Biology subjects

Arumugam, P.

Publications and source records attributed to Arumugam, P..

16 recordsLinked to original sources

bioRxiv2026

GM-CSF Programs Alveolar Macrophages to Maintain Pulmonary Lymphocyte Homeostasis

Pulmonary lymphocytosis is a common feature of chronic lung disease, but the mechanisms governing lymphocyte accumulation within the lung remain poorly understood. Here, we identify granulocyte-macrophage colony-stimulating factor (GM-CSF) as a central regulator of pulmonary lymphocyte homeostasis. Using genetic models, single-nucleus and spatial transcriptomics, cell trafficking analyses, and pulmonary macrophage replacement studies, we show that loss of GM-CSF signaling causes profound pulmonary lymphocytosis characterized by lymphoid follicle formation, local immunoglobulin production, and expansion of multiple lymphocyte lineages. Mechanistically, GM-CSF signaling deficiency blocks terminal alveolar macrophage differentiation and reprograms alveolar macrophages into inflammatory states marked by reduced PU.1 and PPAR{gamma} expression, enhanced NF-{kappa}B signaling, and expression of lymphocyte-recruiting chemokines. These changes promote lymphocyte recruitment, local proliferation, and lymphoid lineage commitment. Restoration of GM-CSF-responsive alveolar macrophages re-established macrophage identity, suppressed inflammatory chemokine programs, and reversed pulmonary lymphocytosis. Together, these findings identify alveolar macrophages as gatekeepers of adaptive immune homeostasis in the lung.

immunology↗

bioRxiv2026

Autophagy prevents ER stress-induced Tight Junction barrier disruption via claudin-2 homeostasis

Defective intestinal epithelial tight junction (TJ) barrier function and endoplasmic reticulum (ER) stress are central pathological features of inflammatory bowel disease (IBD), yet the molecular mechanisms ER stress to TJ disruption remains poorly understood. Here, we investigated the role of autophagy in regulating intestinal TJ homeostasis during ER stress. ER stress was elevated in inflamed Crohns disease tissue and chronic dextran sulfate sodium (DSS) colitis. In human intestinal epithelial Caco-2 monolayers, murine colon, and human colonic explants, induction of ER stress with tunicamycin, thapsigargin, or brefeldin A disrupted TJ barrier integrity, as demonstrated by reduced transepithelial electrical resistance and increased paracellular permeability. ER stress selectively increased the pore-forming TJ protein claudin-2 and altered occludin localization without significantly affecting other claudins. Pharmacologic activation of autophagy with rapamycin attenuated ER stress, restored TJ barrier function, reduced claudin-2 accumulation, and preserved occludin localization. Conversely, CRISPR-Cas9-mediated deletion of autophagy gene ATG7 exacerbated ER stress, apoptosis, and TJ barrier dysfunction in vitro, while intestinal epithelial-specific Atg7 knockout mice exhibited enhanced ER stress-induced intestinal permeability in-vivo. Mechanistically, prolonged ER stress impaired autophagic flux through IRE1 kinase signaling, resulting in accumulation of p62 and claudin-2. Inhibition of IRE1 kinase activity restored autophagy, reduced claudin-2 levels, and preserved TJ barrier function. We further identified adaptor-associated kinase 1 (AAK1) as a downstream mediator of IRE1 signaling during ER stress, with increased AP2M1 phosphorylation and altered claudin-2 trafficking. Claudin-2 overexpression alone induced ER stress and lysosomal damage, suggesting a feed-forward mechanism amplifying epithelial injury. Finally, enteric rapamycin administration reduced ER stress and restored autophagy in murine DSS colitis. Collectively, these findings identify an IRE1-AAK1-autophagy axis as a critical regulator of intestinal TJ barrier integrity during ER stress.

physiology↗

bioRxiv2026

Perturbing glycosylphosphatidylinositol (GPI)-anchor biosynthesis alters cell wall architecture and modulates fungal morphology

Filamentous fungi are widely used for industrial protein production but their tendency to form dense mycelial pellets limits nutrient transfer, oxygen uptake and fermentation efficiency. While cell wall components are known to influence fungal aggregation, the molecular mechanisms linking cell wall biosynthesis to macroscopic morphology remain poorly defined. Using Aspergillus oryzae as a model, we show that perturbation of glycosylphosphatidylinositol (GPI)-anchored protein biosynthesis influences fungal morphology by altering cell wall composition. Disruption of the GPI ethanolamine phosphate transferase 1 (mcd4) or inhibition of glucosaminyl phosphatidylinositol acyltransferase Gwt1 using antifungal drug manogepix (MGX) induced hyper-branching and weakened the cell wall. Notably, chemical inhibition of Gwt1 by MGX causes a marked transition from pelleted to dispersed mycelial growth. Solid-state NMR (ssNMR) analysis revealed reorganisation of galactosaminogalactan (GAG) and galactomannan (GM), including complete loss of cationic galactosamine (GalN), a key determinant of hyphal adhesion. Transcriptomic analysis revealed downregulation of genes involved in somatic cell fusion, linking altered wall composition to impaired germling aggregation. Strikingly, MGX treatment induced distinct morphological outcomes in other industrially relevant fungi, indicating species-specific cell- wall dependencies. Together, these findings identify GPI-anchored proteins as key regulators of fungal morphology and provide a framework for rational morphology engineering to improve industrial fermentation.

molecular biology↗

bioRxiv2025

Genome-Wide CRISPR/Cas9 Screening Reveals Lipid Metabolism and Inflammatory Signalling as Modulators of Ganoderic Acid DM Cytotoxicity

Ganoderic Acid DM (GA-DM), a triterpenoid derived from Ganoderma lucidum, exhibits anti-cancer and anti-diabetic activities, but the underlying mechanisms of action remain unclear. To identify genetic modulators of GA-DM response, we conducted a genome-wide CRISPR/Cas9 knockout screen in human melanoma cells. The screen revealed key roles for genes regulating lipid metabolism and inflammatory signalling, particularly the SREBP (Sterol Regulatory Element-binding Protein) and NF-{kappa}B (Nuclear Factor kappa-light-chain-enhancer of activated B cells) pathways, in the cellular response to GA-DM. While loss of genes involved in regulation of cholesterol biosynthesis conferred resistance to GA-DM, the disruption of ubiquitin-mediated proteolysis and the Hippo pathway genes sensitised cells to GA-DM. Inflammatory genes enriched at later time points suggests that a delayed cellular response contributes to cytotoxicity. Our findings propose a mechanistic model wherein GA-DM perturbs lipid and inflammatory pathways to exert cytotoxic effects and highlights potential targets to enhance its therapeutic efficacy. This work demonstrates the utility of functional genomics in elucidating natural product mechanisms and guiding rational drug development.

genomics↗

bioRxiv2025

Metabolic engineering approach to boost γ-decalactone production in S. cerevisiae identifies a critical role for the peroxisomal Fatty Acyl-CoA Synthetase Faa2

Lactones constitute a family of aroma compounds found in fruits, flowers and vegetables and are in high demand in the food industry. Previous studies have reported biotransformation of castor beans-extracted ricinoleic acid to {gamma}-decalactone using oleaginous yeast Yarrowia lipolytica. Given the potential toxicities associated with castor beans, we have used a metabolic flux-engineering approach to produce {gamma}-decalactone from oleic acid in Saccharomyces cerevisiae. Intracellular conversion of oleic acid to ricinoleic acid was achieved by the expression of oleate hydroxylase Fah12 from ergot fungus Claviceps purpurea. Glycerol-3-phosphate dehydrogenase-mediated glycerol synthesis was identified as the major metabolic diversion of oleic acid that negatively impacts {gamma}-decalactone yields. Chemogenomic profiling analysis revealed that the tryptophan biosynthetic pathway provides resistance to {gamma}-decalactone-mediated toxicity in yeast. Overexpression of tryptophan transporter Tat1 enhanced {gamma}-decalactone production by about 3- fold. Deficiency of genes encoding the cytoplasmic fatty acyl CoA synthetases FAA1 or FAA4 alone did not significantly influence {gamma}-decalactone production. However, deficiency of peroxisomal FAA2 drastically diminished the yield of {gamma}-decalactone. Thus, this study uncovers the metabolic barriers to oleic acid-to-{gamma}-decalactone conversion and identifies Faa2 as an essential element in this biotransformation.

synthetic biology↗

bioRxiv2022

Metabolism of glucose activates TORC1 through multiple mechanisms in Saccharomyces cerevisiae

Target of Rapamycin Complex 1 (TORC1) is a conserved eukaryotic protein complex that links the presence of nutrients with cell growth. In Saccharomyces cerevisiae, TORC1 activity is positively regulated by the presence of amino acids and glucose in the medium. However, mechanisms underlying nutrient-induced TORC1 activation remain poorly understood. By utilizing a TORC1 activation assay, we demonstrate that differential metabolism of glucose activates TORC1 through three distinct pathways in yeast. The first canonical Rag GTPase-dependent pathway requires conversion of glucose to fructose 1,6-bisphosphate which activates TORC1 via the Rag GTPase heterodimer Gtr1GTP/Gtr2GDP. The second non-canonical Rag GTPase-dependent pathway requires conversion of glucose to glucose 6-phosphate which activates TORC1 via Gtr1GTP/Gtr2GTP. The third Rag GTPase-independent pathway requires complete glycolysis and vacuolar ATPase reassembly for TORC1 activation. Glucose-induced TORC1 activation can be uncoupled from glucose-induced AMPK inactivation. We have established a roadmap to deconstruct the link between glucose metabolism and TORC1 activation.

genetics↗

bioRxiv2022

Floating Sphere Assay: A rapid qualitative method for microvolume analysis of gelation

1A huge, unprecedented demand for gelatin coupled with its implications on global sustainability has resulted in the need to discover novel proteins with gelling attributes for applications in the food industry. Currently used gelation assays require large sample volumes and thus the screening for novel gelling proteins is a formidable technical challenge. In this paper, we report the Floating Sphere Assay which is a simple, economical, and miniaturized assay to detect minimum gelling concentration with volumes as low as 50 l. Results from the Floating Sphere Assay are consistent with currently used methods for gelation tests and accurately estimate the Minimum Gelling Concentrations (MGCs) of gelatin, {kappa}-carrageenan and gellan gum. The assay was also able to differentiate the strengths of strong and weak gellan gum gels prepared at pH 3.5 and pH 7.0 respectively. The Floating Sphere Assay can be utilized in high-throughput screens for gelling proteins and can accelerate the discovery of gelatin substitutes. 2 HighlightsO_LIWe report the Floating Sphere Assay that can be used to assesses minimum gelling concentration of solutions with volumes as low as 50 l. C_LIO_LIObserving whether a glass sphere placed on the surface of a test solution floats or sinks is diagnostic of gel formation C_LIO_LIFloating Sphere Assay can distinguish a strong gel from a weak gel C_LIO_LIFloating Sphere Assay is a rapid and cost-effective approach to screen for novel plant-based gelatin alternatives. C_LI

biochemistry↗

bioRxiv2021

The mitochondrial gene CMPK2 functions as a rheostat for macrophage homeostasis in inflammation

In addition to their role in cellular energy production, mitochondria are increasingly recognized as regulators of the innate immune response of phagocytes. Here, we demonstrate that altering expression levels of the mitochondria associated enzyme, cytidine monophosphate kinase 2 (CMPK2), disrupts mitochondrial physiology and significantly de-regulates the resting immune homeostasis of macrophages. Both CMPK2 silenced as well as constitutively over expressing macrophage lines portray mitochondrial stress with marked depolarization of their membrane potential, enhanced ROS and disturbed architecture culminating in the enhanced expression of the pro-inflammatory genes-IL1{beta}, TNF and IL8. Interestingly, the long-term modulation of CMPK2 expression resulted in an increased glycolytic flux of macrophages akin to the altered physiological state of activated M1 macrophages. While infection induced inflammation for restricting pathogens is regulated, our observation of a total dysregulation of basal inflammation by bidirectional alteration of CMPK2 expression, only highlights a critical role of this gene in mitochondria mediated control of inflammation.

immunology↗

bioRxiv2021

Inhibition of granuloma triglyceride synthesis imparts control of Mycobacterium tuberculosis through curtailed inflammatory responses

Lipid metabolism plays a complex and dynamic role in host-pathogen interaction during Mycobacterium tuberculosis infection. While bacterial lipid metabolism is key to the success of the pathogen, the host also offers a lipid rich environment in the form of necrotic caseous granulomas, making this association beneficial for the pathogen. Accumulation of the neutral lipid triglyceride, as lipid droplets at the cellular cuff of necrotic granulomas, is a peculiar feature of pulmonary tuberculosis. The role of triglyceride synthesis in the TB granuloma and its impact on the disease outcome has not been studied in detail. Here, we identified diacylglycerol O-acyltransferase 1 (DGAT1) to be essential for accumulation of triglyceride in necrotic TB granulomas using the C3HeB/FeJ murine model of infection. Treatment of infected mice with a pharmacological inhibitor of DGAT1 (T863) led to reduction in granuloma triglyceride levels and bacterial burden. A decrease in bacterial burden was associated with reduced neutrophil infiltration and degranulation, and a reduction in several pro-inflammatory cytokines including IL1{beta}, TNF, IL6, and IFN{beta}. Triglyceride lowering impacted eicosanoid production through both metabolic re-routing and via transcriptional control. Our data suggests that manipulation of lipid droplet homeostasis may offer a means for host directed therapy in Tuberculosis.

immunology↗

bioRxiv2021

Identification and characterization of novel infection associated transcripts in macrophages

Regulated expression of genes in response to internal and external stimuli is primarily responsible for the enormous plasticity and robustness of biological systems. Recent studies have elucidated complex regulatory non protein coding transcript (lncRNA) circuits in coordinated response of immune cells. By analysis of lncRNA expression profiles of macrophages in response to Mtb infection, we identified novel highly expressed transcripts, unique in encompassing one functional protein coding transcript- CMPK2 and a previously identified type I IFN responsive lncRNA- NRIR. While these RNA are induced by virulent Mtb early, the complete absence of expression in non-viable Mtb infected cells coupled to a more protracted expression profile in the case of BCG suggest an important role in macrophage response to mycobacteria. Moreover, enhanced expression was observed in macrophages from TB patients. The elevated expression by 1h in response to fast growing bacteria further emphasizes the importance of these RNAs in the macrophage infection response. These transcripts (TILT1, 2,3 - TLR4 and Infection induced Long Transcript) are triggered exclusively by TLR4 stimulation (LPS) with faster and stronger kinetics in comparison to the lncRNA - NRIR. Overall, we provide evidence for the presence of numerous transcripts that is a part of the early infection response program of macrophages.

immunology↗

bioRxiv2021

Role of GM-CSF in regulating metabolism and mitochondrial functions critical to macrophage proliferation

Granulocyte-macrophage colony-stimulating factor (GM-CSF) exerts pleiotropic effects on macrophages and is required for self-renewal but the mechanisms responsible are unknown. Using GM-CSF receptor-{beta}-chain deficient (Csf2rb-/-) mice, we show GM-CSF is critical for mitochondrial turnover, functions, and integrity. GM-CSF signaling is essential for fatty acid {beta}-oxidation and markedly increased tricarboxylic acid cycle activity, oxidative phosphorylation, and ATP production. GM-CSF also regulated cytosolic pathways including glycolysis, pentose phosphate pathway, and amino acid synthesis. We conclude that GM-CSF regulates macrophages in part through a critical role in maintaining mitochondria, which are necessary for cellular metabolism as well as proliferation and self-renewal.

cell biology↗

bioRxiv2020

High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next generation sequencing

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance and for determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for detection of SARS-CoV-2, with an additional advantage of enabling genetic epidemiology of SARS-CoV-2.

genomics↗

bioRxiv2020

The antidepressant sertraline provides a novel host directed therapy module for augmenting TB therapy

A prolonged therapy, primarily responsible for development of drug resistance by Mycobacterium tuberculosis (Mtb), obligates any new TB regimen to not only reduce treatment duration but also escape pathogen resistance mechanisms. With the aim of harnessing the host response in providing support to existing regimens, we used sertraline (SRT) to stunt the pro-pathogenic type I IFN response of macrophages to infection. While SRT alone could only arrest bacterial growth, it effectively escalated the bactericidal activities of Isoniazid (H) and Rifampicin (R) in macrophages. This strengthening of antibiotic potencies by SRT was more evident in conditions of ineffective control by these frontline TB drug, against tolerant strains or dormant Mtb. SRT, could significantly combine with standard TB drugs to enhance early pathogen clearance from tissues of mice infected with either drug sensitive/ tolerant strains of Mtb. Further, we demonstrate an enhanced protection in acute TB infection of the highly susceptible C3HeB/FeJ mice with the combination therapy signifying the use of SRT as a potent adjunct to standard TB therapeutic regimens against bacterial populations of diverse physiology. This study advocates a novel host directed adjunct therapy regimen for TB with a clinically approved anti-depressant to achieve quicker and greater control of infection.

immunology↗

bioRxiv2019

TORC1 regulates the transcriptional response to glucose and developmental cycle via the Tap42-Sit4-Rrd1/2 pathway in Saccharomyces cerevisiae

Target of Rapamycin Complex 1 (TORC1) is a highly conserved eukaryotic protein complex that couples the presence of growth factors and nutrients in the environment with cellular proliferation. TORC1 is primarily implicated in linking amino acid levels with cellular growth in yeast and mammals. Although glucose deprivation has been shown to cause TORC1 inactivation in yeast, the precise role of TORC1 in glucose signaling and the underlying mechanisms remain unclear. In this paper, we demonstrate that the presence of glucose in the growth medium is both necessary and sufficient for TORC1 activation. TORC1 activity increases upon addition of glucose to yeast cells growing in a non-fermentable carbon source. Conversely, shifting yeast cells from glucose to a non-fermentable carbon source reduces TORC1 activity. Analysis of transcriptomic data revealed that glucose and TORC1 co-regulate about 27% (1668/6004) of yeast genes. We demonstrate that TORC1 orchestrates the expression of glucose-response genes mainly via the Tap42-Sit4-Rrd1/Rrd2 pathway. To confirm TORC1s role in glucose-signaling, we tested its role in spore germination, a glucose-dependent developmental state transition in yeast. TORC1 regulates the glucose-responsive genes during spore germination and inhibition of TORC1 blocks spore germination. We propose that a regulatory loop that involves activation of TORC1 by glucose and regulation of glucose-responsive genes by TORC1, mediates nutritional control of growth and development in yeast.

genetics↗

bioRxiv2019

Macrocyclization of an all-D linear peptide improves target affinity and imparts cellular activity: A novel stapled α-helical peptide modality

Peptide-based inhibitors hold great potential for targeted modulation of intracellular protein-protein interactions (PPIs) by leveraging vast chemical space relative to primary structure via sequence diversity as well as conformationally through varying secondary and tertiary structures. However, the development of peptide therapeutics has been hindered because of their limited conformational stability, proteolytic sensitivity and cell permeability. Several contemporary peptide design strategies address these issues to varying degrees. Strategic macrocyclization through optimally placed chemical braces such as olefinic hydrocarbon crosslinks, commonly referred to as staples, may address these issues by i) restricting conformational freedom to improve target affinities, ii) improving proteolytic resistance, and iii) enhancing cell permeability. Conversely, molecules constructed entirely from D-amino acids are hyper-resistant to proteolytic cleavage, but generally lack conformational stability and membrane permeability. Since neither approach is a complete solution, we have combined these strategies to identify the first examples of all-D -helical stapled and stitched peptides. As a template, we used a recently reported all D-linear peptide that is a potent inhibitor of the p53-Mdm2 interaction, but is devoid of cellular activity. To design both stapled and stitched all-D-peptide analogues, we used computational modelling to predict optimal staple placement. The resultant novel macrocyclic all D-peptide was determined to exhibit increased -helicity, improved target binding, complete proteolytic stability and, most notably, cellular activity.

biochemistry↗

bioRxiv2019

Modern clinical Mtb strains leverage the type I IFN pathway for a pro-inflammatory response in the host

Host phagocytes respond to infections by innate defense mechanisms through metabolic shuffling in order to restrict the invading pathogen. However, this very plasticity of the host provides an ideal platform for pathogen mediated manipulation. By employing the macrophage model of Mtb infection, we identify an important strategy employed by modern clinical lineages in regulating the host immune-metabolism axis. The potent ability of these strains to specifically elicit a strong and early macrophage type I IFN response (in contrast to the protracted response to ancient Mtb), was dependent on an increased ability to localize in acidified phagosomes; this higher transit via acidified compartments is important for stimulation of the DNA dependent signaling in infected macrophages. The augmented IFN signaling provided a positive regulatory loop for enhanced expression of the cellular oxysterol-CH25H which in turn facilitated higher levels of IL6 in macrophages infected with the modern Mtb strains. Requirement of type I IFN signaling in mycobacterial intracellular growth highlights another unique ability of Mtb to manipulate host cell physiology and proinflammatory responses.\n\nSignificance StatementCo-evolution with humans has enabled the development of novel adaptive mechanisms for survival in host specific environments in the human TB pathogen-Mtb. We present one such mechanism of modern Mtb strains harnessing the type I IFN immune axis to regulate the host pro-inflammatory response. Our results highlight the use of host intracellular endosomal transit as a mechanism by these strains to ensure a strong type I IFN response in macrophages. We also demonstrate the ability of Mtb to regulate macrophage cholesterol metabolism in order to fine tune the host innate responses. These findings lay the foundation of the future development of a host axis directed intervention strategy against this pathogen.

immunology↗