Contributions of Guanylate-Binding Proteins and the Very Large Inducible GTPase in the Restriction of Actin-Based Motility of Burkholderia thailandensis

Abstract

There is a group of intracellular bacterial pathogens that hijack the host cell’s actin cytoskeleton system to develop actin-based motility, which is crucial for their infection and dissemination. To counter these intracellular pathogens, human cell-autonomous immune responses, particularly those activated by interferon-gamma (IFNγ), have been shown to interfere with such microbial actin-based motility. Among the four families of IFNγ-inducible GTPases that mediate many host-resistance programs, the guanylate-binding proteins (GBPs) are known to form coatomers on the surface of Shigella flexneri, thereby disrupting the polar localization of Shigella’s actin nucleator IcsA to restrict actin tail formation. While GBP coatomers have also been observed on the surface of another actin-nucleating intracellular pathogen, Burkholderia thailandensis, their role in restricting B. thailandensis actin-based motility remains poorly understood. In this work, I demonstrate that although human GBP1 is capable of restricting the actin-based motility of B. thailandensis, this effect is not achieved by GBP1 alone. Instead, it requires additional, as-yet-unidentified IFNγ-inducible genes (ISGs), whose expression is limited to specific cell lines, thus providing an explanation for the cell-line-specific restriction of B. thailandensis actin-based motility. In parallel, I identified the GTPase, Very Large Interferon Inducible 1 (GVIN1), an IFNγ-inducible very large GTPase previously annotated as a pseudogene, as a novel host defense factor in human cells. GVIN1 assembles coatomers around B. thailandensis and, in concert with other ISGs, effectively restricts B. thailandensis actin-based motility. This novel GVIN1-dependent pathway requires additional, as-yet-unidentified ISGs that are distinct from those involved in the GBP1-dependent pathway. Despite the GBP1-dependent pathway and GVIN1-dependent pathway sharing a strategy of forming antimicrobial coatomers on the surface of B. thailandensis, I demonstrated that these two IFNγ-inducible pathways function in parallel and operate independently. Intriguingly, while GBP1 disrupts the polar localization of IcsA on S. flexneri by inducing its diffusion, I revealed that both the GBP1-dependent pathway and GVIN1-dependent pathway provoke a complete disappearance, rather than mislocalization, of the B. thailandensis actin nucleator BimA from the bacterial pole. In addition, I investigated the functional and structural properties of mGbp2, the murine homolog of human GBP1. Given that GBP1’s ability to form coatomers on bacterial pathogens depends on its C‑terminal polybasic motif (PBM), which generates a positively charged ring, I identified an analogous triple-lysine motif at the C‑terminus of mGbp2 that is similarly essential for its pathogen‑targeting activity. Together, this work defines two parallel, IFNγ-inducible defense pathways against the intracellular pathogen B. thailandensis, expanding our understanding of GBP1-mediated antimicrobial activity and providing the first functional characterization of GVIN1 protein products. Furthermore, by characterizing the C-terminal triple-lysine motif in murine Gbp2 and demonstrating its essential role in targeting Gram-negative bacteria, I have enhanced our understanding of the structural and mechanistic principles underlying GBP-LPS interactions. In summary, these findings illuminate the diversity, specificity, and mechanistic complexity of interferon‑stimulated GTPase-mediated antimicrobial responses and paves the way for future studies to identify the unknown cofactors involved in GVIN1-dependent and GBP1-dependent inhibition of actin-based motility.