Human giant GTPase GVIN1 forms an antimicrobial coatomer around the intracellular bacterial pathogen Burkholderia thailandensis

  1. Weilun Guo
  2. Shruti S Apte
  3. Mary S Dickinson
  4. So Young Kim
  5. Miriam Kutsch
  6. Jörn Coers

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    eLife Assessment

    This fundamental study identifies GVIN as a previously unrecognized interferon-induced antimicrobial GTPase and establishes its role in cell-autonomous immunity against intracellular Burkholderia. The compelling data demonstrate that GVIN assembles into coat-like structures on the bacterial surface and restricts BimA-dependent actin-based motility through a mechanism independent of GBP1. These findings expand our understanding of interferon-induced host defense mechanisms and reveal a distinct pathway by which human cells restrict the intracellular spread of bacterial pathogens.

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Abstract

Several human pathogens exploit the kinetic forces generated by polymerizing actin to power their intracellular motility. Human cell-autonomous immune responses activated by the cytokine interferon-gamma (IFNγ) interfere with such microbial actin-based motility, yet the underlying molecular mechanisms are poorly defined. Here, we identify the IFNγ-inducible human giant GTPases GVIN1 as a novel host defense protein that blocks the bacterial pathogen Burkholderia thailandensis from high-jacking the host’s actin polymerization machinery. We found that GVIN1 proteins form a coatomer around cytosolic bacteria and prevent Burkholderia from establishing force-generating actin comet tails. Coatomers formed by a second IFNγ-inducible GTPase, human guanylate binding protein 1 (GBP1), constitute a GVIN1-independent but mechanistically related anti-motility pathway. We show that coating with either GVIN1 or GBP1 displaces the Burkholderia outer membrane protein BimA, an actin nucleator that is essential for actin tail formation. Both GVIN1 and GBP1 coatomers require additional IFNγ-inducible co-factors to disrupt the membrane localization of BimA, demonstrating the existence of two parallel-acting IFNγ-inducible defense modules that evolved to target a virulence trait critical for the pathogenesis of numerous bacterial infectious agents.

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  1. Version published to 10.7554/elife.106896.2 on eLife
  2. Version published to 10.7554/elife.106896 on eLife
  3. eLife

    eLife Assessment

    This fundamental study identifies GVIN as a previously unrecognized interferon-induced antimicrobial GTPase and establishes its role in cell-autonomous immunity against intracellular Burkholderia. The compelling data demonstrate that GVIN assembles into coat-like structures on the bacterial surface and restricts BimA-dependent actin-based motility through a mechanism independent of GBP1. These findings expand our understanding of interferon-induced host defense mechanisms and reveal a distinct pathway by which human cells restrict the intracellular spread of bacterial pathogens.

  4. eLife

    Reviewer #1 (Public review):

    This is a very elegant and experimentally convincing study. Using systematic screening of actin tail formation in two bacterial strains and employing a panel of CRISPR-CAS ko cell lines, the authors identify the dynamin-related GTPases GVIN as antibacterial agent, which forms an oligomeric coat around an intracellular Burkholderia strain. The bacterial O-antigen LPS layer is required for the formation of the GVIN coat, which disturbs the polar localization of the bacterial actin-polymerizing BimA protein.

    The experiments appear of high quality, figures are well prepared, and clean and statistically significant results are provided. I have no criticism on the presented approaches.

    The identification of a novel GBP1-independent pathway targeting intracellular bacteria is not only of fundamental importance for the immunity field, but also of high interest to researchers in other areas, for example, evolutionary or structural biologists.

    Comments on revised version.

    This is a beautiful study.

  5. eLife

    Reviewer #2 (Public review):

    Public summary

    Summary:

    The authors wanted to investigate how cells defend against intracellular pathogens, such as Shigella and Burkholderia species, that coopt the host actin machinery to spread from cell-to-cell. Previous work has identified IFNg-inducible GTPase of the Guanylate Binding Protein (GBP) family in cytosolic defence against Gram-negative bacteria. By forming a coat around Shigella, human GBP1 suppresses actin-based motility by displacing IcsA, which is the actin-polymerising virulence factor present at bacterial poles. In addition, GBP1 recruits the cytosolic LPS-sensor, caspase-4, to the bacterial surface, which results in the removal of bacterial replicative niches via pyroptotic cell death. Here they followed up their finding that GBP1 can reduce actin-based motility of Shigella in HeLa cells and surprisingly fails to do so during Burkholderia infection. In contrast, in T24 bladder epithelial cells, GBP1 is competent in blocking Burkholderia actin-tails. They therefore wanted to identify the GBP1-independent factor that blocks actin-based motility in IFNg-treated cells that is absent in HeLa cells.

    Major strengths and weaknesses of the methods and results:

    The authors report a second IFNg-dependent pathway involving the protein product of the gene GVIN1, which was previously thought to be a pseudogene. GVIN1 (GTPase, very large interferon inducible 1) is thus the first human member of this family of ~250 kDa putative GTPases to be demonstrated to be functional and have potential antimicrobial roles. The discovery that GVIN1 is indeed functional, forms coats on Burkholderia in an LPS O-antigen-dependent manner, and limits actin-dependent motility are the main strengths of this paper. The authors use CRISPR/Cas9-based knockouts in HeLa and T24 cells, and complement them, to demonstrate GBP1 and GVIN1 are both required to inhibit actin-based motility.

    An appraisal of whether the authors achieved their aims, and whether the results support their conclusions.

    The authors achieved their main goals through well-planned experiments and unbiased screens. They succeeded in finding the factor that blocks actin-based motility independently of GBP1. This is driven by GVIN1, which coats bacteria and limiting actin-tail formation by reducing the expression of BimA through currently unknown mechanisms. Further, they found that an O-antigen mutant can escape coating by GVIN1, indicating the requirement for these polysaccharides in GVIN1-dependent bacterial sensing. However, the authors have not investigated whether GVIN1, which has two GTPase-domains, does indeed have GTPase activity and whether GVIN1 and GBP1 together completely block cell-to-cell spread by Burkholderia and thereby restrict bacterial numbers over the infection time course. They also do not show whether GBP1 and GVIN1 target the same bacterial cell or different populations of bacteria.

    A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community.

    This work uncovers the antimicrobial actions of a member of yet another family of IFNg-induced GTPases, which potentially acts against other intracellular pathogens. GVIN1 appears to operate independently and in parallel to GBP1, pointing to the breadth and complexity of the IFNg-inducible GTPase families.

    Comments on revised version.

    They have addressed my comments.

  6. eLife

    Reviewer #3 (Public review):

    Summary:

    Here, Guo et al. (2025) propose that the IFN-induced GTPase GVIN1 forms a coat on cytosolic Burkholderia thailandensis, blocking actin tail formation through a mechanism analogous to GBP1-mediated restriction of Shigella motility.

    Their study was prompted by the intriguing observation that IFNγ priming and GBP1 coat formation fail to inhibit B. thailandensis actin-based motility in HeLa cells, yet IFNγ restricts the motility of Burkholderia in T24 cells. Further investigation revealed that IFNγ restricts B. thailandensis motility in T24 cells via both GBP1-dependent and -independent mechanisms, suggesting that HeLa cells lack a critical GBP1 co-factor required to inhibit actin tail formation.

    To identify the GBP1-independent mechanism, the authors performed an siRNA screen of interferon-stimulated genes (ISGs) and identified GVIN1, a large IFN-induced GTPase, as essential for restricting B. thailandensis motility. To identify the GBP1-independent mechanism, perform a knock-down screen for ISGs and find that the loss of GVIN, a very large IFN-induced GTPase, results in higher actin tail positive B. thailandensis in T24 cells. They further demonstrate that GVIN forms coats on the surface of B. thailandensis which prevent the polar localization of BimA and thus actin tail formation. In summary, the data reveal two independent IFNγ-induced pathways that restrict bacterial motility: one GBP1-dependent and the other GVIN1-dependent, each relying on distinct host co-factors.

    Global assessment:

    This is a well-executed study that convincingly demonstrates how GVIN1 restricts actin-based motility of B. thailandensis through the assembly of coatomers. The results are clearly described, the manuscript is easy to follow, and the data are overall compelling and well presented. I have only a few suggestions on how the manuscript could be further improved.

    Comments on revised version.

    The authors have answered all my points.

  7. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Public Reviews:

    Reviewer #1 (Public review):

    This is a very elegant and convincing study. Using systematic screening of actin tail formation in two bacterial strains and employing a panel of CRISPR-CAS ko cell lines, the authors identify a novel dynamin-related GTPase GVIN, which forms an oligomeric coat around an intracellular Burkholderia strain. The bacterial O-antigen LPS layer is required for the formation of the GVIN coat, which disturbs the polar localization of the bacterial actin-polymerizing BimA protein.

    I am not an expert in infection studies, but the experiments appear to be of high quality, the figures are well prepared, and clean and statistically significant results are provided. I have no criticism of the presented approaches.

    The identification of a novel GBP1-independent pathway targeting intracellular bacteria is not only of fundamental importance for the immunity field but also of high interest to researchers in other areas, for example, evolutionary or structural biologists.

    We sincerely thank the reviewer for their generous appraisal of our work. We appreciate their encouraging feedback.

    Reviewer #2 (Public review):

    Summary:

    The authors wanted to investigate how cells defend against intracellular pathogens, such as Shigella and Burkholderia species, that co-opt the host actin machinery to spread from cell-to-cell. Previous work has identified IFNg-inducible GTPase of the Guanylate Binding Protein (GBP) family in cytosolic defence against Gram-negative bacteria. By forming a coat around Shigella, human GBP1 suppresses actin-based motility by displacing IcsA, which is the actin-polymerising virulence factor present at bacterial poles. In addition, GBP1 recruits the cytosolic LPS-sensor, caspase-4, to the bacterial surface, which results in the removal of bacterial replicative niches via pyroptotic cell death. Here, they followed up their finding that GBP1 can reduce actin-based motility of Shigella in HeLa cells and, surprisingly, fails to do so during Burkholderia infection. In contrast, in T24 bladder epithelial cells, GBP1 is competent in blocking Burkholderia actin-tails. They therefore wanted to identify the GBP1-independent factor that blocks actin-based motility in IFNγ-treated cells that is absent in HeLa cells.

    Major strengths and weaknesses of the methods and results:

    The authors report a second IFNγ-dependent pathway involving the protein product of the gene GVIN1, which was previously thought to be a pseudogene. GVIN1 (GTPase, very large interferon inducible 1) is thus the first human member of this family of ~250 kDa putative GTPases to be demonstrated to be functional and have potential antimicrobial roles. The discovery that GVIN1 is indeed functional, forms coats on Burkholderia in an LPS O-antigen-dependent manner, and limits actin-dependent motility are the main strengths of this paper. The authors use CRISPR/Cas9-based knockouts in HeLa and T24 cells, and complement them to demonstrate that GBP1 and GVIN1 are both required to inhibit actin-based motility.

    An appraisal of whether the authors achieved their aims and whether the results support their conclusions:

    The authors achieved their main goals through well-planned experiments and unbiased screens. They succeeded in finding the factor that blocks actin-based motility independently of GBP1. This is driven by GVIN1, which coats bacteria and limits actin-tail formation by reducing the expression of BimA through currently unknown mechanisms. Further, they found that an O-antigen mutant can escape coating by GVIN1, indicating the requirement for these polysaccharides in GVIN1-dependent bacterial sensing. However, the authors have not investigated whether GVIN1, which has two GTPase domains, does indeed have GTPase activity and whether GVIN1 and GBP1 together completely block cell-to-cell spread by Burkholderia and thereby restrict bacterial numbers over the infection time course. They also do not show whether GBP1 and GVIN1 target the same bacterial cell or different populations of bacteria.

    A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

    This work uncovers the antimicrobial actions of a member of yet another family of IFNg-induced GTPases, which potentially acts against other intracellular pathogens. GVIN1 appears to operate independently and in parallel to GBP1, pointing to the breadth and complexity of the IFNγ-inducible GTPase families.

    We thank the reviewer for their overall positive assessment of our work and their suggestions for additional studies. We addressed all the comments and in response we have added additional experimental data to the revised manuscript. However, while we agree that a detailed biochemical characterization of GVIN1 protein is warranted, we consider such experiments outside of the scope of the current work.

    Reviewer #3 (Public review):

    Summary:

    Here, Guo et al. (2025) propose that the IFN-induced GTPase GVIN1 forms a coat on cytosolic Burkholderia thailandensis, blocking actin tail formation through a mechanism analogous to GBP1-mediated restriction of Shigella motility.

    Their study was prompted by the intriguing observation that IFNγ priming and GBP1 coat formation fail to inhibit B. thailandensis actin-based motility in HeLa cells, yet IFNγ restricts the motility of Burkholderia in T24 cells. Further investigation revealed that IFNγ restricts B. thailandensis motility in T24 cells via both GBP1-dependent and -independent mechanisms, suggesting that HeLa cells lack a critical GBP1 co-factor required to inhibit actin tail formation.

    To identify the GBP1-independent mechanism, the authors performed an siRNA screen of interferon-stimulated genes (ISGs) and identified GVIN1, a large IFN-induced GTPase, as essential for restricting B. thailandensis motility. To identify the GBP1-independent mechanism, perform a knockdown screen for ISGs and find that the loss of GVIN, a very large IFN-induced GTPase, results in higher actin tail-positive B. thailandensis in T24 cells. They further demonstrate that GVIN forms coats on the surface of B. thailandensis, which prevent the polar localization of BimA and thus actin tail formation. In summary, the data reveal two independent IFNγ-induced pathways that restrict bacterial motility: one GBP1-dependent and the other GVIN1-dependent, each relying on distinct host co-factors.

    Global assessment:

    This is a well-executed study that convincingly demonstrates how GVIN1 restricts the actin-based motility of B. thailandensis through the assembly of coatomers. The results are clearly described, the manuscript is easy to follow, and the data are overall compelling and well presented. I have only a few suggestions on how the manuscript could be further improved.

    We thank the reviewer for a clear summary of our findings, and we appreciate the reviewer’s thoughtful suggestions on our manuscript, which we addressed in our revised manuscript.

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    GVIN/VLIG was previously described in the mouse, mostly. Why was it considered a pseudogene in humans? Some more background should be provided.

    Major genome annotation projects (Ensembl, GENCODE, and RefSeq) rely on automated pipelines that flag loci as pseudogenes when they contain features incompatible with a single canonical open reading frame. These pipelines score in-frame premature stop codons, frameshifting indels, or the absence of a full-length matching transcript or protein in reference databases. The human GVINP1 locus displays several such features when compared against a naive single-ORF model of the mouse Vlig1 mRNA, and on that basis GVINP1 was likely categorized as a pseudogene by all three consortia, an assignment that has propagated through UniProt, HGNC, GeneCards, and OMIM ever since (UniProt Q7Z2Y8; GeneCards GVINP1). Importantly, these pipelines are not designed to detect loci that are expressed from more than one ORF or that produce a full-length protein through non-canonical translation, and they do not incorporate mass-spectrometry peptide evidence, ribosome profiling, or interferon-inducible expression data. Therefore, these automated pipelines can draw incorrect conclusions, as is the case here. We have added to the Introduction to provide a bit more background, as requested.

    Is GVIN restricting the proliferation of the Burkholderia strain in the tested human cell line(s)? Is it restricting the intra-host mobility of the bacteria?

    GVIN1 limits intracellular bacterial replication, albeit only modestly (see new data in Fig. S5B). We found that although GBP1 and GVIN1 block actin tail formation through independent, non-overlapping mechanisms, control of intracellular B. thailandensis burden required both proteins: IFN-γ failed to restrict bacterial replication in either GBP1KO or GVIN1KO T24 cells, and the loss-of-restriction phenotypes were quantitatively similar between the two knockouts (Fig. S5B). Together, these data identify GBP1 and GVIN1 as the effectors of two cooperating IFN-γ–induced defense pathways that jointly control B. thailandensis.

    P 10: Remarkably, GVIN1 coatomers formed only infrequently on the surface of the O-antigen-deficient delta wbpm B. thailandensis mutant (Figure 5F). This information is not included in Figure 5F (only the quantification in E) and could be added.

    We have added representative images of the corresponding microscopy data for both WT and DwbpM in a new Figure S8B.

    Reviewer #2 (Recommendations for the authors):

    Major points:

    (1) The finding of GVIN1 as a protein is important and deserves further characterisation. For instance, it would be useful to investigate the transcripts they find in RNAseq, start sites, length of transcript, etc. There are two GTPase domains in this protein, but is either of them active as a GTPase if key GTP-binding and/or hydrolysis residues are mutated?

    We agree that biochemical and enzymatic characterization of the GVIN1 protein is an important future direction. Similarly, careful investigations of GVIN1 expression, ribosome profiling, etc. ought to be conducted. However, these represent substantial, self-contained lines of investigation, and we will address them in a dedicated follow-up studies.

    (2) Throughout the paper, the authors have only investigated the proximal steps of actin-tail formation. They do not go on to measure the impact of bacterial targeting by GBP1 or GVIN1, specifically in terms of bacterial spread cell-to-cell and replication (e.g., CFU measurements over time) and the impact on pyroptosis. Does GBP1 and/or GVIN1 targeting reduce bacterial CFUs over time?

    re: 1) bacterial burden (CFU data); 2) host cell death (pyroptosis), and 3) cell-to-cell spread: 1) Our revised manuscript now provides CFU data (Fig. S5B) that identifies a modest impact on bacterial burden by GVIN1 expression – please, see also our response to Reviewer #1. 2) We already provided data in our original submission (Fig. S2D) demonstrating that IFNγ priming of either T24 WT or GBP1KO cells did not appear to change the degree of host cell death in infected cells, as measured by Lactate Dehydrogenase (LDH) release assays. Accordingly, no further studies into host cell death pathways seem warranted at this time. 3) B. thailandensis is unusual among cytosolic bacterial pathogens in that it can spread between host cells not only through actin tail–driven propulsion but also via flagellar motility (PMID: 21730143). Therefore actin-based motility is not essential for cell-to-cell spread for this specific organism (PMID: 21730143). Consistent with this dual-mode dissemination, IFN-γ priming of WT T24 cells had no measurable impact on plaque-based cell-to-cell spread (new data in Fig. S5A), despite sharply reduced actin tail formation inside IFN-γ primed WT T24.

    (3) The enumeration of microscopy could be improved by providing the % of GBP1+ve or GVIN1+ve bacteria that are actin-ve.

    We added those data presentations as new panels Fig. S2G for GBP1 and Fig. S7B. As expected, these data show that under IFN-γ primed conditions GBP1-coated or GVIN1-coated bacteria fail to form actin tails.

    (4) The authors show a schematic of GBP1 and GVIN1 targeting the same bacterial cell, but they have not demonstrated this experimentally. For many of their claims, it is important to show GVIN1 and GBP1 co-staining during infection with WT and the wbpM mutant Burkholderia.

    The schematic depicted GBP1 and GVIN1 on opposite sides of the same bacterium purely for illustrative clarity and was not intended to convey that GBP1 and GVIN1 necessarily coat the same bacterial cell. We agree, however, that whether GBP1 and GVIN1 coatomers can co-decorate a single bacterium is a mechanistically interesting question, and we have now addressed it experimentally. Dual immunostaining in IFNγ-primed cells showed that most targeted bacteria were encased by either a GBP1 or a GVIN1 coatomer, but not both (Fig. S7C). A small subpopulation of GBP1/GVIN1 double-positive bacteria was nonetheless detectable (Fig. S7C–D), indicating that the two coat types are largely, but not strictly, mutually exclusive. The rarity of dual-coated bacteria reinforces our conclusion that the two coatomers operate as predominantly independent effector systems, consistent with their ability to restrict actin tail formation independently of one another.

    (5) The authors make points about GBP1 and GVIN1 as being independent pathways and the need for a cofactor. To be certain about these conclusions, have they co-expressed both GBP1 and GVIN1 together in double knockout HeLa & T24 and assessed outcomes? They started out to look for a GBP1 cofactor that is required in HeLa cells, but they found GVIN1, which seems to operate independently of GBP1 (which is not inherently an issue, but I feel this needs to be clarified better). In T24 cells, expression of (mCh-)GBP1 alone without IFNg priming does reduce Actin+ve bacteria (Figure 2C, green bars), so GBP1 can use a factor that is expressed in naïve cells? But the authors have not shown that this factor is GVIN1. The same is true for GVIN1 expressed without IFNγ in T24-GVIN1-/- (Fig 5C). What happens in a similar experiment with an O-antigen mutant?

    We believe the individual experiments the reviewer proposes have, in fact, already been performed in some fashion and are presented in the manuscript. Because these experiments are distributed across several figures, we have taken this opportunity to summarize the logic here and to point the reviewer to the relevant panels.

    First, we would like to clarify the origin of GVIN1 in our study, as the reviewer's framing suggests that we set out to identify a GBP1 co-factor and instead landed on GVIN1. Our siRNA screen (Fig. 4A–B) was in fact designed to identify ISGs required for the GBP1-independent anti-actin-tail pathway and was therefore conducted in a GBP1KO T24 background. GVIN1 emerged as a factor whose depletion restored actin tail formation in the absence of GBP1, and it is therefore, by design of the screen, an effector of a GBP1-independent pathway rather than a candidate GBP1 co-factor.

    Second, we show that single deletion of either GBP1 or GVIN1 produces a partial and quantitatively similar de-restriction phenotype, whereas only the DKO fully abolishes IFNγ-mediated restriction of actin tail formation (Fig. 4C). Thus, we are seeing the classical additive signature of two non-redundant pathways acting on the same phenotype. In the gain-of-function direction, ectopic re-expression of GVIN1 in the DKO background restores IFNγ-dependent restriction to a level indistinguishable from the GBP1KO single knockout (Fig. 5D). A reciprocal experiment co-expressing both GBP1 and GVIN1 in the DKO would only test for additivity of two effects we have already shown to occur independently and would not provide an orthogonal test of independence beyond what Fig. 4C and Fig. 5D already establish.

    Third, we do not think GVIN1 is a likely candidate for the basal GBP1 co-factor that the reviewer infers from Fig. 2C. GVIN1 is IFNγ-inducible and effectively absent from unprimed T24 cells (the putative co-factor, while IFNγ-inducible, would be expected to be expressed at appreciable levels in naïve cells). Indeed, GVIN1 entered our screen precisely because of that inducibility (Fig. 4A and RNA-seq data in Fig. S3A–B). More decisively, we show that IFNγ-primed HeLa cells allow for ectopically expressed GVIN1 but not for ectopically expressed GBP1 to restrict actin tail formation. In other words, the ISG co-factor for the GVIN1 pathway must be distinct from the ISG co-factor for the GBP1 pathway. Lastly, we now show that GBP1 and GVIN1 coatomers are largely mutually exclusive, further arguing against GVIN1 acting as the GBP1 co-factor or vice versa in blocking actin tail formation.

    Fourth, the O-antigen mutant experiment the reviewer asked about has also been performed and provides another point of mechanistic separation between the two pathways. Ectopically expressed GVIN1 assembles a coatomer on WT B. thailandensis but only rarely on the ΔwbpM O-antigen-deficient mutant (Fig. 5F). This observation parallels (and thus mechanistically explains) why ΔwbpM is resistant specifically to the GBP1-independent (i.e., GVIN1-dependent) branch of IFNγ defense (Fig. 3E). GBP1, by contrast, engages both B. thailandensis WT and the O-antigen ΔwbpM mutant efficiently (new Fig. S8C).

    Taken together, four convergent lines of evidence (additive genetics in the DKO (Fig. 4C), cross-complementation of GVIN1 in the GBP1-null background (Fig. 5D), non-overlapping co-factor requirements in HeLa versus T24 cells (Fig. 2, Fig. 5), and distinct O-antigen dependencies for the two coatomers (Fig. 5E, Fig. 8C)) establish that GBP1 and GVIN1 constitute two parallel, independently operating antimicrobial modules, each likely with its own set of IFN-inducible co-factors.

    (6) The discussion does not currently discuss GBP1/Caspase-4-driven pyroptosis as a host-defence mechanism, which should be included. The authors should also discuss why Shigella and Burkholderia are differentially targeted, given the differences in the mechanism of actin polymerisation by IcsA and BimA.

    We previously showed that a GBP1 polybasic motif mutant that is unable to form coatomers on bacteria remains able to activate Caspase-4-driven pyroptosis and thus demonstrated that coatomers are not strictly required for GBP1-dependent Caspase-4 activation (PMID: 37023136). We now provide this information in the Discussion of the revised manuscript.

    We provide new data showing that GVIN1 forms coatomers on BimA-deficient and WT B. thailandensis with similar efficiency (new Fig. S8A) and we previously demonstrated that icsA plays no role in GBP1 coatomer formation (PMID: 33885766). Therefore, actin polymerization appears to play no role in either GVIN1 or GBP1 coatomer formation.

    Minor points:

    (1) Have the authors used anti-actin antibodies or phalloidin staining? Please clarify.

    We used phalloidin staining and this information is now also provided in the Results section (lines 118-119) in addition to the Materials and Methods section of the paper.

    (2) Provide the Gene ID, Ensembl ID, etc., for GVIN1 at first mention.

    We inserted the text: “Because automated annotation pipelines flag predicted in-frame stop codons, GVIN1 has long been catalogued as the pseudogene GVINP1 (GTPase, Very large IFN-Inducible Pseudogene-1) by Ensembl, GENCODE, and RefSeq (HGNC 25813; NCBI Gene 387751; OMIM 616121).” in the Introduction (lines 104 – 107)

    (3) The bond linking the core sugars with the O-antigen is missing in the schematic of LPS in Figure 3A. Please mention which sugars these are, if it is known for Burkholderia.

    We have corrected the schematic (Fig.3A) to include the bond linking the core sugars with the O-antigen.

    (4) RNA-seq datasets should be deposited to GEO or similar public databases, and project IDs provided.

    The RNA-seq datasets was deposited to the NCBI Gene Expression Omnibus (GEO), and the project accession IDs are is cited in the Data Availability statement.

    (5) The description of the transcriptomics analyses was not clear - are the heatmaps in Figure S3 of genes that were induced by IFNg in these cell lines, but not HeLa or A594, but the description states fold-change over naïve cells?

    The reviewer is correct and we apologize for the error in the figure legends, which we edited to clarify that the heatmap compares gene expression across IFNγ-primed cell lines.

    Reviewer #3 (Recommendations for the authors):

    Major comments:

    (1) It would be nice to include some additional analysis on GVIN: GBP1 and GVIN1 seem to recognize different structures and need different cofactors. Are they structurally similar (alignment)? Can you map functionally important GBP1 regions on GVIN1, such as a polybasic motif, similar to motifs found in GBP1? Or does GVIN have a lipidic membrane anchor or a CAAX box? In other words, is GVIN binding directly to the outer membrane or through the unknown cofactor? Is GVIN an effector of another protein that binds to the OM?

    Detailed structural studies including the mapping and biochemical characterization of GVIN1's membrane-targeting domain(s) represent substantial, self-contained lines of investigation that are beyond the scope of this study and that we hope to present in a future follow-up study.

    (2) In T24 cells, GVIN1 and GBP1 (and their cofactors) get induced upon IFNg priming. Upon B. thailandensis infection, is it possible to see mixed or double coats? Does GBP1 have a higher affinity for Shigella and GVIN for B. thailandensis? Is GVIN1 B. thailandensis-specific? It would be a nice addition to discuss other virulent Burkholderia species that make actin tails (such as B. pseudomallei) to further enhance the possible relevance of GVIN1.

    Please see our response to reviewer #2: dual immunostaining in IFNγ-primed cells showed that most targeted bacteria were encased by either a GBP1 or a GVIN1 coatomer, but not both (Fig. S7C). A small subpopulation of GBP1/GVIN1 double-positive bacteria was nonetheless detectable (Fig. S7C–D), indicating that the two coat types are largely, but not strictly, mutually exclusive. Re: the measurement of GBP1/ GVIN1 affinities for different bacteria – we agree that this is an area of future exploration but not within the scope of this study. Extending research on GVIN1 to other bacterial organisms including other Burkholderia species is indeed among our future research goals. We more broadly speculate on the potential importance of GVIN1-mediated host defense targeting other Burkholderia species in the Discussion (see lines 363 ff)

    (3) In panel F of Figure 6, It appears that GVIN forms full coats in the absence of IFNγ priming, but relocalizes to the bacterial poles upon IFNγ treatment. Is this shift in localization a representative and consistent observation, or could it be an artifact of the specific image selected? Additionally, could this relocalization be driven by the presence of IFNγ-induced cofactors? For example, might GVIN interact directly with BimA, or could an IFNγ-dependent cofactor mediate its recruitment to the poles?

    We believe the author is commenting on Figure 5F instead of 6 based on the question. The perceived shift in GVIN1 localization to the bacterial pole under IFNγ-primed conditions was not something we routinely observe and the original image shown in Fig. 5F is likely an outlier. We therefore replaced the original image with a more representative image.

    (4) The authors conclude that BimA is eliminated from the outer membrane, but it is unclear what evidence this conclusion is based on. Could it be that BimA is not eliminated, but rather becomes diffusely distributed in the membrane in the presence of GVIN1, and is therefore more difficult to detect by microscopy? One way to test this hypothesis would be to delete BimC. If BimA localization is indeed dependent on BimC, its signal should become diffuse. If a BimA signal can be detected in BimC-deficient bacteria, but not in GVIN-positive bacteria, it would indeed strengthen the claim that BimA is removed from the membrane.

    Prior literature demonstrates that on a bimC mutant, BimA remains detectable on the bacterial surface in a dispersed manner and that this dispersed BimA localization correlates with an "actin cloud" surrounding the bacteria. In our GVIN1-coated bacteria, we do not detect an actin cloud or residually localized BimA signals. We have now mentioned this in the Results section line 305 - 308 referencing this published phenotype to strengthen our argument that BimA is likely cleared from the membrane rather than merely redistributed.

    (5) The siRNA mini-screen would benefit from appropriate controls demonstrating that the targeted genes were effectively knocked down. Without this, it's difficult to interpret negative results with confidence. It remains possible that the co-factor of GVIN1 is among the ISGs tested, but that the knockdown was insufficient to produce a detectable phenotype.

    We now acknowledge this limitation. We have updated the text on lines 221-223 to explicitly state that our mini-screen does not rule out potential false negatives due to varying knockdown efficiencies.

    (6) The authors convincingly demonstrate that GVIN1 coats prevent actin tail formation by B. thailandensis, but they do not explore the broader biological consequences of this observation. An important next step would be to assess whether GVIN1 coating restricts B. thailandensis cell-to-cell spread. By 8 hours post-infection, a clear difference in spread should be observable if actin-based motility is impaired. Additionally, it would be valuable to determine whether GVIN1-coated bacteria are still capable of intracellular replication. These functional assays would provide important insight into the biological relevance of GVIN1-mediated coating.

    We have now assessed the effect of GVIN1 on intracellular bacterial replication (see new CFU data in Fig. S5B) and cell-cell motility (see new data in Fig. S5A). Please, also refer to our comments in response to Reviewer 1 for more details.

    (7) As far as I am aware, the exact function of the GBP1 coat remains unclear. The authors refer to their earlier work (Kutsch et al., 2020) and claim to have demonstrated an "emulsifying activity." However, this overinterpretes the findings of that publication, since Kutsch et al. (2020) showed that the GBP1 coat disrupts the barrier function of the O-antigen, rather than compromising the integrity of the outer membrane itself. This disruption increases bacterial susceptibility to polymyxin B, but does not show an 'emulsifying effect'. Furthermore, the same group later reported that GBP1 exhibits an "aggregating" activity on LPS micelles (Dickinson et al., 2023), which seems to contradict the notion of an emulsifying function. I would recommend tempering the current claims about GBP1's activity. The precise impact of the GBP1 coat on the bacterial outer membrane remains unresolved and should be presented as an open question.

    Our previous work demonstrated GBP1 coatomers on the bacterial surface disrupt the membrane-stiffening and membrane-shielding activities of O-antigen. We stand by the interpretation of our results and think it is likely that GBP1 coatomers act like surface-active agents or emulsifiers. However, we agree with the reviewer that more biophysical studies are needed to definitively test this model. Accordingly, we have revised some of the language and clearly articulated that GBP1 coatomers acting as a surfactant is a model that we are proposing.

    As the reviewer correctly stated we have also shown that GBP1 can aggregate “free” LPS, i.e. to act more like a flocculant. Substances that are both flocculants and surfactants exist (e.g. long-chain fatty acid soaps). However, these substances typically do not act as flocculants and surfactants simultaneously under the exact same conditions. Similarly, we propose that GBP1’s role depends on context: we propose that GBP1 coatomers on the bacterial surface act as surfactants, while polymerizing GBPs bound to LPS acts a flocculant.

    Minor comments:

    (1) Figure 3: Can a GBP1 coat assemble on the delta wbpM mutant of B. thailandensis? The authors suggest GBP binds to the O-antigen, but other publications suggest that the O-antigen is dispensable in certain bacteria.

    We conducted these experiments – see also our response to reviewer #2. Briefly, ectopically expressed GVIN1 assembles a coatomer on WT B. thailandensis but only rarely on the ΔwbpM O-antigen-deficient mutant (Fig. 5F). GBP1, by contrast, engages both B. thailandensis WT and the O-antigen ΔwbpM mutant efficiently (new Fig. S8C). We do not argue that GBP1 requires O-antigen binding for coatomer formation on Burkholderia. However, direct or indirect interactions between GVIN1 and O-antigen may be required for GVIN1 coatomer formation.

    (2) Figure 4: GBP6 appears as a hit in the T24 screening but turned out to be a false positive. How does that look in the other cell lines that were part of the screen (U937, THP1)?

    the characterization of T24 GBP6KO cells clearly demonstrated that GBP6 is dispensable for either the GBP1 or the GVIN1 pathway in these cells. While we cannot formally rule out that GBP6 would be required for the GBP1/GVIN1 pathways in other cell lines, there are no compelling arguments in favor of this model – as far as we can tell - and we therefore did not investigate this further.

    (3) Figure 5F: Please show the blots confirming exogenous GVIN expression.

    We have provided an immunoblot showing the expression of exogenous GVIN1in HEK293T cells in new Fig. S7A

    (4) Figure 6: Can you induce GVIN expression after actin tails have been formed and stop disassembling them?

    This is an interesting idea and will require a technically challenging video microscopy approach that we will consider for future investigations.

    (5) Figure 6: Are the bacteria showing no BimA signal GVIN1-coated? Eventually, stain for LPS to see if bacteria are lacking BimA also lack LPS.

    Our preliminary studies indeed support this and thus indicate that the elimination of BimA from the bacterial surface requires the concerted effort of the GVIN1 coatomer and its putative co-factor (or the GBP1 coatomer and its putative co-factor, respectively). A detailed characterization of the mechanism by which BimA is removed from the bacterial surface will be part of future studies.

  8. Version published to 10.1101/2025.03.24.645074v2 on bioRxiv
  9. eLife

    eLife Assessment

    This is a fundamental discovery revealing two independent IFNγ-induced pathways that restrict bacterial motility: one GBP1-dependent and the other GVIN1-dependent. The findings are supported by compelling evidence. While the paper is already very strong, there are a few points that could be addressed editorially or through the addition of a few key experiments.

  10. eLife

    Reviewer #1 (Public review):

    This is a very elegant and convincing study. Using systematic screening of actin tail formation in two bacterial strains and employing a panel of CRISPR-CAS ko cell lines, the authors identify a novel dynamin-related GTPase GVIN, which forms an oligomeric coat around an intracellular Burkholderia strain. The bacterial O-antigen LPS layer is required for the formation of the GVIN coat, which disturbs the polar localization of the bacterial actin-polymerizing BimA protein.

    I am not an expert in infection studies, but the experiments appear to be of high quality, the figures are well prepared, and clean and statistically significant results are provided. I have no criticism of the presented approaches.

    The identification of a novel GBP1-independent pathway targeting intracellular bacteria is not only of fundamental importance for the immunity field but also of high interest to researchers in other areas, for example, evolutionary or structural biologists.

  11. eLife

    Reviewer #2 (Public review):

    Summary:

    The authors wanted to investigate how cells defend against intracellular pathogens, such as Shigella and Burkholderia species, that co-opt the host actin machinery to spread from cell-to-cell. Previous work has identified IFNg-inducible GTPase of the Guanylate Binding Protein (GBP) family in cytosolic defence against Gram-negative bacteria. By forming a coat around Shigella, human GBP1 suppresses actin-based motility by displacing IcsA, which is the actin-polymerising virulence factor present at bacterial poles. In addition, GBP1 recruits the cytosolic LPS-sensor, caspase-4, to the bacterial surface, which results in the removal of bacterial replicative niches via pyroptotic cell death. Here, they followed up their finding that GBP1 can reduce actin-based motility of Shigella in HeLa cells and, surprisingly, fails to do so during Burkholderia infection. In contrast, in T24 bladder epithelial cells, GBP1 is competent in blocking Burkholderia actin-tails. They therefore wanted to identify the GBP1-independent factor that blocks actin-based motility in IFNg-treated cells that is absent in HeLa cells.

    Major strengths and weaknesses of the methods and results:

    The authors report a second IFNg-dependent pathway involving the protein product of the gene GVIN1, which was previously thought to be a pseudogene. GVIN1 (GTPase, very large interferon inducible 1) is thus the first human member of this family of ~250 kDa putative GTPases to be demonstrated to be functional and have potential antimicrobial roles. The discovery that GVIN1 is indeed functional, forms coats on Burkholderia in an LPS O-antigen-dependent manner, and limits actin-dependent motility are the main strengths of this paper. The authors use CRISPR/Cas9-based knockouts in HeLa and T24 cells, and complement them to demonstrate that GBP1 and GVIN1 are both required to inhibit actin-based motility.

    An appraisal of whether the authors achieved their aims and whether the results support their conclusions:

    The authors achieved their main goals through well-planned experiments and unbiased screens. They succeeded in finding the factor that blocks actin-based motility independently of GBP1. This is driven by GVIN1, which coats bacteria and limits actin-tail formation by reducing the expression of BimA through currently unknown mechanisms. Further, they found that an O-antigen mutant can escape coating by GVIN1, indicating the requirement for these polysaccharides in GVIN1-dependent bacterial sensing. However, the authors have not investigated whether GVIN1, which has two GTPase-domains, does indeed have GTPase activity and whether GVIN1 and GBP1 together completely block cell-to-cell spread by Burkholderia and thereby restrict bacterial numbers over the infection time course. They also do not show whether GBP1 and GVIN1 target the same bacterial cell or different populations of bacteria.

    A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

    This work uncovers the antimicrobial actions of a member of yet another family of IFNg-induced GTPases, which potentially acts against other intracellular pathogens. GVIN1 appears to operate independently and in parallel to GBP1, pointing to the breadth and complexity of the IFNg-inducible GTPase families.

  12. eLife

    Reviewer #3 (Public review):

    Summary:

    Here, Guo et al. (2025) propose that the IFN-induced GTPase GVIN1 forms a coat on cytosolic Burkholderia thailandensis, blocking actin tail formation through a mechanism analogous to GBP1-mediated restriction of Shigella motility.

    Their study was prompted by the intriguing observation that IFNγ priming and GBP1 coat formation fail to inhibit B. thailandensis actin-based motility in HeLa cells, yet IFNγ restricts the motility of Burkholderia in T24 cells. Further investigation revealed that IFNγ restricts B. thailandensis motility in T24 cells via both GBP1-dependent and -independent mechanisms, suggesting that HeLa cells lack a critical GBP1 co-factor required to inhibit actin tail formation.

    To identify the GBP1-independent mechanism, the authors performed an siRNA screen of interferon-stimulated genes (ISGs) and identified GVIN1, a large IFN-induced GTPase, as essential for restricting B. thailandensis motility. To identify the GBP1-independent mechanism, perform a knock-down screen for ISGs and find that the loss of GVIN, a very large IFN-induced GTPase, results in higher actin tail-positive B. thailandensis in T24 cells. They further demonstrate that GVIN forms coats on the surface of B. thailandensis, which prevent the polar localization of BimA and thus actin tail formation. In summary, the data reveal two independent IFNγ-induced pathways that restrict bacterial motility: one GBP1-dependent and the other GVIN1-dependent, each relying on distinct host co-factors.

    Global assessment:

    This is a well-executed study that convincingly demonstrates how GVIN1 restricts the actin-based motility of B. thailandensis through the assembly of coatomers. The results are clearly described, the manuscript is easy to follow, and the data are overall compelling and well presented. I have only a few suggestions on how the manuscript could be further improved.

  13. Version published to 10.7554/elife.106896.1 on eLife
  14. Version published to 10.1101/2025.03.24.645074v1 on bioRxiv