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 that generates NOD2-stimulating muropeptides. NOD2 activation in myeloid cells induced interleukin-1ß (IL-1ß) secretion to increase the proportion of IL-22-producing CD4+ T helper cells and innate lymphoid cells that promote tissue repair. 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.

Functional glycoproteomics by integrated network assembly and partitioning.

The post-translational modification (PTM) of proteins by O-linked ß-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.

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 and 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.

Chemoproteomics reveals microbiota-derived aromatic monoamine agonists for GPRC5A.

The microbiota generates diverse metabolites to modulate host physiology and disease, but their protein targets and mechanisms of action have not been fully elucidated. To address this challenge, we explored microbiota-derived indole metabolites and developed photoaffinity chemical reporters for proteomic studies. We identified many potential indole metabolite-interacting proteins, including metabolic enzymes, transporters, immune sensors and G protein-coupled receptors. Notably, we discovered that aromatic monoamines can bind the orphan receptor GPRC5A and stimulate ß-arrestin recruitment. Metabolomic and functional profiling also revealed specific amino acid decarboxylase-expressing microbiota species that produce aromatic monoamine agonists for GPRC5A-ß-arrestin recruitment. Our analysis of synthetic aromatic monoamine derivatives identified 7-fluorotryptamine as a more potent agonist of GPRC5A. These results highlight the utility of chemoproteomics to identify microbiota metabolite-interacting proteins and the development of small-molecule agonists for orphan receptors.

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.

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-1ß 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.

Peptidoglycan NlpC/P60 peptidases in bacterial physiology and host interactions.

The bacterial cell wall is composed of a highly crosslinked matrix of glycopeptide polymers known as peptidoglycan that dictates bacterial cell morphology and protects against environmental stresses. Regulation of peptidoglycan turnover is therefore crucial for bacterial survival and growth and is mediated by key protein complexes and enzyme families. Here, we review the prevalence, structure, and activity of NlpC/P60 peptidases, a family of peptidoglycan hydrolases that are crucial for cell wall turnover and division as well as interactions with antibiotics and different hosts. Understanding the molecular functions of NlpC/P60 peptidases should provide important insight into bacterial physiology, their interactions with different kingdoms of life, and the development of new therapeutic approaches.

Tools for mammalian glycoscience research.

Cellular carbohydrates or glycans are critical mediators of biological function. Their remarkably diverse structures and varied activities present exciting opportunities for understanding many areas of biology. In this primer, we discuss key methods and recent breakthrough technologies for identifying, monitoring, and manipulating glycans in mammalian systems.

Microbial mechanisms to improve immune checkpoint blockade responsiveness.

The human microbiota acts as a diverse source of molecular cues that influence the development and homeostasis of the immune system. Beyond endogenous roles in the human holobiont, host-microbial interactions also alter outcomes for immune-related diseases and treatment regimens. Over the past decade, sequencing analyses of cancer patients have revealed correlations between microbiota composition and the efficacy of cancer immunotherapies such as checkpoint inhibitors. However, very little is known about the exact mechanisms that link specific microbiota with patient responses, limiting our ability to exploit these microbial agents for improved oncology care. Here, we summarize current progress towards a molecular understanding of host-microbial interactions in the context of checkpoint inhibitor immunotherapies. By highlighting the successes of a limited number of studies focused on identifying specific, causal molecules, we underscore how the exploration of specific microbial features such as proteins, enzymes, and metabolites may translate into precise and actionable therapies for personalized patient care in the clinic.

Improving immunotherapy response through the use of designer bacteria.

Reprogramming the tumor microenvironment may be a key strategy to broaden the efficacy of current cancer immunotherapies. In a recent Nature paper, Canale et al. use synthetic biology to alter intratumoral arginine levels via engineered bacteria, leading to improved responsiveness to anti-PD-L1 checkpoint blockade in a murine model of cancer.

Enterococcus peptidoglycan remodeling promotes checkpoint inhibitor cancer immunotherapy.

The antitumor efficacy of cancer immunotherapy can correlate with the presence of certain bacterial species within the gut microbiome. However, many of the molecular mechanisms that influence host response to immunotherapy remain elusive. In this study, we show that members of the bacterial genus Enterococcus improve checkpoint inhibitor immunotherapy in mouse tumor models. Active enterococci express and secrete orthologs of the NlpC/p60 peptidoglycan hydrolase SagA that generate immune-active muropeptides. Expression of SagA in nonprotective E. faecalis was sufficient to promote immunotherapy response, and its activity required the peptidoglycan sensor NOD2. Notably, SagA-engineered probiotics or synthetic muropeptides also augmented anti-PD-L1 antitumor efficacy. Taken together, our data suggest that microbiota species with specialized peptidoglycan remodeling activity and muropeptide-based therapeutics may enhance cancer immunotherapy and could be leveraged as next-generation adjuvants.

RecT Recombinase Expression Enables Efficient Gene Editing in Enterococcus spp.

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 that 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 short palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scarless 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 a 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. IMPORTANCE Enterococcus 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 underlying 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 the rapid and efficient integration of mutagenic DNA templates to generate substitutions, deletions, and insertions in the genomic DNA of E. faecium. These improved recombineering methods should facilitate functional and mechanistic studies of Enterococcus.

Frontiers in cancer immunotherapy-a symposium report.

Cancer immunotherapy has dramatically changed the approach to cancer treatment. The aim of targeting the immune system to recognize and destroy cancer cells has afforded many patients the prospect of achieving deep, long-term remission and potential cures. However, many challenges remain for achieving the goal of effective immunotherapy for all cancer patients. Checkpoint inhibitors have been able to achieve long-term responses in a minority of patients, yet improving response rates with combination therapies increases the possibility of toxicity. Chimeric antigen receptor T cells have demonstrated high response rates in hematological cancers, although most patients experience relapse. In addition, some cancers are notoriously immunologically "cold" and typically are not effective targets for immunotherapy. Overcoming these obstacles will require new strategies to improve upon the efficacy of current agents, identify biomarkers to select appropriate therapies, and discover new modalities to expand the accessibility of immunotherapy to additional tumor types and patient populations.

Sulfated glycans engage the Ang-Tie pathway to regulate vascular development.

The angiopoietin (Ang)-Tie pathway is essential for the proper maturation and remodeling of the vasculature. Despite its importance in disease, the mechanisms that control signal transduction through this pathway are poorly understood. Here, we demonstrate that heparan sulfate glycosaminoglycans (HS GAGs) regulate Ang-Tie signaling through direct interactions with both Ang ligands and Tie1 receptors. HS GAGs formed ternary complexes with Ang1 or Ang4 and Tie2 receptors, resulting in potentiation of endothelial survival signaling. In addition, HS GAGs served as ligands for the orphan receptor Tie1. The HS-Tie1 interaction promoted Tie1-Tie2 heterodimerization and enhanced Tie1 stability within the mature vasculature. Loss of HS-Tie1 binding using CRISPR-Cas9-mediated mutagenesis in vivo led to decreased Tie protein levels, pathway suppression and aberrant retinal vascularization. Together, these results reveal that sulfated glycans use dual mechanisms to regulate Ang-Tie signaling and are important for the development and maintenance of the vasculature.

Translation of peptidoglycan metabolites into immunotherapeutics.

The discovery of defined peptidoglycan metabolites that activate host immunity and their specific receptors has revealed fundamental insights into host-microbe recognition and afforded new opportunities for therapeutic development against infection and cancer. In this review, we summarise the discovery of two key peptidoglycan metabolites, γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP) and muramyl dipeptide and their respective receptors, Nod1 and Nod2, and review progress towards translating these findings into therapeutic agents. Notably, synthetic derivatives of peptidoglycan metabolites have already yielded approved drugs for chemotherapy-induced leukopenia and paediatric osteosarcoma; however, the broad effects of peptidoglycan metabolites on host immunity suggest additional translational opportunities for new therapeutics towards other cancers, microbial infections and inflammatory diseases.

Peptidoglycan Metabolite Photoaffinity Reporters Reveal Direct Binding to Intracellular Pattern Recognition Receptors and Arf GTPases.

The peptidoglycan fragments γ-d-glutamyl- meso-diaminopimelic acid (iE-DAP) and muramyl-dipeptide (MDP) are microbial-specific metabolites that activate intracellular pattern recognition receptors and stimulate immune signaling pathways. While extensive structure-activity studies have demonstrated that these bacterial cell wall metabolites trigger NOD1- and NOD2-dependent signaling, their direct binding to these innate immune receptors or other proteins in mammalian cells has not been established. To characterize these fundamental microbial metabolite-host interactions, we synthesized a series of peptidoglycan metabolite photoaffinity reporters and evaluated their cross-linking to NOD1 and NOD2 in mammalian cells. We show that active iE-DAP and MDP photoaffinity reporters selectively cross-linked NOD1 and NOD2, respectively, and not their inactive mutants. We also discovered MDP reporter cross-linking to Arf GTPases, which interacted most prominently with GTP-bound Arf6 and coimmunoprecipitated with NOD2 upon MDP stimulation. Notably, MDP binding to NOD2 and Arf6 was abrogated with loss-of-function NOD2 mutants associated with Crohn's disease. Our studies demonstrate peptidoglycan metabolite photoaffinity reporters can capture their cognate immune receptors in cells and reveal unpredicted ligand-induced interactions with other cellular cofactors. These photoaffinity reporters should afford useful tools to discover and characterize other peptidoglycan metabolite-interacting proteins.

Methods for the Detection, Study, and Dynamic Profiling of O-GlcNAc Glycosylation.

The addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins is a ubiquitous posttranslational modification found in all multicellular organisms. Like phosphorylation, O-GlcNAc glycosylation (O-GlcNAcylation) is inducible and regulates a myriad of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are essential to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease. In this chapter, we describe a set of chemoenzymatic labeling methods to (1) detect O-GlcNAcylation on proteins of interest, (2) monitor changes in both the total levels of O-GlcNAcylation and its stoichiometry on proteins of interest, and (3) enable mapping of O-GlcNAc to specific serine/threonine residues within proteins to facilitate functional studies. First, we outline a procedure for the expression and purification of a multiuse mutant galactosyltransferase enzyme (Y289L GalT). We then describe the use of Y289L GalT to modify O-GlcNAc residues with a functional handle, N-azidoacetylgalactosamine (GalNAz). Finally, we discuss several applications of the copper-catalyzed azide-alkyne cycloaddition "click" reaction to attach various alkyne-containing chemical probes to GalNAz and demonstrate how this functionalization of O-GlcNAc-modified proteins can be used to realize (1)-(3) above. Overall, these methods, which utilize commercially available reagents and standard protein analytical tools, will serve to advance our understanding of the diverse and important functions of O-GlcNAcylation.

Comprehensive mapping of O-GlcNAc modification sites using a chemically cleavable tag.

The post-translational modification of serine or threonine residues of proteins with a single N-acetylglucosamine monosaccharide (O-GlcNAcylation) is essential for cell survival and function. However, relatively few O-GlcNAc modification sites have been mapped due to the difficulty of enriching and detecting O-GlcNAcylated peptides from complex samples. Here we describe an improved approach to quantitatively label and enrich O-GlcNAcylated proteins for site identification. Chemoenzymatic labelling followed by copper(i)-catalysed azide-alkyne cycloaddition (CuAAC) installs a new mass spectrometry (MS)-compatible linker designed for facile purification of O-GlcNAcylated proteins from cell lysates. The linker also allows subsequent quantitative release of O-GlcNAcylated proteins for downstream MS analysis. We validate the approach by unambiguously identifying several established O-GlcNAc sites on the proteins α-crystallin and O-GlcNAc transferase (OGT), as well as discovering new, previously unreported sites on OGT. Notably, these novel sites on OGT lie in key functional domains of the protein, underscoring how this site identification method may reveal important biological insights into protein activity and regulation.

Glycan Engineering for Cell and Developmental Biology.

Cell-surface glycans are a diverse class of macromolecules that participate in many key biological processes, including cell-cell communication, development, and disease progression. Thus, the ability to modulate the structures of glycans on cell surfaces provides a powerful means not only to understand fundamental processes but also to direct activity and elicit desired cellular responses. Here, we describe methods to sculpt glycans on cell surfaces and highlight recent successes in which artificially engineered glycans have been employed to control biological outcomes such as the immune response and stem cell fate.

Long-lived engineering of glycans to direct stem cell fate.

Glycans mediate many critical, long-term biological processes, such as stem cell differentiation. However, few methods are available for the sustained remodeling of cells with specific glycan structures. A new strategy that enables the long-lived presentation of defined glycosaminoglycans on cell surfaces using HaloTag proteins (HTPs) as anchors is reported. By controlling the sulfation patterns of heparan sulfate (HS) on pluripotent embryonic stem cell (ESC) membranes, it is demonstrated that specific glycans cause ESCs to undergo accelerated exit from self-renewal and differentiation into neuronal cell types. Thus, the stable display of glycans on HTP scaffolds provides a powerful, versatile means to direct key signaling events and biological outcomes such as stem cell fate.