Biology subjects

Visweswariah, S. S.

Publications and source records attributed to Visweswariah, S. S..

7 recordsLinked to original sources

bioRxiv2025

Drosophila blood cells bridge distant injury and gut homeostasis through Upd3-mediated inter-organ signaling

Inter-organ communication is a central feature of host defence and tissue repair. In Drosophila, hemocytes secrete the cytokine-like ligand Unpaired 3 (Upd3, an IL-6 homolog) to activate JAK/STAT signaling in the gut, yet why this axis is essential for survival after injury has remained unclear. Here we show that loss of hemocyte activation by reactive oxygen species (ROS) and the consequent failure to produce Upd3 leads to adherens junction disruption, intestinal barrier dysfunction, and increased lethality following clean injury. Hemocyte-derived Upd3 drives sustained STAT activation in the gut epithelium, promoting enterocyte turnover and survival after injury. Chronic STAT activity further modulates intestinal stem cell fate and differentiation, indicating that hemocyte-gut signaling shapes long-term epithelial homeostasis. Notably, hemocytes home to the gut after distant wounding, where their localized presence enhances resistance to enteric infection. Taken together, these findings reveal that Drosophila blood cells bridge distant injury and gut homeostasis through Upd3-mediated inter-organ signaling that links wound sensing to epithelial integrity and host survival.

immunology↗

bioRxiv2025

GBP1 recruitment to actin-rich pedestals induced by extracellular Gram-negative bacteria promotes pyroptosis.

The IFN{gamma}-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 (Fc{gamma}R-Tir) by combining the intracellular signalling domain of Tir and the extracellular ligand-binding domain of the Fc{gamma} receptor. Clustering of Fc{gamma}R-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 AbstractThe IFN{gamma}-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.

immunology↗

bioRxiv2024

Coevolving Residues Distant from the Ligand Binding Site are Involved in GAF Domain Function

Ligand binding to GAF domains regulates the activity of associated catalytic domains in a wide variety of proteins. For instance, cGMP binding to the GAFa domain of phosphodiesterase 5 (PDE5) activates the cGMP-hydrolyzing catalytic domain in the protein. However, the residues involved and the mechanism of GAF domain function are not entirely clear. Here, combining computational and experimental analysis, we show that two highly coevolving residues distant from the ligand binding site play a critical role in GAF domain allostery. Specifically, Statistical Coupling Analysis (SCA) of GAF domain sequences revealed the highest coevolution score for residues L267 and F295. Molecular dynamics (MD) simulations of both apo and holo forms of the wild type and mutant (L267A and F295A) PDE5 GAFa domains revealed significant alterations in structural dynamics and interaction with cGMP. Incorporation of the mutations in a Bioluminescence Resonance Energy Transfer (BRET)-based biosensor, which reports a ligand-induced conformational change, revealed a change in the conformation of the GAF domain and an increase in the EC50 of cGMP-induced conformational change. Similar results were obtained regarding cGMP-induced conformational change in the full-length PDE5 and in the fluorescence of the GAF domain fluorescent protein, miRFP670nano3. Finally, structural analysis of conformers observed in MD simulations revealed a possible mechanism underlying the impact of mutations of these two coevolving residues in the PDE5 GAFa domain. Our results provide insight into the role of distant, coevolving residues in GAF domain allostery, and may aid in understanding evolution of allostery in proteins.

molecular biology↗

bioRxiv2023

A universal stress protein is essential for the survival of Mycobacterium tuberculosis

Mycobacterium tuberculosis employs several signaling pathways to regulate its cellular physiology and survival within the host. Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways in which these bacteria utilize cAMP. We have earlier identified universal stress proteins (USP), Rv1636 and MSMEG_3811 in M. tuberculosis and M. smegmatis respectively, as abundantly expressed, novel cAMP-binding proteins. In this study, we show that these USPs may function to regulate cAMP signaling by direct sequestration of the second messenger. In slow-growing mycobacteria, concentrations of Rv1636 were equivalent to the amounts of cAMP present in the cell, and overexpression of Rv1636 in M. smegmatis increased levels of bound cAMP. Rv1636 is secreted via the SecA2 secretion system in M. tuberculosis but is not directly responsible for the efflux of cAMP from the cell. While msmeg_3811 could be readily deleted from the genome of M. smegmatis, we find that the rv1636 gene is essential for growth of M. tuberculosis, and this functionality depends on the cAMP-binding ability of Rv1636. This is the first evidence of a sink for any second messenger in bacterial signaling that would allow mycobacterial cells to regulate the available intracellular free pool of cAMP.

microbiology↗

bioRxiv2021

Particle Uptake Driven Phagocytosis in Macrophages and Neutrophils Enhances Bacterial Clearance

Humans are exposed to numerous synthetic foreign particulates in the form of environmental pollutants and diagnostic or therapeutic agents. Specialized immune cells (phagocytes) clear these particulates by phagocytosing and attempting to degrade them. The process of recognition and internalization of the particulates may trigger changes in the function of phagocytes. Some of these changes, especially the ability of a particle-loaded phagocyte to take up and neutralize pathogens, remains poorly studied. Herein, we demonstrate that the uptake of non-stimulatory cargo-free particles enhances the phagocytic ability of monocytes, macrophages and neutrophils. The enhancement in phagocytic ability was independent of particle properties, such as size or the base material constituting the particle. Additionally, we show that the increased phagocytosis was not a result of cellular activation or cellular heterogeneity but was driven by changes in cell membrane fluidity and cellular compliance. A consequence of the enhanced phagocytic activity was that particulate-laden immune cells neutralize E. coli faster in culture. Moreover, when administered in mice as a prophylactic, particulates enable faster clearance of E. coli and S. epidermidis. Together, we demonstrate that the process of uptake induces cellular changes that favor additional phagocytic events. This study provides insights into using non-stimulatory cargo-free particles to engineer immune cell functions for applications involving faster clearance of phagocytosable particulates.

immunology↗

bioRxiv2020

An evolutionarily conserved metallophosphodiesterase is a determinant of lifespan in Drosophila

Evolutionarily conserved genes usually have a critical role to play during organismal aging and longevity. Here, we show that a previously uncharacterized Class III metallophosphoesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins in mammals, is necessary for optimal lifespan. dMPPED is the product of the gene CG16717 and hydrolyzed a variety of phosphodiester substrates in a metal-dependent manner. dMPPED was expressed widely during development and in the adult fly. Deletion of the gene in flies dramatically reduced lifespan, without affecting development or fecundity. Longevity was restored on ubiquitous expression of the protein, and neuronal expression of both wild type and the catalytically inactive form of dMPPED was also able to restore normal lifespan. Overexpression of the protein, both ubiquitously and neuronally in wild type flies extended lifespan by ~ 20%. RNA-seq analysis of dMPPEDKO flies revealed mis-regulation of innate immune pathways, a number of transcription factors and genes earlier reported to affect aging and lifespan. Importantly, neuronal expression of mammalian MPPED2 was able to rescue lifespan in dMPPEDKO flies, but not extend lifespan in wild type flies. This reports the first description of the biological role of an evolutionarily conserved metallophosphoesterase that may serve as a scaffolding protein in diverse signaling pathways to modulate longevity in the fly.

developmental biology↗

bioRxiv2017

Receptor guanylyl cyclase C confers protection during oral Salmonella Typhimurium infection by attenuating ileal damage and enhancing cytokine and antimicrobial peptide production

Non-typhoidal Salmonella disease contributes towards significant morbidity and mortality across the world. Host factors including IFN-{gamma}, TNF- and gut microbiota, significantly influence the outcome of Salmonella pathogenesis. However, the entire repertoire of host protective mechanisms contributing to Salmonella pathogenicity is not completely appreciated. Here, we have investigated the roles of receptor guanylyl cyclase C (GC-C) that is predominantly expressed in the intestine, and regulates intestinal cell proliferation and fluid-ion homeostasis. Mice deficient in GC-C (Gucy2c-/-) displayed accelerated mortality following infection via the oral route, in spite of possessing comparative systemic Salmonella infection burden. Survival following intra-peritoneal infection remained similar, indicating that GC-C offered protection via a gut-mediated response. Serum cortisol was higher in Gucy2c-/- mice, in comparison to wild type (Gucy2c+/+) mice, and an increase in infection-induced thymic atrophy, with loss in immature CD4+CD8+ double positive thymocytes, was observed. Accelerated and enhanced damage in the ileum, including submucosal edema, epithelial cell damage, focal tufting and distortion of villus architecture, was seen in Gucy2c-/- mice, concomitant with a larger number of ileal tissue-associated bacteria. Transcription of key mediators in Salmonella-induced inflammation (IL-22/Reg3{beta}) were altered in Gucy2c-/- mice in comparison to Gucy2c+/+ mice. A reduction in fecal Lactobacilli, which are protective against Salmonella infection, was observed in Gucy2c-/- mice. Gucy2c-/- mice cohoused with wild type mice continued to show reduced Lactobacilli and increased susceptibility to infection. Our study therefore suggests that receptor GC-C confers a survival advantage during gut-mediated S. Typhimurium pathogenesis, presumably by regulating Salmonella-effector mechanisms and maintaining a beneficial microbiome.

microbiology↗