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

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.

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.

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.