The gut-to-brain axis for toxin-induced defensive responses.

After ingestion of toxin-contaminated food, the brain initiates a series of defensive responses (e.g., nausea, retching, and vomiting). How the brain detects ingested toxin and coordinates diverse defensive responses remains poorly understood. Here, we developed a mouse-based paradigm to study defensive responses induced by bacterial toxins. Using this paradigm, we identified a set of molecularly defined gut-to-brain and brain circuits that jointly mediate toxin-induced defensive responses. The gut-to-brain circuit consists of a subset of Htr3a+ vagal sensory neurons that transmit toxin-related signals from intestinal enterochromaffin cells to Tac1+ neurons in the dorsal vagal complex (DVC). Tac1+ DVC neurons drive retching-like behavior and conditioned flavor avoidance via divergent projections to the rostral ventral respiratory group and lateral parabrachial nucleus, respectively. Manipulating these circuits also interferes with defensive responses induced by the chemotherapeutic drug doxorubicin. These results suggest that food poisoning and chemotherapy recruit similar circuit modules to initiate defensive responses.

Presynaptic Excitation via GABAB Receptors in Habenula Cholinergic Neurons Regulates Fear Memory Expression.

Fear behaviors are regulated by adaptive mechanisms that dampen their expression in the absence of danger. By studying circuits and the molecular mechanisms underlying this adaptive response, we show that cholinergic neurons of the medial habenula reduce fear memory expression through GABAB presynaptic excitation. Ablating these neurons or inactivating their GABAB receptors impairs fear extinction in mice, whereas activating the neurons or their axonal GABAB receptors reduces conditioned fear. Although considered exclusively inhibitory, here, GABAB mediates excitation by amplifying presynaptic Ca(2+) entry through Cav2.3 channels and potentiating co-release of glutamate, acetylcholine, and neurokinin B to excite interpeduncular neurons. Activating the receptors for these neurotransmitters or enhancing neurotransmission with a phosphodiesterase inhibitor reduces fear responses of both wild-type and GABAB mutant mice. We identify the role of an extra-amygdalar circuit and presynaptic GABAB receptors in fear control, suggesting that boosting neurotransmission in this pathway might ameliorate some fear disorders.

Glucose-induced CRL4COP1-p53 axis amplifies glycometabolism to drive tumorigenesis.

The diabetes-cancer association remains underexplained. Here, we describe a glucose-signaling axis that reinforces glucose uptake and glycolysis to consolidate the Warburg effect and overcome tumor suppression. Specifically, glucose-dependent CK2 O-GlcNAcylation impedes its phosphorylation of CSN2, a modification required for the deneddylase CSN to sequester Cullin RING ligase 4 (CRL4). Glucose, therefore, elicits CSN-CRL4 dissociation to assemble the CRL4COP1 E3 ligase, which targets p53 to derepress glycolytic enzymes. A genetic or pharmacologic disruption of the O-GlcNAc-CK2-CSN2-CRL4COP1 axis abrogates glucose-induced p53 degradation and cancer cell proliferation. Diet-induced overnutrition upregulates the CRL4COP1-p53 axis to promote PyMT-induced mammary tumorigenesis in wild type but not in mammary-gland-specific p53 knockout mice. These effects of overnutrition are reversed by P28, an investigational peptide inhibitor of COP1-p53 interaction. Thus, glycometabolism self-amplifies via a glucose-induced post-translational modification cascade culminating in CRL4COP1-mediated p53 degradation. Such mutation-independent p53 checkpoint bypass may represent the carcinogenic origin and targetable vulnerability of hyperglycemia-driven cancer.

A signalling pathway for transcriptional regulation of sleep amount in mice.

In mice and humans, sleep quantity is governed by genetic factors and exhibits age-dependent variation1-3. However, the core molecular pathways and effector mechanisms that regulate sleep duration in mammals remain unclear. Here, we characterize a major signalling pathway for the transcriptional regulation of sleep in mice using adeno-associated virus-mediated somatic genetics analysis4. Chimeric knockout of LKB1 kinase-an activator of AMPK-related protein kinase SIK35-7-in adult mouse brain markedly reduces the amount and delta power-a measure of sleep depth-of non-rapid eye movement sleep (NREMS). Downstream of the LKB1-SIK3 pathway, gain or loss-of-function of the histone deacetylases HDAC4 and HDAC5 in adult brain neurons causes bidirectional changes of NREMS amount and delta power. Moreover, phosphorylation of HDAC4 and HDAC5 is associated with increased sleep need, and HDAC4 specifically regulates NREMS amount in posterior hypothalamus. Genetic and transcriptomic studies reveal that HDAC4 cooperates with CREB in both transcriptional and sleep regulation. These findings introduce the concept of signalling pathways targeting transcription modulators to regulate daily sleep amount and demonstrate the power of somatic genetics in mouse sleep research.

A mitochondrial SCF-FBXL4 ubiquitin E3 ligase complex degrades BNIP3 and NIX to restrain mitophagy and prevent mitochondrial disease.

Mitophagy is a fundamental quality control mechanism of mitochondria. Its regulatory mechanisms and pathological implications remain poorly understood. Here, via a mitochondria-targeted genetic screen, we found that knockout (KO) of FBXL4, a mitochondrial disease gene, hyperactivates mitophagy at basal conditions. Subsequent counter screen revealed that FBXL4-KO hyperactivates mitophagy via two mitophagy receptors BNIP3 and NIX. We determined that FBXL4 functions as an integral outer-membrane protein that forms an SCF-FBXL4 ubiquitin E3 ligase complex. SCF-FBXL4 ubiquitinates BNIP3 and NIX to target them for degradation. Pathogenic FBXL4 mutations disrupt SCF-FBXL4 assembly and impair substrate degradation. Fbxl4-/- mice exhibit elevated BNIP3 and NIX proteins, hyperactive mitophagy, and perinatal lethality. Importantly, knockout of either Bnip3 or Nix rescues metabolic derangements and viability of the Fbxl4-/- mice. Together, beyond identifying SCF-FBXL4 as a novel mitochondrial ubiquitin E3 ligase restraining basal mitophagy, our results reveal hyperactivated mitophagy as a cause of mitochondrial disease and suggest therapeutic strategies.

Mixed Lineage Kinase Domain-like Protein MLKL Breaks Down Myelin following Nerve Injury.

Successful regeneration of severed peripheral nerves requires the breakdown and subsequent clearance of myelin, tightly packed membrane sheaths of Schwann cells that protect nerve fibers and harbor nerve growth-inhibitory proteins. How Schwann cells initiate myelin breakdown in response to injury is still largely unknown. Here we report that, following sciatic nerve injury, MLKL, a pseudokinase known to rupture cell membranes during necroptotic cell death, is induced and targets the myelin sheath membrane of Schwann cells to promote myelin breakdown. The function of MLKL in disrupting myelin sheaths requires injury-induced phosphorylation of serine 441, an activation signal distinct from the necroptosis-inducing phosphorylation by RIP3 kinase. Mice with Mlkl specifically knocked out in Schwann cells showed delayed myelin sheath breakdown. Lack of MLKL reduced nerve regeneration following injury, whereas overexpression of MLKL accelerated myelin breakdown and promoted the regeneration of axons.

TMEM225 Is Essential for Sperm Maturation and Male Fertility by Modifying Protein Distribution of Sperm in Mice.

Nonobstructive azoospermia is the leading cause of male infertility. Abnormal levels of transmembrane protein 225 (TMEM225), a testis-specific protein, have been found in patients with nonobstructive azoospermia, suggesting that TMEM225 plays an essential role in male fertility. Here, we generated a Tmem225 KO mouse model to explore the function and mechanism of TMEM225 in male reproduction. Male Tmem225 KO mice were infertile. Surprisingly, Tmem225 deletion did not affect spermatogenesis, but TMEM225-null sperm exhibited abnormalities during epididymal maturation, resulting in reduced sperm motility and an abnormal hairpin-loop configuration. Furthermore, proteomics analyses of cauda sperm revealed that signaling pathways related to mitochondrial function, the glycolytic pathway, and sperm flagellar morphology were abnormal in Tmem225 KO sperm, and spermatozoa lacking TMEM225 exhibited high reactive oxygen species levels, reduced motility, and flagellar folding, leading to typical asthenospermia. These findings suggest that testicular TMEM225 may control the sperm maturation process by regulating the expression of proteins related to mitochondrial function, glycolysis, and sperm flagellar morphology in epididymal spermatozoa.

c-JUN-mediated transcriptional responses in lymphatic endothelial cells are required for lung fluid clearance at birth.

Fluid clearance mediated by lymphatic vessels is known to be essential for lung inflation and gas-exchange function during the transition from prenatal to postnatal life, yet the molecular mechanisms that regulate lymphatic function remain unclear. Here, we profiled the molecular features of lymphatic endothelial cells (LECs) in embryonic and postnatal day (P) 0 lungs by single-cell RNA-sequencing analysis. We identified that the expression of c-JUN is transiently upregulated in P0 LECs. Conditional knockout of Jun in LECs impairs the opening of lung lymphatic vessels at birth, leading to fluid retention in the lungs and neonatal death. We further demonstrated that increased mechanical pressure induces the expression of c-JUN in LECs. c-JUN regulates the opening of lymphatic vessels by modulating the remodeling of the actin cytoskeleton in LECs. Our study established the essential regulatory function of c-JUN-mediated transcriptional responses in facilitating lung lymphatic fluid clearance at birth.

Near-room-temperature water-mediated densification of bulk van der Waals materials from their nanosheets.

The conventional fabrication of bulk van der Waals (vdW) materials requires a temperature above 1,000 °C to sinter from the corresponding particulates. Here we report the near-room-temperature densification (for example, ∼45 °C for 10 min) of two-dimensional nanosheets to form strong bulk materials with a porosity of <0.1%, which are mechanically stronger than the conventionally made ones. The mechanistic study shows that the water-mediated activation of van der Waals interactions accounts for the strong and dense bulk materials. Initially, water adsorbed on two-dimensional nanosheets lubricates and promotes alignment. The subsequent extrusion closes the gaps between the aligned nanosheets and densifies them into strong bulk materials. Water extrusion also generates stresses that increase with moulding temperature, and too high a temperature causes intersheet misalignment; therefore, a near-room-temperature moulding process is favoured. This technique provides an energy-efficient alternative to design a wide range of dense bulk van der Waals materials with tailored compositions and properties.

P62 promotes FSH-induced antral follicle formation by directing degradation of ubiquitinated WT1.

In females, the pathophysiological mechanism of poor ovarian response (POR) is not fully understood. Considering the expression level of p62 was significantly reduced in the granulosa cells (GCs) of POR patients, this study focused on identifying the role of the selective autophagy receptor p62 in conducting the effect of follicle-stimulating hormone (FSH) on antral follicles (AFs) formation in female mice. The results showed that p62 in GCs was FSH responsive and that its level increased to a peak and then decreased time-dependently either in ovaries or in GCs after gonadotropin induction in vivo. GC-specific deletion of p62 resulted in subfertility, a significantly reduced number of AFs and irregular estrous cycles, which were same as pathophysiological symptom of POR. By conducting mass spectrum analysis, we found the ubiquitination of proteins was decreased, and autophagic flux was blocked in GCs. Specifically, the level of nonubiquitinated Wilms tumor 1 homolog (WT1), a transcription factor and negative controller of GC differentiation, increased steadily. Co-IP results showed that p62 deletion increased the level of ubiquitin-specific peptidase 5 (USP5), which blocked the ubiquitination of WT1. Furthermore, a joint analysis of RNA-seq and the spatial transcriptome sequencing data showed the expression of steroid metabolic genes and FSH receptors pivotal for GCs differentiation decreased unanimously. Accordingly, the accumulation of WT1 in GCs deficient of p62 decreased steroid hormone levels and reduced FSH responsiveness, while the availability of p62 in GCs simultaneously ensured the degradation of WT1 through the ubiquitin‒proteasome system and autophagolysosomal system. Therefore, p62 in GCs participates in GC differentiation and AF formation in FSH induction by dynamically controlling the degradation of WT1. The findings of the study contributes to further study the pathology of POR.

Blockage of MLKL prevents myelin damage in experimental diabetic neuropathy.

SignificanceDiabetic neuropathy is a commonly occurring complication of diabetes that affects hundreds of millions of patients worldwide. Patients suffering from diabetic neuropathy experience abnormal sensations and have damage in their peripheral nerve axons as well as myelin, a tightly packed Schwann cell sheath that wraps around axons to provide insulation and increases electrical conductivity along the nerve fibers. The molecular events underlying myelin damage in diabetic neuropathy are largely unknown, and there is no efficacious treatment for the disease. The current study, using a diabetic mouse model and human patient nerve samples, uncovered a molecular mechanism underlying myelin sheath damage in diabetic neuropathy and provides a potential treatment strategy for the disease.

SP1 impacts the primordial to primary follicle transition by regulating cholesterol metabolism in granulosa cells.

The primordial to primary follicle transition (PPT) in the ovary is critical to maintain sustainable reproductive resources in female mammals. However, it is unclear how granulosa cells (GCs) of the primary follicle participate in regulating PPT. This study focused on exploring the role of transcription factor Sp1 (SP1) in regulating PPT based on the fact that SP1 is pivotal for pregranulosa cell proliferation before primordial follicle formation. The results showed that mice fertility was prolonged when Sp1 was specifically depleted from GCs (GC- Sp1 -/- ). Besides, the PPT in GC- Sp1 -/- mice was reduced, resulting in more primordial follicles being preserved. Single-cell RNA-seq also indicated that the level of cholesterol metabolism was downregulated in GC- Sp1 -/- mice. Additionally, the PPT was promoted by either overexpression of ferredoxin-1 (FDX1), one of the key genes in mediating cholesterol metabolism or supplementing cholesterol for cultured fetal ovaries. Collectively, SP1 in GCs participates in the metabolism of cholesterol partially by regulating the transcription of Fdx1 during the PPT.

Preclinical and phase I studies of an antisense oligonucleotide drug targeting IGF-1R in liver cancer.

Aim: To evaluate a novel antisense oligonucleotide drug targeting human IGF-1R in preclinical and phase I studies of liver cancer. Materials & methods: The tolerability and safety of an investigational new drug were evaluated in a dose-escalation trial involving 17 patients with advanced liver cancer after preclinical assessment of pharmacokinetics and pharmacodynamics. Results: The drug exposure levels in the phase I trial were determined by the in vivo efficacy with pharmacokinetics evaluation in rats and rhesus monkeys. This clinical study showed that the maximum tolerated dose was 3.96 mg/kg, and the dose-limiting toxicity dose was 4.4 mg/kg. Conclusion: The drug was safe and tolerable in patients with advanced liver cancer.Clinical Trial Registration: ChiCTR2100044235 (www.chictr.org.cn).

CT102 is a potential new drug for liver cancer treatment. It belongs to a new form of medicine using gene therapy technology called antisense oligonucleotides. There are some antisense oligonucleotides approved for treating rare diseases. This study evaluated the antitumor effect, metabolism and safety of CT102 in preclinical and clinical trials. The results showed that CT102 could inhibit tumor growth in mice with liver cancer and maintain high levels in the liver. It was found that CT102 was safe and tolerable in patients with advanced liver cancer. This suggests that CT102 has therapeutic potential for liver cancer treatment. The good tolerability and safety of CT102 in patients supports further studies on liver cancer treatment.

The role of silver nanoparticles alone and combined with imipenem on carbapenem-resistant Klebsiella pneumoniae.

AIMS: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections poses a significant threat to human health, necessitating urgent development of new antimicrobial agents. Silver nanoparticles (AgNPs), which are among the most widely used engineered nanomaterials, have been extensively studied. However, the impact of AgNPs on CRKP and the potential for drug resistance development remain inadequately explored. METHODS AND RESULTS: In this study, broth dilution method was used to determine the minimum inhibitory concentration (MIC) was determined using the broth dilution method. Results indicated MIC values of 93.1 ± 193.3 µg ml-1 for AgNPs, 2.3 ± 5.1 µg ml-1 for AgNO3, and 25.1 ± 48.3 µg ml-1 for imipenem (IMI). The combined inhibitory effect of AgNPs and IMI on CRKP was assessed using the checkerboard method. Moreover, after 6-20 generations of continuous culture, the MIC value of AgNPs increased 2-fold. Compared to IMI, resistance of Kl. pneumoniae to AgNPs developed more slowly, with a higher fold increase in MIC observed after 20 generations. Whole-genome sequencing revealed four nonsynonymous single nucleotide polymorphism mutations in CRKP after 20 generations of AgNP treatment. CONCLUSION: We have demonstrated that AgNPs significantly inhibit CRKP isolates and enhance the antibacterial activity of imipenem against Kl. pneumoniae. Although the development of AgNP resistance is gradual, continued efforts are necessary for monitoring and studying the mechanisms of AgNP resistance.

High-throughput extraction and automatic purification of alternariol from edible and medicinal herbs based on aptamer-functionalized magnetic nanoparticles.

Mycotoxin contamination is widespread in plants and herbs, posing serious threats to the consumer and human health. Of them, alternariol (AOH) has attracted great attention as an "emerging" mycotoxin in medicinal herbs. However, a specific and high-throughput extraction method for AOH is currently lacking. Thus, developing an efficient pre-treatment technique for AOH detection is extremely vital. Here, a novel automated magnetic solid-phase extraction method was proposed for the highly efficient extraction of AOH. Combining the aptamer-functionalized magnetic nanoparticles (AMNPs) and the automatic purification instrument, AOH could be extracted in medicinal herbs in high throughput (20 samples) and a short time (30 min). The main parameters affecting extraction were optimized, and the method was finally carried out by incubation AMNPs with 3 mL of sample solution for 10 min, and then desorption in 75% methanol for liquid-phase detection. Under optimal conditions, good reproducibility, stability, and selectivity were realized with an adsorption capacity of 550.84 ng/mg. AOH extraction in three edible herbs showed good resistance to matrix interference with recovery rates from 86% to 111%. In combination with AMNPs and the automatic purification instrument, high-throughput and labor-free extraction of AOH in different complex matrices was achieved, which could be extended in other complex matrices.

Mitophagy protects against silver nanoparticle-induced hepatotoxicity by inhibiting mitochondrial ROS and the NLRP3 inflammasome.

Silver nanoparticles (AgNPs) have wide clinical applications because of their excellent antibacterial properties; however, they can cause liver inflammation in animals. Macrophages are among the main cells mediating inflammation and are also responsible for the phagocytosis of nanomaterials. The NLRP3 inflammasome is a major mechanism of inflammation, and its activation both induces cytokine release and triggers inflammatory cell death (i.e., pyroptosis). In previous studies, we demonstrated that mitophagy activation plays a protective role against AgNP-induced hepatotoxicity. However, the exact molecular mechanisms underlying these processes are not fully understood. In this study, we demonstrate that AgNP exposure induces NLRP3 inflammasome activation, mitochondrial damage and pyroptosis in vivo and in vitro. NLRP3 silencing or inhibiting mitochondrial reactive oxygen species (ROS) overproduction reduces PINK1-Parkin-mediated mitophagy. Meanwhile, the inhibition of mitophagy ROS production, mitochondrial, NLRP3-mediated inflammation, and pyroptosis in RAW264.7 cells were more pronounced than in the control group. These results suggest that PINK1-Parkin-mediated mitophagy plays a protective role by reducing AgNP-induced mitochondrial ROS and subsequent NLRP3 inflammasome activation.

AnCMBR-AFB-integrated process for the treatment of high nitrogen and phosphorus wastewater.

Improving the nitrogen and phosphorus removal rates and efficiently controlling membrane fouling are the keys to fully exploiting the applicability of anaerobic membrane bioreactor (AnMBR) process in high-concentration wastewater treatment. To that purpose, an integrated reactor composed of an anaerobic ceramic membrane bioreactor and N anaerobic fluidized bed (AnCMBR-AFB) was built and pollutant removal efficiency, nitrogen and phosphorus recovery characteristics, and membrane pollution features of this integrated reactor were investigated. The results revealed that the integrated reactor had good pollutant removal efficiency, with turbidity, chromaticity, and UV254 average values of the effluent being 0.470 NTU, 0.011 A, and 0.057 cm-1, respectively, and the average CODCr removal rate was 80%. The nitrogen and phosphorus recoveries were significantly higher than the nitrogen and phosphorus removal rates of conventional AnMBR at 23.20 ± 1.17% and 43.34 ± 1.54%, respectively. Microscopic analysis revealed the formation of magnesium ammonium phosphate (MAP) crystals on the carrier's surface, and friction between the carrier and the membrane surface could delay membrane fouling while allowing the contaminated membrane surface to retain significant roughness. Membrane fouling was mostly brought on by amides and saturated hydrocarbons, and inorganic metal ions also played a role to some extent.

Somatic genetics analysis of sleep in adult mice.

Classical forward and reverse mouse genetics require germline mutations and, thus, are unwieldy to study sleep functions of essential genes or redundant pathways. It is also time-consuming to conduct electroencephalogram/electromyogram-based mouse sleep screening owing to labor-intensive surgeries and genetic crosses. Here, we describe a highly accurate SleepV (video) system and adeno-associated virus (AAV)-based adult brain chimeric (ABC)-expression/knockout (KO) platform for somatic genetics analysis of sleep in adult male or female mice. A pilot ABC screen identifies CREB and CRTC1, of which constitutive or inducible expression significantly reduces quantity and/or quality of non-rapid eye movement sleep. Whereas ABC-KO of exon 13 of Sik3 by AAV-Cre injection in Sik3-E13flox/flox adult mice phenocopies Sleepy (Sik3Slp/+) mice, ABC-CRISPR of Slp/Sik3 reverses hypersomnia of Sleepy mice, indicating a direct role of SLP/SIK3 kinase in sleep regulation. Multiplex ABC-CRISPR of both orexin/hypocretin receptors causes narcolepsy episodes, enabling one-step analysis of redundant genes in adult mice. Therefore, this somatic genetics approach should facilitate high-throughput analysis of sleep regulatory genes, especially for essential or redundant genes, in adult mice by skipping mouse development and minimizing genetic crosses.SIGNIFICANCE STATEMENTThe molecular mechanisms of mammalian sleep regulation remain unclear. Classical germline mouse genetics are unwieldy to study sleep functions of essential genes or redundant pathways. The EEG/EMG-based mouse sleep screening is time-consuming owing to labor-intensive surgeries and lengthy genetic crosses. To overcome these "bottlenecks", we developed a highly accurate video-based sleep analysis system and adeno-associated virus-mediated ABC-expression/knockout platform for somatic genetics analysis of sleep in adult mice. These methodologies facilitate rapid identification of sleep regulatory genes, but also efficient mechanistic studies of the molecular pathways of sleep regulation in mice.

Self-Referenced Synthetic Urinary Biomarker for Quantitative Monitoring of Cancer Development.

Urinary monitoring of diseases has gained considerable attention due to its simple and non-invasive sampling. However, urinalysis remains limited by the dearth of reliable urinary biomarkers and the intrinsically enormous heterogeneity of urine samples. Herein, we report, to our knowledge, the first renal-clearable Raman probe encoded by an internal standard (IS)-conjugated reporter that acts as a quantifiable urinary biomarker for reliable monitoring of cancer development, simultaneously eliminating the impact of sample heterogeneity. Upon delivery of the probes into tumor microenvironments, the endogenously overexpressed ß-glucuronidase (GUSB) can cleave the target-responsive residues of the probes to produce IS-retained gold nanoclusters, which were excreted into host urine and analyzed by Au growth-based surface-enhanced Raman spectroscopy. As a result, the in vivo GUSB activity was transformed into in vitro quantitative urinary signals. Based on this IS-encoded synthetic biomarker, both the cancer progression and therapy efficacy were quantitatively monitored, potentiating clinical implications.

Ubiquitination and degradation of GBPs by a Shigella effector to suppress host defence.

Interferon-inducible guanylate-binding proteins (GBPs) mediate cell-autonomous antimicrobial defences. Shigella flexneri, a Gram-negative cytoplasmic free-living bacterium that causes bacillary dysentery, encodes a repertoire of highly similar type III secretion system effectors called invasion plasmid antigen Hs (IpaHs). IpaHs represent a large family of bacterial ubiquitin-ligases, but their function is poorly understood. Here we show that S. flexneri infection induces rapid proteasomal degradation of human guanylate binding protein-1 (hGBP1). We performed a transposon screen to identify a mutation in the S. flexneri gene ipaH9.8 that prevented hGBP1 degradation. IpaH9.8 targets hGBP1 for degradation via Lys48-linked ubiquitination. IpaH9.8 targets multiple GBPs in the cytoplasm independently of their nucleotide-bound states and their differential function in antibacterial defence, promoting S. flexneri replication and resulting in the death of infected mice. In the absence of IpaH9.8, or when binding of GBPs to IpaH9.8 was disrupted, GBPs such as hGBP1 and mouse GBP2 (mGBP2) translocated to intracellular S. flexneri and inhibited bacterial replication. Like wild-type mice, mutant mice deficient in GBP1-3, 5 and 7 succumbed to S. flexneri infection, but unlike wild-type mice, mice deficient in these GBPs were also susceptible to S. flexneri lacking ipaH9.8. The mode of IpaH9.8 action highlights the functional importance of GBPs in antibacterial defences. IpaH9.8 and S. flexneri provide a unique system for dissecting GBP-mediated immunity.