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• W3033854583 abstract "Article8 June 2020Open Access Source DataTransparent process Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions Miriam Kutsch orcid.org/0000-0002-9346-2196 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Linda Sistemich Department of Physical Chemistry I, Ruhr-University Bochum, Bochum, Germany Search for more papers by this author Cammie F Lesser Division of Infectious Diseases, Center for Bacterial Pathogenesis, Massachusetts General Hospital, Boston, MA, USA Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Marcia B Goldberg Division of Infectious Diseases, Center for Bacterial Pathogenesis, Massachusetts General Hospital, Boston, MA, USA Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Christian Herrmann Department of Physical Chemistry I, Ruhr-University Bochum, Bochum, Germany Search for more papers by this author Jörn Coers Corresponding Author [email protected] orcid.org/0000-0001-8707-4608 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Department of Immunology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Miriam Kutsch orcid.org/0000-0002-9346-2196 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Linda Sistemich Department of Physical Chemistry I, Ruhr-University Bochum, Bochum, Germany Search for more papers by this author Cammie F Lesser Division of Infectious Diseases, Center for Bacterial Pathogenesis, Massachusetts General Hospital, Boston, MA, USA Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Marcia B Goldberg Division of Infectious Diseases, Center for Bacterial Pathogenesis, Massachusetts General Hospital, Boston, MA, USA Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA Search for more papers by this author Christian Herrmann Department of Physical Chemistry I, Ruhr-University Bochum, Bochum, Germany Search for more papers by this author Jörn Coers Corresponding Author [email protected] orcid.org/0000-0001-8707-4608 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Department of Immunology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Author Information Miriam Kutsch1, Linda Sistemich2, Cammie F Lesser3,4, Marcia B Goldberg3,4, Christian Herrmann2 and Jörn Coers *,1,5 1Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA 2Department of Physical Chemistry I, Ruhr-University Bochum, Bochum, Germany 3Division of Infectious Diseases, Center for Bacterial Pathogenesis, Massachusetts General Hospital, Boston, MA, USA 4Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA 5Department of Immunology, Duke University Medical Center, Durham, NC, USA *Corresponding author. Tel: +1 919 684 7109; E-mail: [email protected] EMBO J (2020)39:e104926https://doi.org/10.15252/embj.2020104926 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In the outer membrane of gram-negative bacteria, O-antigen segments of lipopolysaccharide (LPS) form a chemomechanical barrier, whereas lipid A moieties anchor LPS molecules. Upon infection, human guanylate binding protein-1 (hGBP1) colocalizes with intracellular gram-negative bacterial pathogens, facilitates bacterial killing, promotes activation of the lipid A sensor caspase-4, and blocks actin-driven dissemination of the enteric pathogen Shigella. The underlying molecular mechanism for hGBP1's diverse antimicrobial functions is unknown. Here, we demonstrate that hGBP1 binds directly to LPS and induces “detergent-like” LPS clustering through protein polymerization. Binding of polymerizing hGBP1 to the bacterial surface disrupts the O-antigen barrier, thereby unmasking lipid A, eliciting caspase-4 recruitment, enhancing antibacterial activity of polymyxin B, and blocking the function of the Shigella outer membrane actin motility factor IcsA. These findings characterize hGBP1 as an LPS-binding surfactant that destabilizes the rigidity of the outer membrane to exert pleiotropic effects on the functionality of gram-negative bacterial cell envelopes. Synopsis Human Guanylate Binding Protein 1 (hGBP1) provides intracellular host defense to gram-negative bacteria expressing lipopolysaccharide (LPS) on their surfaces. Using novel in vitro binding assays as well as cell biological approaches this study reveals that hGBP1 operates as an LPS-binding and -clustering surfactant to destabilize the gram-negative outer membrane, thereby rendering hGBP1-bound bacteria susceptible to attacks by other antimicrobial molecules. Polymerizing hGBP1 binds directly to LPS and to gram-negative bacteria. A stable hGBP1 protein coat is formed on bacteria expressing O-antigen. hGBP1 clusters LPS molecules and disrupts the O-antigen barrier function. hGBP1 disturbs O-antigen-dependent function of the Shigella virulence protein IcsA. Introduction Cell-autonomous immunity describes the ability of individual cells within a multicellular organism to activate a wide range of cell-intrinsic host defense programs directed at intracellular pathogens. These ancient, hard-wired defense programs are commonly under spatial and temporal control and often require inducing signals to launch (Howard, 2007; Randow et al, 2013). Microbe-associated molecular patterns such as the gram-negative bacterial outer membrane molecule lipopolysaccharide (LPS) can act as such inducing signals in infected cells by activating specialized pattern recognition receptors that include the cytosolic LPS sensor caspase-4 (Hagar et al, 2013; Kayagaki et al, 2013; Shi et al, 2014). Alternatively, proinflammatory cytokines released by sentinel and immune effector cells instruct host cells to transition into a state of heightened antimicrobial resistance. Perhaps the most potent inducer of cell-autonomous immunity against bacterial pathogens is the lymphocyte-derived cytokine gamma-interferon (IFNγ), which controls the expression of hundreds of antimicrobial proteins encoded by IFN-stimulated genes (ISGs). The specific functions of many of these ISGs are either unknown or only poorly characterized (MacMicking, 2012). Among the most highly expressed ISGs are guanylate binding proteins (GBPs) which have important roles in antibacterial host defense (Coers, 2017; Man et al, 2017; Praefcke, 2018; Santos & Broz, 2018; Huang et al, 2019). GBPs promote the lysis of gram-negative bacteria inside infected macrophages (Man et al, 2015; Meunier et al, 2015; Li et al, 2017; Liu et al, 2018) and aid in the activation of the LPS sensor caspase-4 in response to infections, bacterial outer membrane vesicles or cytoplasmic LPS (Meunier et al, 2014; Pilla et al, 2014; Finethy et al, 2017; Lagrange et al, 2018; Santos et al, 2018). Human GBP1 also interferes with the ability of the cytosol-invading bacterial pathogen Shigella flexneri to usurp the host actin polymerization machinery for intracellular bacterial motility and cellular dissemination (Piro et al, 2017; Wandel et al, 2017). The molecular mechanisms by which GBPs can exert these seemingly distinct cellular functions are undetermined; yet, they appear linked to the capacity of GBPs to specifically associate with intracellular bacterial pathogens. Human GBP1 (hGBP1) colocalizes with the cytosol-resident gram-negative bacterial pathogens Burkholderia thailandensis and S. flexneri but not gram-positive Listeria monocytogenes (Piro et al, 2017). Four of the six additional hGBP paralogs also associate with cytosolic S. flexneri, but in a strictly hGBP1-dependent manner (Li et al, 2017; Piro et al, 2017; Wandel et al, 2017). Together, these reported observations hinted at the intriguing yet untested model that hGBP1 is unique among hGBPs in its ability to operate as a bona fide cytosolic receptor for gram-negative bacteria. Here, we demonstrate that polymerizing hGBP1 attaches directly to gram-negative bacteria via LPS binding. Following the initial attachment, hGBP1 transitions into a stable protein coat encapsulating exclusively “smooth” bacterial strains, i.e., bacteria expressing the outermost O-antigen polysaccharide segment of LPS. We find that hGBP1 coating of bacteria disrupts the O-antigen barrier protective against sublethal concentrations of the antimicrobial peptide polymyxin B, enables the recognition of lipid A by caspase-4 on the bacterial surface, and also interferes with the function of the Shigella outer membrane protein IcsA critical for intracellular bacterial motility. Together, our observations designate hGBP1 as an LPS-binding and LPS-clustering surfactant that disrupts critical functions of the gram-negative bacterial outer membrane and thereby promotes diverse antibacterial host defense pathways. Results Farnesylated hGBP1 binds directly to Shigella flexneri in a GTPase-dependent manner As typical for a dynamin superfamily protein (Daumke & Praefcke, 2016; Ramachandran & Schmid, 2018), hGBP1 consists of a large N-terminal globular G domain (LG domain) and a helical C-terminus that segregates into the middle domain (MD) and the GTPase effector domain (GED) (Fig 1A). In the presence of its substrate GTP, hGBP1 dimerizes (Ince et al, 2017) and binds to membranes via hydrophobic interactions mediated by its C-terminal farnesyl moiety that is otherwise buried within a hydrophobic pocket when hGBP1 is nucleotide-free (Fres et al, 2010; Shydlovskyi et al, 2017; Ji et al, 2019). Therefore, in order to test whether hGBP1 is able to bind to the outer membrane of the cytosolic gram-negative bacterium S. flexneri, we mixed fluorescently labeled farnesylated hGBP1 (hGBP1F) with either formaldehyde-fixed or live bacteria in the presence of guanine nucleotides and captured confocal images following varying incubation times (Fig 1B). We observed direct binding of hGBP1F to both fixed and live S. flexneri in the presence of GTP but not GDP (Fig 1C and Appendix Fig S1A) across a physiological range (Naschberger et al, 2006) of hGBP1 protein concentrations (Appendix Fig S1B). Consistent with colocalization studies performed in tissue culture (Li et al, 2017; Piro et al, 2017; Wandel et al, 2017), we found that mutations disrupting nucleotide binding (hGBP1FK51A), GTP hydrolysis (hGBP1FR48A) or GDP hydrolysis (hGBP1FH74A) (Praefcke et al, 2004), or lack of protein farnesylation stopped hGBP1 from binding to S. flexneri in vitro (Fig 1C). These data demonstrate that hGBP1F binds directly to S. flexneri through a GTP-hydrolysis-dependent process. Figure 1. Farnesylated hGBP1 binds directly to Shigella flexneri in a GTPase-dependent manner A. Structure of full-length, nucleotide-free, farnesylated hGBP1 (PDB entry 6k1z) and GDP·AlFX-bound LG-domain dimer (PDB entry 2B92). Insert (i) shows the farnesyl moiety and the triple arginine stretch (3R = R584–586). Insert (ii) highlights residues required for nucleotide binding and hydrolysis. B. Experimental design: fluorescently labeled recombinant hGBP1 variants and nucleotides were mixed with broth-cultured live or fixed bacteria; bacterial binding was monitored by confocal microscopy. C. Confocal images of formaldehyde-fixed GFP+ S. flexneri following 20 min of incubation with 2 mM GTP and 10 μM Alexa-Fluor647-labeled protein. Bacteria associated with hGBP1 mutants after 20 min were quantified. Mean frequencies ± SEM of combined data from two independent experiments are shown. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ***P ≤ 0.001. Scale bars equal 5 μm. Flow diagram depicts effects of K51A, R48A, and H74A hGBP1 mutations on nucleotide binding and hydrolysis. Source data are available online for this figure. Source Data for Figure 1 [embj2020104926-sup-0010-SDataFig1.xlsx] Download figure Download PowerPoint hGBP1 polymerization is required for bacterial binding When expressed in cells, hGBP1 forms discrete granular structures (Britzen-Laurent et al, 2010). In time-lapse microscopy experiments using a S. flexneri mutant strain deficient for the bacterial hGBP1 antagonist IpaH9.8 shown to dramatically reduce hGBP1 recruitment to cytosolic bacteria (Li et al, 2017; Piro et al, 2017; Wandel et al, 2017), we observed the appearance of these hGBP1 granular structures in close proximity to cytosolic bacteria and furthermore recorded the transition of these granules into a hGBP1 protein coat encapsulating entire bacteria (Fig 2A and Movie EV1). Similar to the intracellular dynamics of hGBP1 targeting to S. flexneri, we found that upon GTP supplementation hGBP1F granular structures formed rapidly in vitro and associated with bacterial surfaces. In remarkable symmetry to our live cell imaging data, these bacteria-associated hGBP1 granules then transformed into a protein coat encasing individual bacteria in vitro (Figs 2B and EV1A, and Movie EV2). Figure 2. hGBP1 polymerization is required for bacterial binding A. Translocation of ectopically expressed mCherry-hGBP1 to cytosolic GFP+ Shigella flexneri ΔipaH9.8 in HeLa hGBP1-KO cells was monitored by time-lapse microscopy. Individual time frames of Movie EV1 starting at 55 min post-infection (mpi) are shown. B. Confocal time-lapse microscopy was used to image 10 μM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP in the presence or absence of formaldehyde-fixed GFP+ S. flexneri. Individual time frames of Movie EV2 depict hGBP1F fluorescence intensity. Merged images of hGBP1F and S. flexneri fluorescence are shown for the 60 min time points. C. Images were taken at 45 min after addition of 10 μM Alexa-Fluor647-hGBP1F to formaldehyde-fixed GFP+ S. flexneri in the presence of indicated nucleotides (GTP, natural substrate; GppNHp, non-hydrolysable GTP analog; GTPγS, slowly hydrolysable GTP analog; GDP·AlFX, GTP transition state analog). hGBP1-associated bacteria after 45 min were quantified. Combined data from two independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****P ≤ 0.0001. D. Model: hGBP1 polymers bind to S. flexneri directly and transition into a bacterium-encapsulating protein coat. Data information: All scale bars equal 5 μm. Source data are available online for this figure. Source Data for Figure 2 [embj2020104926-sup-0011-SDataFig2.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Polymerizing hGBP1F binds directly to Shigella flexneri across a physiological range of hGBP1F protein concentrations A. Time-lapse microscopy frames of formaldehyde-fixed GFP+ S. flexneri following admixture of varying concentrations of Alexa-Fluor647-labeled hGBP1F and 2 mM GTP. Individual time frames depict hGBP1F fluorescence intensity. Merged images of hGBP1F and S. flexneri fluorescence are shown for the 60 min time points. B. Polymerization of 10 μM hGBP1F monitored over time by absorption spectroscopy at 350 nm after addition of 2 mM (GTP, GppNHp, GTPγS) or 250 μM (GDP·AlFX) nucleotide. C. Confocal images taken of formaldehyde-fixed GFP+ S. flexneri and rhodamine-labeled lipid vesicles 20 min after addition of 10 μM Alexa-Fluor647-hGBP1F and 2 mM GTP, 2 mM GTPγS, or 250 μM GDP·AlFX. D. Model: In its GTP-bound conformation, the C-terminal farnesyl tail is released from its hydrophobic pocket allowing hGBP1 to bind to lipid vesicles in vitro and to host membranes inside human cells. Direct binding to bacteria on the other hand requires hGBP1 to self-assemble over several GTP turnover cycles into polymers. These hGBP1 polymers bind to S. flexneri directly and then transition into a bacteria-encapsulating protein coat. Data information: All scale bars equal 5 μm. Source data are available online for this figure. Download figure Download PowerPoint Although hGBP1F granules detected inside cells were initially interpreted as membrane-associated vesicle-like structures (Britzen-Laurent et al, 2010), our recent in vitro studies showed that hGBP1F not only binds to membranes but also self-assembles into large polymers independent of exogenous lipids (Shydlovskyi et al, 2017; Sistemich et al, 2020). We therefore surmised that the hGBP1F granules that were forming both in the presence and in the absence of exogenously added bacteria in vitro (Fig 2B) constituted hGBP1F polymers. In support of this notion and in confirmation of previous work (Shydlovskyi et al, 2017), we found that the build-up of supramolecular hGBP1F particles measured by spectroscopy occurred only in the presence of hydrolysable GTP (Fig EV1B). We further observed that the poorly hydrolysable analog GTPγS—while sufficient to direct the attachment of hGBP1F to artificial vesicles (Fig EV1C), as previously reported (Shydlovskyi et al, 2017)—failed to engender in vitro hGBP1F attachment to bacteria (Figs 2C and EV1C, and Movies EV3 and EV4). Similarly, mimicking the hGBP1 GTP hydrolysis transition state through admixture of GDP·aluminum fluoride (GDP·AlFX) triggered hGBP1F binding to vesicles but not bacteria (Figs 2C and EV1C, and Movie EV5). Because hGBP1F forms irreversible short polymers in the presence of GDP·AlFX (Shydlovskyi et al, 2017; Sistemich et al, 2020), these results indicate that reversible hGBP1F polymerization is required for direct binding of hGBP1F to S. flexneri (Figs 2D and EV1D). The C-terminal hGBP1 polybasic motif is required for sustained binding to Shigella flexneri Human GBP1 polymers have a ring-like structure with individual hGBP1 molecules assembled around the hydrophobic core formed by their farnesyl moieties (Shydlovskyi et al, 2017). Immediately adjacent to the C-terminal farnesyl group exists a short polybasic motif containing a stretch of three arginines (3R) (Fig 1A). We previously demonstrated that the C-terminal polybasic motif containing the 3R stretch is unique to hGBP1 among the human GBP family and required for efficient colocalization of hGBP1 with intracytosolic S. flexneri (Piro et al, 2017). To determine the mechanisms by which 3R drives hGBP1 translocation to S. flexneri, we assessed the ability of recombinant hGBP1FR584-586A, a mutant lacking the 3R stretch, to form polymers and to bind to bacteria in vitro. First, we monitored protein polymerization by absorption spectroscopy and found that hGBP1FR584-586A could still form large particulates, albeit with delayed kinetics (Figs 3A and EV2A). Because hGBP1 undergoes polymerization-accelerated cooperative hydrolysis (Praefcke et al, 2004; Shydlovskyi et al, 2017), the apparent slowdown in polymerization kinetics likely explains the moderately reduced rate of GTP hydrolysis that we observed in reactions with the hGBP1FR584-586A mutant (Figs 3A and C, and EV2B). This conclusion is supported by our observation that the R584-586A mutation has no impact on the GTP hydrolysis rates of non-farnesylated and thus non-polymerizing hGBP1 (Fig EV2C). Figure 3. A C-terminal hGBP1 polybasic motif is required for sustained binding to Shigella flexneri A. Polymerization of 10 μM hGBP1F and hGBP1FR584-586A in the presence of 2 mM GTP was monitored over time by absorption spectroscopy at 350 nm. Absorbance signals were superimposed with nucleotide composition of the same solution analyzed at defined time points, revealing the characteristic first phase of slow polymer nucleation and the second phase of fast polymer growth and cooperative hydrolysis. B. Scanning electron micrographs of live S. flexneri incubated with no protein or with 5 μM of either hGBP1F, hGBP1FR584-586A, or hGBP1FR48A (non-polymerizing mutant) in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces. C. Confocal time-lapse microscopy frames of formaldehyde-fixed GFP+ S. flexneri following admixture of 10 μM Alexa-Fluor647-labeled hGBP1F or hGBP1FR584-586A and 2 mM GTP. Binding of hGBP1F polymers to bacteria at 5 min and enclosure of bacteria with hGBP1F protein coats after 60 min were quantified. Mean frequencies ± SEM of combined data from at least three independent experiments are shown. Significance was determined by unpaired t-tests, two-tailed. ***P ≤ 0.001; ****P ≤ 0.0001. Data information: All scale bars equal 5 μm. Source data are available online for this figure. Source Data for Figure 3 [embj2020104926-sup-0012-SDataFig3.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. hGBP1F polymerization is dependent on GTP, and subsequent GDP hydrolysis and its dynamics are modified by the C-terminal 3R stretch A. Polymerization of 10 μM hGBP1FR584-586A was monitored over time by absorption spectroscopy at 350 nm after admixture of 2 mM (GTP, GppNHp, GTPγS) or 250 μM (GDP·AlFX) nucleotide. B. Polymerization of different hGBP1 variants (at 10 μM) induced by 2 mM GTP was monitored over time by absorption spectroscopy at 350 nm. Absorbance signals were superimposed with nucleotide composition analyzed at defined time points of the corresponding sample. Blots for hGBP1F and hGBP1FR584-586A are the same as shown in Fig 3A. C. GTP turnover numbers were determined for different hGBP1 variants. Turnover numbers for both the slow and the fast phases of GTP hydrolysis (1st phase [1.], 2nd phase [2.]) were quantified during hGBP1F and hGBP1FR584-586A polymerization. Combined data from three independent experiments are shown as mean turnover numbers ± SEM. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; **P ≤ 0.01; ****P ≤ 0.0001. Source data are available online for this figure. Download figure Download PowerPoint To interrogate the function of the C-terminal polybasic motif further, we visualized hGBP1F polymer formation using scanning electron microscopy. After 4 min of incubation with GTP, we observed polymeric structures in the presence of hGBP1F or hGBP1FR584-586A but not in the presence of the non-polymerizing mutant hGBP1FR48A (Fig 3B). Notably, the polymers formed by hGBP1FR584-586A appeared morphologically distinct from hGBP1F polymers. Moreover, hGBP1F but not hGBP1FR584-586A polymers appeared to form continuous connections with the outer surface of S. flexneri (Fig 3B), suggesting that hGBP1FR584-586A polymers could be functionally distinct from hGBP1F polymers. We therefore tested whether bacterial binding, or alternatively bacterial hGBP1 coating, was impacted by the R584-586A mutation. We noticed that hGBP1FR584-586A polymers were found in direct contact with bacteria at 5 min post-hGBP1FR584-586A and GTP supplementation, albeit with a two-fold reduction in the total number of hGBP1-bound bacteria compared to hGBP1F (Fig 3C). Although initial bacterial binding appeared relatively intact, hGBP1FR584-586A failed to form a protein coat enclosing individual bacteria at any point during the 1 h incubation time (Fig 3C). Collectively, these observations suggest that the C-terminal polybasic motif alters the dynamics of hGBP1F polymerization and is required for the tight association of hGBP1F with the bacterial surface. Direct binding of hGBP1F to LPS mediates its association with gram-negative bacteria Our finding that hGBP1F polymers attached directly to S. flexneri led us to hypothesize that hGBP1F binds a non-self substrate exposed on bacterial outer membranes. To test this hypothesis, we first monitored binding of hGBP1F to a diverse set of pathogenic bacterial species. We found that hGBP1F polymers bound not only to S. flexneri but also to other gram-negative human bacterial pathogens that we tested, namely Salmonella enterica Typhimurium (ST), Legionella pneumophila (Lp), and uropathogenic Escherichia coli (UPEC). However, we did not observe any binding of hGBP1F to the gram-positive bacteria Listeria monocytogenes (Lm) and Staphylococcus aureus (Sa) (Fig 4A). These results indicated that hGBP1F polymers recognized a molecule present in gram-negative but not in gram-positive bacterial cell envelopes. Because the surface-exposed, lipidated sugar LPS is a highly expressed component of gram-negative outer membranes but absent from gram positives (Simpson & Trent, 2019), we asked whether hGBP1F could bind directly to LPS. To test this hypothesis, we mixed fluorescently labeled, smooth (O-antigen+) E. coli LPS (O55:B55) or rough (O-antigen−) Salmonella minnesota LPS (SM) with hGBP1F in the presence of GTP and monitored colocalization of hGBP1F with LPS over time. As expected, GTP induced the formation of hGBP1F polymers, apparent as granular structures (Fig 4B). Notably, these hGBP1F granules colocalized with LPS clusters that formed simultaneously with hGBP1F granules and were also dependent on farnesylation as well as hGBP1F polymerization, since polymerization-deficient hGBP1F mutants (K51AF, R48AF, H74AF) and non-farnesylated hGBP1 did not induce LPS clusters (Figs 4B and EV3A, and Movie EV6). Notably, hGBP1 polymerization facilitated the clustering of both smooth LPS O55:B55 and rough LPS SM (Figs 4B and EV3A), demonstrating that O-antigen is dispensable for LPS binding by polymerizing hGBP1. We next confirmed hGBP1F binding to LPS in dot-blot assays (Fig EV3B). The polymerization-competent but 3R-deficient mutant hGBP1FR584-586A was able to bind and cluster LPS in suspension (Fig 4B) but failed to adhere to nitrocellulose-bound LPS in dot-blot assays (Fig EV3B), further underscoring the importance of the 3R stretch in enabling sustained hGBP1F binding to an LPS-decorated surface. To further characterize the dynamics of hGBP1F-LPS interactions, we measured hGBP1F polymerization and GTP hydrolysis in the presence and absence of LPS O55:B5. We observed faster initiation of hGBP1F polymerization and accelerated GTP hydrolysis kinetics in the presence of LPS (Fig 4C), revealing a role for LPS as a potential nucleation-promoting factor for hGBP1 polymerization. LPS is an amphipathic molecule that forms micelles in aqueous solution (Santos et al, 2003), and we can therefore conclude from the combined data that hGBP1F binds directly to LPS micelles, which results in accelerated hGBP1F polymerization as well as the assembly of large LPS aggregates in a polymerization-dependent manner. Figure 4. Direct binding of hGBP1F to LPS mediates its association with gram-negative bacteria A. Representative confocal images taken at 60 min after admixture of 2 mM GTP and 10 μM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F to formaldehyde-fixed gram-negative and gram-positive bacteria expressing GFP (Salmonella enterica Typhimurium [ST], L. monocytogenes [Lm]), or RFP (Shigella flexneri [Sf], uropathogenic E. coli [UPEC]), or dsRed (L. pneumophila [Lp], S. aureus [Sa]). B. (Upper panel) Confocal time-lapse microscopy frames of 5 μM Alexa-Fluor488-LPS-O55:B5 after addition of 5 μM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP. (Lower panel) Confocal images taken of 5 μM Alexa-Fluor488-LPS-O55:B5 20 min after addition of 5 μM Alexa-Fluor647-hGBP1F or Alexa-Fluor647-hGBP1FR48A supplemented with 2 mM GTP. Graphs depict average aggregate area of Alexa-Fluor488-LPS-O55:B5 supplemented with 2 mM GTP and 5 μM of the indicated hGBP1 variant. Mean area ± SEM of combined data from three independent experiments. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ***P ≤ 0.001; ****P ≤ 0.0001. C. Polymerization of 5 μM hGBP1F induced by addition of 2 mM GTP in the presence and absence of 5 μM LPS-O55:B5 was monitored by absorption spectroscopy at 350 nm. The absorbance signal was superimposed with nucleotide composition of the same solution analyzed at defined time points. Maximal hydrolysis rates (turnover numbers) were determined for hGBP1F variants in the presence and absence of 5 μM LPS-O55:B5. Graphs show mean turnover numbers ± SEM of combined data from at least three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ***P ≤ 0.001; ****P ≤ 0.0001. D. Formaldehyde-fixed GFP+ S. flexneri supplemented with varying LPS-O55:B5 concentrations were mixed with 5 μM hGBP1F and 2 mM GTP. After 60 min, hGBP1F-enclosed S. flexneri were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****P ≤ 0.0001. E. Confocal images of formaldehyde-fixed GFP-, RFP-, or dsRed-expressing gram-negative bacteria 60 min after addition of 5 μM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F and 2 mM GTP in the presence and absence of 100 μM LPS-O55:B5. After 60 min, hGBP1F-enclosed gram-negative bacteria were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by two-way A" @default.
• W3033854583 created "2020-06-12" @default.
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• W3033854583 date "2020-06-08" @default.
• W3033854583 modified "2023-10-15" @default.
• W3033854583 title "Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions" @default.
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