Biology subjects

Griffin, M. E.

Publications and source records attributed to Griffin, M. E..

9 recordsLinked to original sources

bioRxiv2026

One-step glycan photoaffinity probe synthesis enables identification of human milk oligosaccharide-interacting microbiota

Glycans are found on the surfaces of all living cells and engage in cell signaling through the binding of various molecular targets. However, chemical probes to detect and identify carbohydrate interactions remain difficult to produce, due in large part to the challenges of late-stage, site-specific functionalization of oligosaccharides. Here, we report the synthesis and application of a minimal bifunctional diazirine/alkyne linker utilizing an N,O-dialkylhydroxylamine (NODHA) moiety that can readily be installed on the anomeric position of unprotected sugars. Our linker functionalizes glycans with complete {beta}-stereoselectivity, appends less mass than a single hexose unit, and maintains the closed-ring structure of the reducing end sugar. With this molecule, we generate glycan photoaffinity probes in a single step using unprotected human milk oligosaccharides (HMOs). We then validate binding selectivity of our compounds using lectins with known affinity towards our sugars. Finally, we demonstrate the utility of these glycan photoaffinity probes by performing photocrosslinking on a complex microbiota community and use cell sorting paired with metagenomic sequencing to identify potential new HMO-interacting microbes. Together, our work provides a one-step route to synthesize photoaffinity probes from unprotected, reducing glycans for interrogating carbohydrate interactions at the host-microbiota interface.

microbiology↗

bioRxiv2026

Capsular Polysaccharide Safeguards a Prophage-Bacterium Symbiosis by Preventing Collateral Attack and Promoting Viral Transmission

Many symbioses exist along a continuum from cooperation to conflict. Latent bacterial viruses known as prophages embody this duality. Acting as both partner and predator, they can enhance bacterial fitness while retaining the capacity to kill their hosts through lytic replication. Yet what determines the balance between cooperation and conflict in prophage-bacterium symbioses, and how these associations avoid collapse, remains poorly characterized. To identify mechanisms that stabilize prophag-bacterium partnerships, we used experimental evolution to perturb a natural association through repeated cycles of transmission and reinfection. This perturbation consistently selected mutant hosts lacking capsular polysaccharide production and exposed a hidden conflict that proved detrimental to both partners. During lytic outbreaks, host cells suffered lethal collateral prophage attack while dispersing virions became entrapped on neighboring cells and lysis debris, severely restricting transmission. Genetic and imaging-based studies revealed that capsular polysaccharides suppress these maladaptive interactions by limiting phage readsorption at the cell surface. We term this host-mediated safeguard the Hyperion effect, after the Greek Titan of light. Hyperion interactions enable phages to radiate outward from host populations, thereby averting collateral attack and promoting viral transmission. Our findings demonstrate that conflicts between prophages and their hosts can extend beyond individual cells to whole populations, with consequences that scale to shape patterns of prophage spread and microbial community assembly. More broadly, our work illustrates how mutually beneficial prophage-bacterium interactions can arise not only through cooperation, but also through the suppression of mutually detrimental conflicts.

microbiology↗

bioRxiv2023

Functional glycoproteomics by integrated network assembly and partitioning

The post-translational modification (PTM) of proteins by O-linked {beta}-N-acetyl-D-glucosamine (O-GlcNAcylation) is widespread across the proteome during the lifespan of all multicellular organisms. However, nearly all functional studies have focused on individual protein modifications, overlooking the multitude of simultaneous O-GlcNAcylation events that work together to coordinate cellular activities. Here, we describe Networking of Interactors and SubstratEs (NISE), a novel, systems-level approach to rapidly and comprehensively monitor O-GlcNAcylation across the proteome. Our method integrates affinity purification-mass spectrometry (AP-MS) and site-specific chemoproteomic technologies with network generation and unsupervised partitioning to connect potential upstream regulators with downstream targets of O-GlcNAcylation. The resulting network provides a data-rich framework that reveals both conserved activities of O-GlcNAcylation such as epigenetic regulation as well as tissue-specific functions like synaptic morphology. Beyond O-GlcNAc, this holistic and unbiased systems-level approach provides a broadly applicable framework to study PTMs and discover their diverse roles in specific cell types and biological states.

cell biology↗

bioRxiv2023

Antimicrobial overproduction sustains intestinal inflammation by inhibiting Enterococcus colonization

Loss of antimicrobial proteins such as REG3 family members compromises the integrity of the intestinal barrier. Here, we demonstrate that overproduction of REG3 proteins can also be detrimental by reducing a protective species in the microbiota. Patients with inflammatory bowel disease (IBD) experiencing flares displayed heightened levels of secreted REG3 proteins that mediated depletion of Enterococcus faecium (Efm) from the gut microbiota. Efm inoculation of mice ameliorated intestinal inflammation through activation of the innate immune receptor NOD2, which was associated with the bacterial DL-endopeptidase SagA. Microbiota sensing by NOD2 in myeloid cells mediated IL-1{beta} secretion and increased the proportion of IL-22-producing CD4+ T helper cells and innate lymphoid cells. Finally, Efm was unable to protect mice carrying a NOD2 gene variant commonly found in IBD patients. Our findings demonstrate that inflammation self-perpetuates by causing aberrant antimicrobial activity that disrupts symbiotic relationships with gut microbes.

immunology↗

bioRxiv2023

N-arylpyrazole NOD2 agonists promote immune checkpoint inhibitor therapy

The characterization of microbiota mechanisms in health and disease has reinvigorated pattern recognition receptors as prominent targets for immunotherapy. Notably, our recent studies on Enterococcus species revealed peptidoglycan remodeling and activation of NOD2 as key mechanisms for microbiota enhancement of immune checkpoint inhibitor therapy. Inspired by this work and other studies of NOD2 activation, we performed in silico ligand screening and developed N-arylpyrazole dipeptides as novel NOD2 agonists. Importantly, our N-arylpyrazole NOD2 agonist is enantiomer-specific, effective at promoting immune checkpoint inhibitor therapy and requires NOD2 for activity in vivo. Given the significant functions of NOD2 in innate and adaptive immunity, these next-generation agonists afford new therapeutic leads and adjuvants for a variety of NOD2-responsive diseases.

cancer biology↗

bioRxiv2021

Chemoproteomics of microbiota metabolites reveals small-molecule agonists for orphan receptor GPRC5A

The microbiota generates diverse metabolites that can engage multiple pathways to modulate host physiology and disease, but their protein targets and mechanism(s) of action have not been fully elucidated. To address this challenge, we focused on indole-3-acetic acid (IAA), a prominent microbiota metabolite, and developed IAA-based chemical reporters for proteomic studies. We discovered that IAA interacts with many proteins in host cells, including small-molecule transporters, receptors and metabolic enzymes. Notably, our functional studies revealed that IAA binds to orphan G protein-coupled receptors such as GPRC5A, but only aromatic monoamines were capable of inducing GPRC5A signaling. Functional profiling of microbiota uncovered specific bacterial species and enzymes that generate GPRC5A agonists. Finally, biochemical characterization of GPRC5A activation identified more potent synthetic agonists as well as key amino acid residues involved in ligand binding. These studies highlight the utility of chemoproteomics to dissect protein targets and mechanisms of action for microbiota metabolites.

biochemistry↗

bioRxiv2020

RecT recombinase expression enables efficient gene editing in Enterococcus

Enterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Commensal strains of E. faecium can confer beneficial effects on host physiology and immunity, but antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient gene editing methods. To address these limitations, here we report the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Notably, the expression of RecT in combination with clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scar-less single-stranded DNA recombineering to generate specific gene editing mutants in E. faecium. Moreover, we demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic selectable markers to generate gene deletion mutants. As further proof-of-principle, we use CRISPR-Cas9 mediated recombineering to knock out both sortase A genes in E. faecium for downstream functional characterization. The general RecT-mediated recombineering methods described here should significantly enhance genetic studies of E. faecium and other closely related species for functional and mechanistic studies. ImportanceEnterococcus faecium is widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Nevertheless, commensal Enterococcus strains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. This functional dichotomy of Enterococcus species and strains highlights the need for in-depth studies to discover and characterize the genetic components underlining its diverse activities. However, current genetic engineering methods in E. faecium still require passive homologous recombination from plasmid DNA. This involves the successful cloning of multiple homologous fragments into a plasmid, introducing the plasmid into E. faecium, and screening for double-crossover events that can collectively take up to multiple weeks to perform. To alleviate these challenges, we show that RecT recombinase enables rapid and efficient integration of mutagenic DNA templates to generate substitutions, deletions, and insertions in genomic DNA of E. faecium. These improved recombineering methods should facilitate functional and mechanistic studies of Enterococcus.

microbiology↗

bioRxiv2020

Enterococcus peptidoglycan remodeling promotes immune checkpoint inhibitor therapy

The antitumor efficacy of cancer immunotherapy has been correlated with specific species within the gut microbiota. However, molecular mechanisms by which these microbes affect host response to immunotherapy remain elusive. Here we show that specific members of the bacterial genus Enterococcus can promote anti-PD-L1 immunotherapy in mouse tumor models. The active enterococci express and secrete orthologs of the NlpC/p60 peptidoglycan hydrolase SagA that generate immune-active muropeptides. Expression of SagA in non-protective E. faecalis was sufficient to promote antitumor activity of clinically approved checkpoint targets, and its activity required the peptidoglycan sensor Nod2. Notably, SagA-engineered probiotics or synthetic muropeptides also promoted checkpoint inhibitor antitumor activity. Our data suggest that microbiota species with unique peptidoglycan remodeling activity may enhance immunotherapy and could be leveraged for next-generation adjuvants. One Sentence SummaryA conserved family of secreted NlpC/p60 peptidoglycan hydrolases from Enterococcus promote antitumor activity of immune checkpoint inhibitors.

cancer biology↗

bioRxiv2019

Prolonged oral coenzyme Q10-β-cyclodextrin supplementation increases plasma CoQ10 concentration and skeletal muscle complex I+III activity in young, untrained healthy Thoroughbreds

Coenzyme Q10 (CoQ10) is an essential component of the mitochondrial electron transport chain (ETC). Decreased skeletal muscle CoQ10 content may result in decreased ETC activity and energy production. This study aimed to test the hypothesis that prolonged supplementation with oral CoQ10 will increase plasma CoQ10 concentrations and skeletal muscle CoQ10 content in young, healthy untrained Thoroughbreds. Nineteen Thoroughbreds (27.5{+/-}9.7 months old; 11 males, 8 females) from one farm and maintained on a grass pasture with one grain meal per day were supplemented orally once per day for 9 weeks with 1.5 mg/kg body weight of a CoQ10-{beta}-cyclodextrin inclusion complex. Whole-blood and skeletal muscle biopsies were collected before (T0) and after (T1) 9 weeks of supplementation. Plasma CoQ10 concentrations were determined via high-performance liquid chromatography. Skeletal muscle mitochondrial ETC combined complex I+III enzyme activity (an indirect measurement of CoQ10 content) was assessed spectrophotometrically and normalised to mitochondrial abundance. Results were analysed using a paired two-tailed Students t-test with P[≤]0.05 significant. Horses accepted supplementation with no adverse effects. The mean change in plasma CoQ10 concentration from T0 to T1 was significantly greater than zero (0.13{+/-}0.02 vs. 0.25{+/-}0.03 {micro}g/ml, mean difference 0.12{+/-}0.03; P=0.004), although variability in absorbance resulted in only a 58% response rate. The mean change in skeletal muscle complex I+III activity from T0 to T1 was significantly greater than zero (0.36{+/-}0.04 vs. 0.59{+/-}0.05 pmol/min/mg of muscle, mean difference 0.23{+/-}0.05; P=0.0004), although T1 values for 3/19 horses decreased on average by 23% below T0 values. In conclusion, prolonged oral supplementation of the diet of young, healthy untrained Thoroughbreds with CoQ10 increased mean plasma CoQ10 concentration by 99% and mean skeletal muscle complex I+III activity by 65% with variability in absorbance among horses. Additional research is warranted investigating training and exercise effects on skeletal muscle CoQ10 content in CoQ10 supplemented and un-supplemented Thoroughbreds.

biochemistry↗