Biology subjects

Sangwan, N.

Publications and source records attributed to Sangwan, N..

At least 19 recordsLinked to original sources

bioRxiv2026

SETD3 ACTIN HISTIDINE METHYLTRANSFERASE REGULATES INTESTINAL EPITHELIAL BARRIER INTEGRITY AND MUCOSAL INFLAMMATION

Intestinal epithelial barrier is a key feature of the healthy gut, and disruption of this barrier is a driver of mucosal inflammation. Integrity of the intestinal epithelial barrier is controlled by tight junctions (TJs) and adherens junctions (AJs) associated with the underlying actin cytoskeleton. Posttranslational modifications of actins affect the cytoskeletal assembly and dynamics, however whether such modifications regulate epithelial barriers remains unknown. SETD3 methyltransferase methylates actins at the His73 residue, thereby enhancing filamentous (F)-actin polymerization and turnover. This study examines the role of SETD3 in regulating structure and function of normal intestinal epithelial barrier, as well as its involvement in barrier disruption during mucosal inflammation. siRNA mediated knockdown of SETD3 in DLD-1 and HCA-7 human intestinal epithelial cells (IEC) resulted in several functional and morphological cell abnormalities. These include increased permeability of the IEC barrier, diminished assembly of the perijunctional F-actin belt, increased cell size, and delayed TJ reassembly during calcium switch. Consistently, SETD3 knockout (KO) mice displayed increased intestinal permeability and decreased labeling intensity of apical {beta}-actin as compared to wildtype controls. Furthermore, SETD3 KO mice showed increased cell stemness and proliferation in colonic epithelium without defects in AJ/TJ assembly and secretory cell differentiation. In addition, SETD3 KO mice developed more severe disease symptoms during dextran sodium sulfate (DSS)-induced colitis, which were accompanied by accelerated mucosal erosion and increased macrophage, T cell and neutrophil infiltration into colonic tissue. Overall, our data suggests that SETD3 acts as a novel positive regulator of the intestinal epithelial barrier and plays a protective role during intestinal mucosal damage and inflammation.

cell biology↗

bioRxiv2026

CFTR dysfunction in the intestinal epithelium is sufficient to promote pathogenic expansion of E. coli and enhanced barrier permeability in cystic fibrosis

Changes in the gut microbiome in cystic fibrosis (CF) are well characterized, yet their causes and downstream effects remain largely unknown. Well-documented alterations include reduced overall complexity (i.e. alpha diversity) of the gut microbiota and increased relative abundance of E. coli, which are associated with greater inflammation and shorter stature in infants. Previous results from our laboratory using a germ-free cystic fibrosis transmembrane conductance regulator (Cftr) mutant mouse model (CF mouse) demonstrated that the observed fecal microbiome dysbiosis is driven by mutated Cftr independent of factors such as diet or antibiotic treatment. We expand on these results in this report by using the defined 8-member community Altered Schaedler Flora (ASF) with and without E. coli, to show that E. coli is pathogenic in the context of the CF gut microbiome, resulting in increased intestinal permeability. We also show that Cftr deletion in intestinal epithelial cells alone, using a Villin-Cre targeted model, is sufficient to raise E. coli abundance in the fecal microbiome, increase intestinal permeability, and amplify the number of TH17 cells in the mesenteric lymph nodes. Together, our results demonstrate that the intestinal epithelium plays a dominant role in fecal microbiome alterations in CF and that the resultant dysbiosis contributes to CF pathogenesis.

microbiology↗

bioRxiv2026

Noninvasive Diagnostic Ultrasound-Guided Focused Ultrasound Enables Selective, Reversible Inhibition of Peripheral Nociceptive Fibers and Prevents Acute Pain

BackgroundAcute postoperative pain remains a major clinical therapeutic challenge. Current peripheral nerve blockade (PNB) techniques are effective for some patients but are limited by invasiveness, short duration, reliance on highly trained providers, and off-target motor and sensory effects. Focused ultrasound (FUS) is a novel neuromodulatory technology with the potential to achieve noninvasive, selective, reversible, and prolonged inhibition of peripheral nociceptive fibers to prevent and treat acute pain. We hypothesized that noninvasive transcutaneous targeting of the rat sciatic nerve using co-aligned diagnostic ultrasound (dUS) and FUS transducers could produce selective and reversible inhibition of nociceptive pain behaviors while preserving motor and non-nociceptive sensory functions. MethodsIn an in vivo rat hindpaw incisional (HPI) pain model, using a novel, transcutaneous dUS-guided FUS system, the sciatic nerve was located with dUS, and FUS energy was applied to it just prior to hindpaw incision. FUS parameters were iteratively adjusted to achieve reversible, selective inhibition of nociceptive behaviors without changing motor and non-pain sensory behaviors. Animals were randomized into six groups: No Intervention (Control), HPI Only (Disease Control), Sham FUS, FUS Only, FUS+HPI (Intervention), and LA+HPI (Positive Control). Primary outcomes were changes in nociceptive sensory functions, assessed by thermal and mechanical sensitivity. Secondary outcomes were changes in non-nociceptive sensory and motor functions, assessed by hindpaw flexion and extension reflexes. ResultsCompared with the HPI Only group, the FUS+HPI group demonstrated (1) significant attenuation of hindpaw thermal hypersensitivity from day 0 - week 5.0 and week 8.0 - 16.0 (p < 0.05-0.001); (2) significant attenuation of mechanical hypersensitivity from day 0 until week 4.0 (p < 0.05-0.001); (3) no significant attenuation of flexion; and (4) no significant attenuation of extension. ConclusionsTranscutaneous dUS-guided FUS enables selective, reversible inhibition of A{delta} and C nociceptive fiber mediated behaviors while sparing A motor and A{beta} sensory behaviors. FUS-induced PNB prevented both acute and persistent pain behaviors. These findings support FUS as a promising noninvasive peripheral nerve blockade strategy for acute pain management.

neuroscience↗

bioRxiv2026

Multi-Omic Analyses of Dietary Fatty Acid-Microbe-Host Interactions Reveal Metaorganismal Lipid Metabolic Crosstalk Impacting Cardiometabolic Disease

Following a meal, our gut microbiome and human cells collaborate via metaorganismal metabolic circuits to produce diverse nutrient metabolites that systemically circulate to influence health and disease. Although there are now several examples of bacterial fiber-, amino acid-, and micronutrient-derived metabolites impacting cardiometabolic disease, very little is known in regards to how diet-microbe-host interactions impact lipid homeostasis. Here we address this by defining dietary fatty acid substrate availability in germ-free versus conventionally-raised mice coupled to deep multi-omic metabolic phenotyping. Our data demonstrate that the effects of dietary saturated (SFA), monounsaturated (MUFA), and polyunsaturated fatty acids (PUFA) on the host lipidome, transcriptome, proteome and metabolome are uniquely impacted by resident microbiota. Also, the hepatic levels of both pro-inflammatory and pro-resolving lipid mediators are strongly influenced by dietary fatty acid-microbe interactions. This study presents a unique resource to the nutrition and metabolism research community to advance our understanding of metaorganismal lipid metabolism. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/705588v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@4fc7e3org.highwire.dtl.DTLVardef@1cc1dc8org.highwire.dtl.DTLVardef@1b7648dorg.highwire.dtl.DTLVardef@12a8088_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG

biochemistry↗

bioRxiv2025

Environmental Regulation of Toxin Production in Bacillus anthracis

Pathogenic Bacillus anthracis strains carry two plasmids - pX01, which encodes a tripartite protein exotoxin complex (PA, LF, and EF); and pX02, which encodes a poly-D-gamma-glutamic acid capsule. A multidomain transcription factor, AtxA, regulates the expression of these virulence genes. AtxA has two DNA-binding Helix-Turn-Helix (HTH) domains, two phosphoenolpyruvate:carbohydrate phosphotransferase system regulatory domains (PRD1 and PRD2), and a putative EIIB domain (a component of PTS sugar transport EII-complexes). Previous studies showed that glucose and CO2 increase AtxA-dependent toxin gene transcription, along with histidine phosphorylation of PRD1 and PRD2. Our transcriptional profiling of virulence factors, PA secretion, and fluorescent reporter strain analyses confirms a synergistic effect of glucose and CO2 on AtxA-dependent toxin production. Deletion of AtxA ({Delta}atxA) significantly reduced glucose uptake in bacteria, suggesting that AtxA may act within the glucose-PTS system. Mutation analysis of the EIIB domain of AtxA identified the cysteine at position 402 as essential for the transcriptional activity of AtxA. Deletion of glucose PTS permease PtsG ({Delta}ptsG) significantly reduced the expression of PA, LF, and EF. Loss of PtsG also caused attenuation in a mouse model of infection. Intracellular imaging using FLIM confirms a physical interaction of PtsG and AtxA through EIIB domain of AtxA. Using phosphomimetic and phosphoablative mutants of AtxA, we confirmed that the physical interaction of PtsG and AtxA is essential for AtxA activity. Finally, the synergy between glucose and CO2 was targeted by deleting pyruvate carboxylase Pyc ({Delta}pyc), which regulates anaplerosis. This deletion confirms that Pyc stimulates the level of phosphoenolpyruvate (PEP) and increases the phosphorelay in glucose-PTS to enhance AtxA activity. Therefore, we propose that a histidine-phosphorelay from PEP regulates AtxA via PTS enzymatic activity, impacting AtxA activity through physical interaction of AtxA and PtsG. Finally, we propose AtxA as an integral component of the glucose-PTS, where transcriptional activity of AtxA is regulated by environmental signals including glucose and CO2.

molecular biology↗

bioRxiv2025

Platelets drive immune suppression and glioblastoma growth in a sex-dependent manner via PAR4 signaling

Sex differences in cancer outcome, including that of glioblastoma (GBM), are shaped by biological, hormonal, and immunological factors and impact disease progression, treatment responses, survival, and tumor microenvironment (TME) interactions. Platelets regulate the immune responses and tumor progression in many cancers, but it is not clear how they contribute to these sex-based differences by affecting the dynamics of the TME. Here, we show that GBM patients exhibit heightened platelet reactivity driven by PAR4 signaling. In murine GBM models, both pharmacological inhibition of PAR4 using BMS986120 and genetic deletion of PAR4 significantly prolong survival in females but not males. This survival advantage is estrogen dependent: it is preserved in chromosomal male-hormonal female mice within the four-core genotype model and is rescued in ovariectomized mice treated with estrogen. The survival benefit is TME specific and is mediated by platelet-driven enhancement of CD8 T cell infiltration into the tumor. Inhibition of platelet PAR4 signaling increases calcium signaling through an estrogen-dependent interaction between PAR4 and estrogen receptor {beta} (ER{beta})--a receptor interaction not previously described. PAR4-activated platelets within the TME suppress CD8 T cell function, and depletion of CD8 T cells abolishes both the tumor-induced platelet reactivity and the survival benefit conferred by PAR4 inhibition. These findings establish platelet-mediated PAR4 signaling as a critical driver of tumor progression and identify sex-specific immune responses as key to therapeutic efficacy.

cancer biology↗

bioRxiv2025

A novel AAA+ ATPase required for sporulation and stress response in Bacillus anthracis

AAA+ proteins function as molecular machines that utilize ATP to perform diverse cellular functions, including protein homeostasis, stress regulation, and cell cycle/developmental processes. In this study, we identified a novel AAA+ ATPase BAS PrkA in B. anthracis Sterne 34F2 which has 88 % protein homology to Bacillus subtilis PrkA. Conserved domain analysis confirms BAS PrkA has an N-terminal AAA+ ATPase domain with characteristic Walker A and Walker B motifs and a conserved secondary region of homology (SRH) domain, along with a C-terminal cAMP-dependent protein kinase domain. Based on Alpha Fold3 predicted structure, we classified BAS PrkA as part of Clade III of the AAA+ superfamily. Contrary to the reported enzymatic activity in B. subtilis PrkA, we observed that BAS PrkA has negligible protease and kinase activity under in-vitro conditions. Nonetheless, BAS PrkA plays a significant role in regulating sporulation. It is temporally expressed during Stages II to VI during sporulation. A null mutant of BAS PrkA exhibits severe sporulation defects, with reduced spore viability, and down regulation of genes related to spore-coat formation. These phenotypes were restored in a complementation strain expressing BAS PrkA ectopically. Additionally, the null mutant strain showed compromised growth under ionic-osmotic stress conditions. Analysis of the BAS PrkA interactome revealed enrichment of two proteins, ProA and EzrA, that are implicated in osmotic stress response and the sporulation process, respectively. These findings show that BAS PrkA plays a critical role in sporulation and osmotic stress response in B. anthracis.

microbiology↗

bioRxiv2025

The antimicrobial metabolite nisin Z reduces intestinal tumorigenesis and modulates the cecal microbiome in ApcMin/+ mice

Nisin Z, an antimicrobial metabolite produced by Lactococcus lactis spp., has been safely used as a food preservative for many years. Nisin Z also showed promising activity against various cancer types in vitro, and significantly reduced tumor size in an ectopic head and neck cancer model. Here, we investigate the activity of nisin Z for colorectal cancer treatment and observed an in vitro reduction in cellular proliferation, and a moderate enhancement in cell death. We next analyzed the effect of oral nisin Z administration in the Apcmin/+ intestinal adenoma mouse model. We measured tumor burden along the gastrointestinal tract and observed a decrease in tumor burden in the middle region of the small intestine, but not in the lower region or colon. Since tumor progression in the Apcmin/+ model is exacerbated by an inflammatory environment, we next determined whether nisin Z impacts this in a direct or indirect manner. We show that nisin Z can directly reduce NF-{kappa}B activation in a dose-dependent manner. In addition, nisin Z impacted the cecal microbiome composition as well as microbiota-associated plasma metabolites, causing an overall shift towards a more health-associated profile. Interestingly, the Apcmin/+ genotype differentially impacted the nisin Z-mediated differences in cecal microbiome composition and plasma metabolites compared to wildtype animals. In summary, our data suggest that the reduction in small intestinal tumor burden could be due to nisin Zs contribution to a reduced pro-inflammatory environment. Future studies will reveal whether nisins localized effect is due to degradation of the peptidic compound in more distal regions of the gastrointestinal tract and focus on development of delivery systems to increase efficacy. ImportanceWith the increased incidence of colorectal cancer, especially among younger individuals, it is critical to study approaches that help with the prevention and treatment of this debilitating disease. Our study indicates that nisin Z, a bacterially produced peptide antibiotic, decreases the growth of colorectal cancer cells and moderately increases cell death in vitro. Oral administration of nisin Z in an intestinal adenoma mouse model revealed a reduction of tumor burden in the middle region of the small intestine. This decreased tumor burden might in part be attributed to a direct anti-inflammatory effect, as well as an indirect effect on the gut microbiota and their metabolites due to nisin Zs antibacterial activity. Overall, we demonstrate a potential activity for nisin Z in the prevention or amelioration of inflammation-associated colorectal cancer, underscoring the significance of investigating the properties of bacterial natural products in human health.

microbiology↗

bioRxiv2025

Gut microbial conversion of dietary elderberry extract to hydrocinnamic acid improves obesity-associated metabolic disorders

Obesity-associated metabolic disorders such as type 2 diabetes mellitus and metabolic dysfunction associated fatty liver disease are major global health concerns, yet current pharmacological treatments often present with major side-effects. Dietary interventions including polyphenol-rich foods offer a promising complementary option for obesity amelioration, but their efficacy is dependent on specific gut microbial metabolism and the underlying molecular mechanisms mostly remain elusive. Here, we demonstrated that dietary elderberry (Eld) extract abrogates the effects of an obesogenic diet in a gut microbiota-dependent manner, by preventing insulin resistance and reducing hepatic steatosis in mice. We developed a targeted, quantitative liquid chromatography-tandem mass spectrometry method for detection of gut bacterial polyphenol catabolites and identified hydrocinnamic acid as a key microbial metabolite, enriched in the portal vein plasma of Eld supplemented animals. Next, we showed that hydrocinnamic acid potently activates hepatic AMP-activated protein kinase , explaining its role in improved liver lipid homeostasis. Furthermore, we uncovered the metabolic pathway cumulating in hydrocinnamic acid production in the common gut commensal Clostridium sporogenes. Our characterization of hydrocinnamic acid as a diet-derived, and microbiota-dependent metabolite with insulin-sensitizing and anti-steatotic activities will contribute to microbiome-targeted dietary interventions to prevent and treat obesity-associated metabolic diseases.

microbiology↗

bioRxiv2025

A Saccharomyces boulardii synthetic biotic platform for delivery of therapeutic nanobodies to ameliorate gastrointestinal inflammation

Protein-based pharmaceuticals, such as engineered antibodies, form a major drug class of steadily increasing market share. However, these biologic medicines are costly to manufacture, are subject to strict supply chain and storage constraints and often require invasive administration routes. Engineered microbes that secrete bioactive products directly within the microbiome milieu may mitigate these challenges. Here, we describe a cell microfactory platform based on the probiotic yeast Saccharomyces boulardii for the production of nanobody biologics in the gastrointestinal (GI) tract. High-level secretion of nanobodies by S. boulardii was achieved by optimizing promoters, secretion signals, and antibody formats. In mice, oral gavage of S. boulardii allowed efficient and transient colonization of the colonic compartment and in situ production of a therapeutic nanobody directed against tumor necrosis factor (TNF). In a mouse model of chemical-induced colitis, GI-delivery of anti-mTNF nanobody via live S. boulardii improved both survival and disease severity without causing overt perturbation of microbiome composition. These results position S. boulardii as a synthetic biotic platform for the in situ production and delivery of protein-based therapeutics to the GI tract.

synthetic biology↗

bioRxiv2025

Gut Microbe-Derived N-Acyl Serinol Lipids Shape Host Postprandial Metabolic Homeostasis

Although strong evidence links the gut microbiome to metabolic disease, the mechanisms linking microbiota to hormonal and metabolic responses to food are not well understood. After a meal, gut bacteria produce a wide array of small molecule, protein, and lipid metabolites originating from bacterial sources. Annotating physiological function to select gut microbe-derived metabolites is critical to understanding diet-microbe-host interactions, and to developing microbiome-inspired therapies to improve human health. Here, we have investigated the role of a poorly annotated class of gut microbiome-derived lipids called N-acyl amides in postprandial metabolic physiology. Here we show both bacterial overproduction and provision of exogenous N-acyl amides reorganize host hormone-driven metabolic transition after a meal. Moreover, N-acyl amides exert broad effects on the meal- and circadian-related reorganization of gene expression, metabolic hormones, and gut microbiome composition. Collectively, these results demonstrate that microbiota-derived N-acyl amides play a physiologic role in postprandial metabolic homeostasis in the host.

physiology↗

bioRxiv2025

Gut Microbe-Derived Trimethylamine Shapes Circadian Rhythms Through the Host Receptor TAAR5

Elevated levels of the gut microbe-derived metabolite trimethylamine N-oxide (TMAO) are associated with cardiometabolic disease risk. However, the mechanism(s) linking TMAO production to human disease are incompletely understood. Initiation of the metaorganismal TMAO pathway begins when dietary choline and related metabolites are converted to trimethylamine (TMA) by gut bacteria. Gut microbe-derived TMA can then be further oxidized by host flavin-containing monooxygenases to generate TMAO. Previously, we showed that drugs lowering both TMA and TMAO protect mice against obesity via rewiring of host circadian rhythms. Although most mechanistic studies in the literature have focused on the metabolic end product TMAO, here we have instead tested whether the primary metabolite TMA alters host metabolic homeostasis and circadian rhythms via trace amine-associated receptor 5 (TAAR5). Remarkably, mice lacking the host TMA receptor (Taar5-/-) have altered circadian rhythms in gene expression, metabolic hormones, gut microbiome composition, and diverse behaviors. Also, mice genetically lacking bacterial TMA production or host TMA oxidation have altered circadian rhythms. These results provide new insights into diet-microbe-host interactions relevant to cardiometabolic disease, and implicate gut bacterial production of TMA and the host receptor that senses TMA (TAAR5) in the physiologic regulation of circadian rhythms in mice. HIGHLIGHTSO_LIMice lacking the host TMA receptor (Taar5-/-) have altered circadian rhythms. C_LIO_LITaar5-/- mice have altered innate behaviors in a time of day dependent manner. C_LIO_LIThe normal circadian oscillations in the gut microbiome are dysregulated in Taar5-/- mice. C_LIO_LIGenetic deletion of bacterial TMA production or host TMA oxidation shapes circadian rhythms. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=137 HEIGHT=200 SRC="FIGDIR/small/647082v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1608forg.highwire.dtl.DTLVardef@6a809aorg.highwire.dtl.DTLVardef@2131f7org.highwire.dtl.DTLVardef@1575559_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Identification of strain-specific cues that regulate biofilm formation in Bacteroides thetaiotaomicron

Members of the gut microbiome encounter a barrage of host- and microbe-derived microbiocidal factors that must be overcome to maintain fitness in the intestine. The long-term stability of many gut microbiome strains within the microbiome suggests the existence of strain-specific strategies that have evolved to foster resilience to such insults. Despite this, little is known about the mechanisms that mediate this resistance. Biofilm formation represents one commonly employed defense strategy against stressors like those found in the intestine. Here, we demonstrate strain-level variation in the capacity of the gut symbiont Bacteroides thetaiotaomicron to form biofilms. Despite the potent induction of biofilm formation by purified bile in most strains, we show that the specific bile acid species driving biofilm formation differ among strains, and uncover that a secondary bile-acid, lithocholic acid, and its conjugated forms, potently induce biofilm formation in a strain-specific manner. Additionally, we found that the short-chain fatty acid, acetic acid, could suppress biofilm formation. Thus, our data defines the molecular components of bile that promote biofilm formation in B. thetaiotaomicron and reveals that distinct molecular cues trigger the induction or inhibition of this process. Moreover, we uncover strain-level variation in these responses, thus identifying that both shared and strain-specific determinants govern biofilm formation in this species. ImportanceIn order to thrive within the intestine, it is imperative that gut microbes resist the multitude of insults derived from the host immune system and other microbiome members. As such, they have evolved strategies that ensure their survival within the intestine. We investigated one such strategy, biofilm formation, in Bacteroides thetaiotaomicron, a common member of the human microbiome. We uncovered significant variation in natural biofilm formation in the absence of an overt stimulus among different Bacteroides thetaiotaomicron strains, and revealed that different strains adopted a biofilm lifestyle in response to distinct molecular stimuli. Thus our studies provide novel insights into factors mediating gut symbiont resiliency, revealing strain-specific and shared strategies in these responses. Collectively, our findings underscore the prevalence of strain-level differences that should be factored into our understanding of gut microbiome functions.

microbiology↗

bioRxiv2024

Breast tumor microbiome regulates anti-tumor immunity and T cell-associated metabolites

BackgroundBreast cancer, the most common cancer type among women, was recently found to contain a specific tumor microbiome, but its impact on host biology remains unclear. CD8+ tumor-infiltrating lymphocytes (TILs) are pivotal effectors of anti-tumor immunity that influence cancer prognosis and response to therapy. This study aims to elucidate interactions between CD8+ TILs and the breast tumor microbiome and metabolites, as well as how the breast tumor microbiome may affect the tumor metabolome. MethodsWe investigated the interplay among CD8+ TILs, the tumor microbiome, and the metabolome in a cohort of 46 breast cancer patients with mixed subtypes (Cohort A). We characterized the tumor metabolome by mass spectrometry and CD8+ TILs by immunohistochemistry. Microbiome composition and T cell gene transcript levels were obtained from data from our previous study, which utilized 16S rRNA gene sequencing and a targeted mRNA expression panel. To examine interactions between intratumoral Staphylococcus and specific breast cancer subtypes, we analyzed RNA sequencing data from an independent cohort of 370 breast cancer patients (Cohort B). We explored the functions of the tumor microbiome using mouse models of triple-negative breast cancer (TNBC). ResultsIn tumors from Cohort A, the relative abundance of Staphylococcus positively correlated with the expression of T cell activation genes. The abundances of multiple metabolites exhibited significant correlations with CD8+ TILs, of which NADH, {gamma}-glutamyltryptophan, and {gamma}-glutamylglutamate displayed differential abundance in Staphylococcus-positive versus Staphylococcus-negative breast tumors. In a larger breast cancer cohort (Cohort B), we observed positive correlations between tumoral Staphylococcus and CD8+ TIL activity exclusively in TNBC. Preclinical experiments demonstrated that intratumoral administration of S. aureus, the predominant species of Staphylococcus in human breast tumors, resulted in a depletion of total NAD metabolites, and reduced the growth of TNBC tumors by activating CD8+ TILs. ConclusionsWe identified specific metabolites and microbial taxa associated with CD8+ TILs, delineated interactions between the breast tumor microbiome and metabolome, and demonstrated that intratumoral Staphylococcus influences anti-tumor immunity and TIL-associated metabolites. These findings highlight the role of low-biomass microbes in tumor tissues and provide potential biomarkers and therapeutic agents for breast cancer immunotherapy that merit further investigation.

cancer biology↗

bioRxiv2024

Targeting an Olfactory Receptor Mitigates High Residual Platelet Reactivity and Arterial Thrombosis Through Actin Cytoskeleton Depolymerization

BackgroundDespite antiplatelet therapy, some patients remain at high risk for ischemic events due to medication non-responsiveness or High Residual Platelet Reactivity (HRPR). Our goal was to target an orphan G-Protein-Coupled Receptor (GPCR) on platelets belonging to the olfactory receptor family as a new antithrombotic strategy. MethodsUsing an engineered reporter cell line expressing Olfactory Receptor 2L13 (OR2L13) that was recently identified as an orphan GPCR to limit platelet reactivity, we performed a high-throughput screen (HST) of non-odorant bioactive compounds with counter-screen validation, followed by studies to determine changes in platelet function in healthy subjects and in patients with coronary artery disease and peripheral artery disease. Phospho-proteomics identified key signal transduction pathways, and a variety of ex vivo assays and in vivo functional studies examined the impact of an identified non-odorant compound on platelet signaling, platelet biomechanics, and thrombosis. ResultsWe identified 6 ligands specific for OR2L13 that function as receptor agonists, leading to the suppression of platelet aggregation and -granule exocytosis via P2Y12, PAR1, Thromboxane Receptor (TxR) and glycoprotein VI (GPVI) receptors, suggesting involvement of common downstream mediator. The lead OR2L13 agonist identified phosphorylates heat shock protein 27 (HSP27) and depolymerizes the platelet filamentous actin cytoskeleton which was further confirmed in a clot retraction assay (CCF0054500 clot area 70.6 vs. vehicle clot area 5.2, P<0.0001). This anti-thrombotic effect of the OR2L13 agonist was reversed by a HSP27 inhibitor (clot area 3.6, P<0.0001). In a murine cremaster arterial injury model, platelet accumulation at the injury site was reduced by 88.9% by the lead OR2L13 agonist compared to vehicle (P<0.0003) without altering fibrin generation in vivo, or interfering with the coagulation cascade, and without impairing the protective mechanism of platelet hemostasis. ConclusionWe describe and characterize the first non-olfactory tool to target an olfactory receptor for the purpose of inhibiting platelet activation and thrombosis through downstream HSP27 in a comprehensive investigation using a first-of-its kind platelet inhibitor targeting an orphan platelet GPCR.

physiology↗

bioRxiv2023

HMGB1 acts as an agent of host defense at the gut mucosal barrier

Mucosal barriers provide the first line of defense between internal body surfaces and microbial threats from the outside world.1 In the colon, the barrier consists of two layers of mucus and a single layer of tightly interconnected epithelial cells supported by connective tissue and immune cells.2 Microbes colonize the loose, outer layer of colonic mucus, but are essentially excluded from the tight, epithelial-associated layer by host defenses.3 The amount and composition of the mucus is calibrated based on microbial signals and loss of even a single component of this mixture can destabilize microbial biogeography and increase the risk of disease.4-7 However, the specific components of mucus, their molecular microbial targets, and how they work to contain the gut microbiota are still largely unknown. Here we show that high mobility group box 1 (HMGB1), the prototypical damage-associated molecular pattern molecule (DAMP), acts as an agent of host mucosal defense in the colon. HMGB1 in colonic mucus targets an evolutionarily conserved amino acid sequence found in bacterial adhesins, including the well-characterized Enterobacteriaceae adhesin FimH. HMGB1 aggregates bacteria and blocks adhesin-carbohydrate interactions, inhibiting invasion through colonic mucus and adhesion to host cells. Exposure to HMGB1 also suppresses bacterial expression of FimH. In ulcerative colitis, HMGB1 mucosal defense is compromised, leading to tissue-adherent bacteria expressing FimH. Our results demonstrate a new, physiologic role for extracellular HMGB1 that refines its functions as a DAMP to include direct, virulence limiting effects on bacteria. The amino acid sequence targeted by HMGB1 appears to be broadly utilized by bacterial adhesins, critical for virulence, and differentially expressed by bacteria in commensal versus pathogenic states. These characteristics suggest that this amino acid sequence is a novel microbial virulence determinant and could be used to develop new approaches to diagnosis and treatment of bacterial disease that precisely identify and target virulent microbes.

microbiology↗

bioRxiv2022

Miltefosine attenuates inflammation, reduces atherosclerosis, and alters gut microbiota in hyperlipidemic mice.

Excess cholesterol induces foam cell formation, NLRP3 inflammasome activation, and IL-1{beta} release in atherosclerotic plaques. We have shown previously that Miltefosine increased cholesterol release and dampened NLRP3 inflammasome assembly in macrophages. Here, we show that Miltefosine reduced LPS-induced choline uptake by macrophages and attenuated NLRP3 inflammasome assembly in mice. Miltefosine-fed mice showed reduced plasma IL-1{beta} in a polymicrobial cecal slurry injection model of systemic inflammation. Miltefosine-fed mice showed increased reverse cholesterol transport from macrophages to plasma, liver, and feces. Hyperlipidemic apoE-/- mice fed with Miltefosine showed significantly reduced weight gain and markedly reduced atherosclerotic lesions vs. control mice. 16S rDNA sequencing and analysis showed alterations in the gut microbiota profile of Miltefosine-fed hyperlipidemic apoE-/- vs. control mice, with the most notable changes in Romboutsia and Bacteroidetes species. Taken together, these data indicate that Miltefosine causes pleiotropic effects on lipid metabolism, inflammasome activity, atherosclerosis, and the gut microbiota.

cell biology↗

bioRxiv2022

Gut Microbial Trimethylamine is Elevated in Alcohol-Associated Hepatitis and Contributes to Ethanol-Induced Liver Injury in Mice

BACKGROUNDThere is mounting evidence that microbes resident in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcohol-associated hepatitis (AH). However, mechanisms by which gut microbes synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested. METHODSWe used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, finding elevated levels of the microbial metabolite trimethylamine (TMA) in AH. In subsequent studies, we treated mice with non-lethal bacterial choline TMA lyase (CutC/D) inhibitors to blunt gut microbe-dependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing. RESULTSWe show the gut microbial choline metabolite trimethylamine (TMA) is elevated in AH patients and correlates with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial CutC/D activity protects mice from ethanol-induced liver injury. CutC/D inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome and host liver transcriptome. CONCLUSIONSThe microbial metabolite TMA is elevated in patients with AH, and inhibition of TMA production from gut microbes can protect mice from ethanol-induced liver injury.

biochemistry↗