced serum Opti-MEM medium (Gibco, cat. no. 15392402). Induction of HeLa-HGS-mScarlet cells was done using 20 ng/mL doxycycline for 18 hours. For fixed samples, cells were treated with 4% PFA in PBS, which was pre-warmed to 37°C, for 10 minutes at room temperature (RT). After fixation, cells were washed three times for 5 minutes in PBS and were stained using DAPI (Thermo Fisher Scientific, cat. no. D1306) at a dilution of 1:1,000 in PBS for 15 minutes at RT. After three washes using PBS, cells were preserved either in PBS at 4 °C. Microscopy acquisitions were performed on either an Andor Dragonfly 200 (Oxford Instruments) spinning disk microscope equipped with a 63x/1.40-0.60 oil Plan Apo objective, and an Andor Zyla 4.2 PLUS sCMOS Camera ( Fig. 3 ; S2), or LSM880 Airyscan (Carl Zeiss, Jena, Germany) equipped with either a Plan-Apochromat 63x/1.4 Oil, DIC M27 ( Fig. 2 ). The LSM880 utilized ZEN Black 2.3 SP1 software with lasers of 405, 561, and 633 nm, and the 488 nm line from an Argon laser. The corresponding filter sets included BP 420-480, LP570, LP645, and BP 495-550, applied with the Airyscan detector in SuperResolution (SR) mode or in full Airyscan. For live-cell imaging, fast Airyscan mode was used. Post-acquisition, images were processed for pixel reassignment and 2D Wiener deconvolution in ZEN Black. Co-localization quantification was performed using ZEISS arivis software. NanoBiT HEK293T cells were cultured in black 96-well tissue culture plates (Greiner, cat. no. 3916) at a seeding density of 15,000 cells per well in complete DMEM. Cells were incubated at 37°C with 5% CO 2 for 16 hours to allow for attachment. Triplicate were prepared in 96-well plates. For each construct, 40 ng plasmid DNA was mixed with a final concentration of 0.25 M CaCl 2 in sterile water per well to be transfected. This mixture was combined with an equal volume of 2X HeBs (Sigma Aldrich, cat. no. 51558), incubated for 10 minutes at room temperature, and vortexed before transferring the combined transfection mixture to the well. After incubating for 20 hours at 37°C, the Nano-Glo® Live Cell Substrate (Promega, cat. no. N2012) was mixed according to the manufacturer’s instructions and added to cells by replacing the culture medium with the substrate mixture. Plates were gently swirled to distribute the substrate, and luminescence measurements were performed at 37°C using EnVision (PerkinElmer, 2102 Multilabel reader). Cells were washed using PBS and frozen at -20 °C for subsequent lysate preparation. SDS-PAGE and Western Blotting VLP and producer cell lysates were supplemented with sample loading buffer (XT sample buffer, Bio-Rad) and reducing agent (XT reducing agent, Bio-Rad) according to the manufacturer’s instructions. Proteins were separated on 4–12% gradient XT precast Criterion gel (Bio-Rad, cat. no. 3450123) using XT-MOPS buffer (Bio-Rad) at 150 V and subsequently transferred onto a PVDF membrane. For NanoBiT expression analysis, cells were lysed in 20 µL LDS loading buffer (4X, diluted in TBS, Genscript, cat. no. M00676) per well. Prior to separation on a 4–20% polyacrylamide gradient gel (GenScript, cat. no. M42015), protein samples were boiled for 10 minutes at 98 °C. Next, the protein samples were transferred onto a PVDF membrane for 3 hours at 60 V in blotting buffer (48 mM Tris, 39 mM glycine, 0.0375 % SDS and 20 % methanol). Membranes were blocked for 30 min in 1:1 Tris-buffered saline (TBS)/Odyssey blocking solution (cat no. 927-40003, Li-COR) and probed using primary antibodies in TBS-T/Odyssey blocking buffer. After three washes of 10 min in TBS-T (0.1% Tween-20), membranes were incubated with secondary antibody (1/5000 dilution; IRDye antibodies; Li-COR) for 30 min in TBS-T/Odyssey blocking buffer. Following three washes in TBS-T and one additional wash in TBS, fluorescent detection was done using an Odyssey infrared Fc imaging system (Li-COR) or Odyssey infrared scanner (Li-COR). Download figure Open in new tab Supplementary Figure 1. Immunoblot analysis of Gag-SopB virus-like particles (VLPs). VLPs from HEK293T cells expressing Gag-SopB constructs were captured from culture supernatants by anti-FLAG immunoprecipitation. Wild-type (WT), catalytic mutant (C460S), coiled-coil deletion mutant (omitted from our study since no expression was obtained), and reduced catalytic mutant (K527A). Download figure Open in new tab Supplementary Figure 2. IAM and SCV formation during SL1344 invasion upon HGS depletion. HeLa cells were subjected to HGS depletion using siRNA for 72 hours and were infected with SL1344-WT(dsRed). All images display infection sites within a few minutes after bacterial entry (i.e., timeframe following ruffle formation). Yellow arrowhead: SCV, Blue arrowhead: IAM. Supplementary Video 1. Cropped view of time-lapse microscopy of HeLa cells expressing mScarlet-HGS upon doxycycline induction (white) and infected with GFP-expressing Salmonella SL1344 WT (green). 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