Structural insights into the activation mechanism of antimicrobial GBP1.

The dynamin-related human guanylate-binding protein 1 (GBP1) mediates host defenses against microbial pathogens. Upon GTP binding and hydrolysis, auto-inhibited GBP1 monomers dimerize and assemble into soluble and membrane-bound oligomers, which are crucial for innate immune responses. How higher-order GBP1 oligomers are built from dimers, and how assembly is coordinated with nucleotide-dependent conformational changes, has remained elusive. Here, we present cryo-electron microscopy-based structural data of soluble and membrane-bound GBP1 oligomers, which show that GBP1 assembles in an outstretched dimeric conformation. We identify a surface-exposed helix in the large GTPase domain that contributes to the oligomerization interface, and we probe its nucleotide- and dimerization-dependent movements that facilitate the formation of an antimicrobial protein coat on a gram-negative bacterial pathogen. Our results reveal a sophisticated activation mechanism for GBP1, in which nucleotide-dependent structural changes coordinate dimerization, oligomerization, and membrane binding to allow encapsulation of pathogens within an antimicrobial protein coat.

Interferon inducible guanylate-binding protein 1 modulates the lipopolysaccharide-induced cytokines/chemokines and mitogen-activated protein kinases in macrophages.

Guanylate-binding proteins (GBPs) are a family of interferon (IFN)-inducible GTPases and play a pivotal role in the host immune response to microbial infections. These are upregulated in immune cells after recognizing the lipopolysaccharides (LPS), the major membrane component of Gram-negative bacteria. In the present study, the expression pattern of GBP1-7 was initially mapped in phorbol 12-myristate 13-acetate-differentiated human monocytes THP-1 and mouse macrophages RAW 264.7 cell lines stimulated with LPS. A time-dependent significant expression of GBP1-7 was observed in these cells. Moreover, among the various GBPs, GBP1 has emerged as a central player in regulating innate immunity and inflammation. Therefore, to study the specific role of GBP1 in LPS-induced inflammation, knockdown of the Gbp1 gene was carried out in both cells using small interfering RNA interference. Altered levels of different cytokines (interleukin [IL]-4, IL-10, IL-12ß, IFN-γ, tumor necrosis factor-α), inducible nitric oxide synthase, histocompatibility 2, class II antigen A, protein kinase R, and chemokines (chemokine (C-X-C motif) ligand 9 [CXCL9], CXCL10, and CXCL11) in GBP1 knockdown cells were reported compared to control cells. Interestingly, the extracellular-signal-regulated kinase 1/2 mitogen-activated protein (MAP) kinases and signal transducer and activator of transcription 1 (STAT1) transcription factor levels were considerably induced in knockdown cells compared to the control cells. However, no change in the level of phosphorylated nuclear factor-kB, c-Jun, and p38 transcription factors was observed in GBP1 knockdown cells compared to the control cells. This study concludes that GBP1 may alter the expression of cytokines, chemokines, and effector molecules mediated by MAP kinases and STAT1 transcription factors.

Advances in the regulation of inflammasome activation by GBP family in infectious diseases.

Guanylate-binding proteins (GBPs) are a subfamily of interferon-inducible proteins that undertake distinct roles in the the context of bacteria, virus, chlamydia and parasites infections. These proteins exert a notable influence on the progression and outcomes of infectious diseases. Within the realm of host cell-autonomous immunity against pathogens, GBPs have been identified as the regulators of pyroptosis through canonical and noncanonical inflammasome activation pathways. In this review, we summarize the structure and evolution of GBP family members, the canonical and noncanonical inflammasome activation pathways, the roles of GBPs in regulating inflammasome activation, and the mechanisms of GBPs affecting infections induced by different pathogens. We hope to provide new basic research clues for the pathogenesis and diagnosis and treatment of infectious diseases.

Guanylate-binding protein-5 is involved in inflammasome activation by bacterial DNA but only the cooperation of multiple GBPs accounts for control of Brucella abortus infection.

Introduction: Guanylate-binding proteins (GBPs) are produced in response to pro-inflammatory signals, mainly interferons. The most studied cluster of GBPs in mice is on chromosome 3. It comprises the genes for GBP1-to-3, GBP5 and GBP7. In humans, all GBPs are present in a single cluster on chromosome 1. Brucella abortus is a Gram-negative bacterium known to cause brucellosis, a debilitating disease that affects both humans and animals. Our group demonstrated previously that GBPs present on murine chromosome 3 (GBPchr3) is important to disrupt Brucella-containing vacuole and GBP5 itself is important to Brucella intracellular LPS recognition. In this work, we investigated further the role of GBPs during B. abortus infection. Methods and results: We observed that all GBPs from murine chromosome 3 are significantly upregulated in response to B. abortus infection in mouse bone marrow-derived macrophages. Of note, GBP5 presents the highest expression level in all time points evaluated. However, only GBPchr3-/- cells presented increased bacterial burden compared to wild-type macrophages. Brucella DNA is an important Pathogen-Associated Molecular Pattern that could be available for inflammasome activation after BCV disruption mediated by GBPs. In this regard, we observed reduced IL-1ß production in the absence of GBP2 or GBP5, as well as in GBPchr3-/- murine macrophages. Similar result was showed by THP-1 macrophages with downregulation of GBP2 and GBP5 mediated by siRNA. Furthermore, significant reduction on caspase-1 p20 levels, LDH release and Gasdermin-D conversion into its mature form (p30 N-terminal subunit) was observed only in GBPchr3-/- macrophages. In an in vivo perspective, we found that GBPchr3-/- mice had increased B. abortus burden and higher number of granulomas per area of liver tissue, indicating increased disease severity. Discussion/conclusion: Altogether, these results demonstrate that although GBP5 presents a high expression pattern and is involved in inflammasome activation by bacterial DNA in macrophages, the cooperation of multiple GBPs from murine chromosome 3 is necessary for full control of Brucella abortus infection.