Avinash Ravindranath Shenoy

Blood feeding in Anopheles mosquitoes is at the root of all malaria infections in humans and is supported by a range of physiological and behavioral adaptations. Since behavior is orchestrated at the level of the central nervous system, we surveyed the cellular diversity and spatial organization of the female Anopheles gambiae brain and compared it to other insect species. We then describe transcriptional changes associated with mosquito life history events with single brain and single-cell RNA sequencing. We did not identify expansion of specific cell populations, but a modulation of neurotransmission in the mated mosquitoes, and a metabolic and immune response associated with glia, Kenyon cells and immune cells after bloodmeal, and detected signatures of aging in the mosquito brain. We did not find an effect of Plasmodium falciparum infection. Our atlas will empower the study of non-model insect neurobiology, neural basis of vector behavior, and mechanisms underlying malaria transmission.

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Activating mutations in GUCY2C, which encodes the receptor guanylyl cyclase C (GC-C), cause early-onset diarrheal disease and gastrointestinal inflammation. Knock-in mice carrying a familial diarrheal syndrome mutation exhibited impaired gut barrier function. Mutant organoids showed defective secretory lineage specification associated with reduced Wnt3 expression. Zinc administration, which lowers epithelial cGMP levels by inhibiting GC-C, reversed most pathological changes in both mice and organoids.

The IFNγ-induced GTPase guanylate-binding protein 1 (GBP1) binds to lipopolysaccharide (LPS) on cytosolic gram-negative bacteria and promotes pyroptosis via the recruitment and activation of caspase-4 on the bacterial outer membrane. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) are extracellular pathogens that adhere to host cells and stimulate dense actin polymerisation underneath their attachment sites, generating structures described as actin-rich pedestals. Here, we show that GBP1 traffics to actin-rich pedestals in human cells infected with EPEC or EHEC in vitro and mouse colonocytes infected with the EPEC-like murine pathogen Citrobacter rodentium in vivo. GBP1 promotes caspase-4 recruitment to actin-rich pedestals, leading to pyroptosis and IL-18 release. GBP1 mutants defective in LPS coatomer formation also localise to EPEC pedestals. A novel assay that mimics pathogenic effector activity reveals GBP1 recruitment to sterile actin polymerisation sites. We conclude that cytosolic GBP1 is mobilised to sites of pathogen-induced actin remodelling independently of LPS. Our study establishes that GBP1 not only operates as a pattern-recognition receptor but also orchestrates effector-triggered immunity against pathogens that hijack the actin cytoskeleton.

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Guanylate-binding proteins (GBPs) are part of a family of large interferon gamma (IFNψ)-inducible GTPases, with ascribed roles in infection control and induction of programmed cell death. While pathogen-specific functions of GBPs have been studied in detail, their broader regulation of frontline immune defences remain unexplored. Here, we analysed the global contribution of human GBP1-5 to cellular metabolism in IFNψ-stimulated macrophages. We found a robust role of GBP2 and GBP5 in macrophage glycolysis. Only GBP5, and not GBP2 deficiency impaired surface expression and cytokine production of classically IFNψ/LPS-activated macrophages. The GTPase activity of GBP5 was required for the regulation of glycolysis and cytokine production. We found that GBP5 deficiency impaired cellular glucose uptake and lactate production specifically. Isotopic tracing with [U- ¹³ C ₆ ]-Glucose confirmed a decrease in several glycolytic intermediates, including glucose 6-phosphate, pyruvate, and lactate, but stable levels of traced tricarboxylic acid cycle (TCA) intermediates. Elevated ribose-5-phosphate and glycerol levels suggest an altered cytosolic redox balance and enhanced breakdown of fatty acids. GBP5 localised predominantly to the cis-Golgi and in the absence of GBP5 we observed increased Golgi fragmentation, however the total Golgi size remained unchanged. Our results underscore the fundamental role of GBP5 in glycolytic fluxes and Golgi integrity in IFNψ-stimulated macrophages, highlighting its significance in immune function in general and immunometabolism specifically.

The IFNγ-induced GTPase guanylate binding protein 1 (GBP1) binds to lipopolysaccharide (LPS) on cytosolic Gram-negative bacteria and promotes pyroptosis via the recruitment and activation of caspase-4 on the bacterial outer membrane. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) are extracellular pathogens that also induce LPS-and caspase-4-dependent pyroptosis. However, whether GBP1 is involved in this process remains unknown. EPEC and EHEC adhere intimately to intestinal epithelial cells via avid interactions between the bacterial adhesin Intimin and Tir (Translocated intimin receptor), a type 3 secretion system effector protein. Intimin-mediated clustering of Tir triggers actin polymerisation, leading to pedestal-like structures at bacterial attachment sites. Here we show that GBP1 is recruited to actin-rich pedestals in human cells infected with EPEC and EHEC in vitro and mouse colonocytes infected with the EPEC-like murine pathogen Citrobacter rodentium in vivo . GBP1-dependent caspase-4 trafficking to these sites leads to pyroptosis and IL-18 release. GBP1 mutants defective in LPS coatomer formation also localised to EPEC pedestals, pointing to LPS-independent mobilisation of GBP1 by actin polymerisation induced by extracellular A/E pathogens. To further dissect the underlying mechanism, we engineered a chimeric receptor (FcγR-Tir) by combining the intracellular signalling domain of Tir and the extracellular ligand-binding domain of the Fcγ receptor. Clustering of FcγR-Tir with IgG-coated beads produced ‘sterile’ actin-rich pedestals that were sufficient to recruit GBP1 independently of bacteria. Our findings reveal that cytosolic GBP1 is mobilised to sites of pathogen-induced actin remodelling independently of LPS. We establish that GBP1 not only operates as a pattern-recognition receptor but also orchestrates effector-triggered immunity against pathogens that hijack the actin cytoskeleton. 175-word The IFNγ-induced GTPase guanylate binding protein 1 (GBP1) binds to lipopolysaccharide (LPS) on cytosolic Gram-negative bacteria and promotes pyroptosis via the recruitment and activation of caspase-4 on the bacterial outer membrane. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) are extracellular pathogens that adhere to host cells and stimulate dense actin polymerisation underneath their attachment sites, generating structures called actin-rich ‘pedestals’. Here we show that GBP1 traffics to actin-rich pedestals in human cells infected with EPEC and EHEC in vitro and mouse colonocytes infected with the EPEC-like murine pathogen Citrobacter rodentium in vivo . GBP1 promotes caspase-4 recruitment to actin-rich pedestals, leading to pyroptosis and IL-18 release. GBP1 mutants defective in LPS coatomer formation also localised to EPEC pedestals. Our novel assay that mimics pathogenic effector activity revealed GBP1 recruitment to ‘sterile’ actin polymerisation sites. We conclude that cytosolic GBP1 is mobilised to sites of pathogen-induced actin remodelling independently of LPS. Our study establishes that GBP1 not only operates as a pattern-recognition receptor but also orchestrates effector-triggered immunity against pathogens that hijack the actin cytoskeleton.

Influenza A virus (IAV) Matrix 2 protein (M2) is an ion channel, required for efficient viral entry and egress. M2 interacts with the small ubiquitin-like LC3 protein through a cytoplasmic C-terminal LC3-interacting region (LIR). Here, we report that M2 is cleaved by caspases, abolishing the M2–LC3 interaction. A crystal structure of the M2 LIR in complex with LC3 indicates the caspase cleavage tetrapeptide motif ( ₈₂ SAVD ₈₅ ) is an unstructured linear motif that does not overlap with the LIR. IAV mutant expressing a permanently truncated M2, mimicking caspase cleavage, exhibit defects in M2 plasma membrane transport, viral filament formation, and virion production. Our results reveal a dynamic regulation of the M2–LC3 interaction by caspases. This highlights the role of host proteases in regulating IAV exit, relating virion production with host cell state.

Enterobacter cloacae is a Gram-negative nosocomial pathogen of the ESKAPE ( Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas , and Enterobacter spp.) priority group with increasing multi-drug resistance via the acquisition of resistance plasmids. However, E. cloacae can also display forms of antibiotic refractoriness, such as heteroresistance and tolerance. Here, we report that E. cloacae displays transient heteroresistance to aminoglycosides, which is accompanied with the formation of small colony variants (SCVs) with increased minimum inhibitor concentration (MIC) of gentamicin and other aminoglycosides used in the clinic, but not other antibiotic classes. To explore the underlying mechanisms, we performed RNA sequencing of heteroresistant bacteria, which revealed global gene expression changes and a signature of the CpxRA cell envelope stress response. Deletion of the cpxRA two-component system abrogated aminoglycoside heteroresistance and SCV formation, pointing to its indispensable role in these processes. The introduction of a constitutively active allele of cpxA led to high aminoglycoside MICs , consistent with cell envelope stress response driving these behaviors in E. cloacae . Cell envelope stress can be caused by environmental cues, including heavy metals. Indeed, bacterial exposure to copper increased gentamicin MIC in the wild-type but not in the Δ cpxRA mutant. Moreover, copper exposure also elevated the gentamicin MICs of clinical isolates from bloodstream infections, suggesting that CpxRA- and copper-dependent aminoglycoside resistance is broadly conserved in E. cloacae strains. Altogether, we establish that E. cloacae relies on transcriptional reprogramming via the envelope stress response pathway for transient resistance to a major class of frontline antibiotic. IMPORTANCE: Enterobacter cloacae is a bacterium that belongs to the WHO high-priority group and an increasing threat worldwide due its multi-drug resistance. E. cloacae can also display heteroresistance, which has been linked to treatment failure. We report that E. cloacae shows heteroresistance to aminoglycoside antibiotics. These are important frontline microbicidal drugs used against Gram-negative bacterial infections; therefore, understanding how resistance develops among sensitive strains is important. We show that aminoglycoside resistance is driven by the activation of the cell envelope stress response and transcriptional reprogramming via the CpxRA two-component system. Furthermore, heterologous activation of envelope stress via copper, typically a heavy metal with antimicrobial actions, also increased aminoglycoside MICs of the E. cloacae type strain and clinical strains isolated from bloodstream infections. Our study suggests aminoglycoside recalcitrance in E. cloacae could be broadly conserved and cautions against the undesirable effects of copper.

Influenza A virus (IAV) Matrix 2 (M2) protein is an ion channel, required for efficient viral entry and egress. M2 interacts with the small ubiquitin-like LC3 protein through a cytoplasmic C-terminal LC3 interacting region (LIR). Here, we report that M2 is cleaved by caspases, abolishing the M2-LC3 interaction. A crystal structure of the M2 LIR in complex with LC3 indicates the caspase cleavage tetrapeptide motif ( ₈₂ SAVD ₈₅ ) is an unstructured linear motif that does not overlap with the LIR. Furthermore, an IAV mutant expressing a permanently truncated M2, mimicking caspase cleavage, is impaired in M2 plasma membrane transport and produces a significantly attenuated virus. Our results reveal a dynamic regulation of the M2-LC3 interaction by caspases. This highlights the role of host proteases in regulating IAV exit, relating virion production with host cell state.