Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor cancer therapy.

Enterococcus faecium is a microbiota species in humans that can modulate host immunity, but has also acquired antibiotic resistance and is a major cause of hospital-associated infections. Notably, diverse strains of E. faecium produce SagA, a highly conserved peptidoglycan hydrolase that is sufficient to promote intestinal immunity and immune checkpoint inhibitor antitumor activity. However, the functions of SagA in E. faecium were unknown. Here we report that deletion of sagA impaired E. faecium growth and resulted in bulged and clustered enterococci due to defective peptidoglycan cleavage and cell separation. Moreover, Δ sagA showed increased antibiotic sensitivity, yielded lower levels of active muropeptides, displayed reduced activation of the peptidoglycan pattern-recognition receptor NOD2, and failed to promote cancer immunotherapy. Importantly, plasmid-based expression of SagA, but not its catalytically-inactive mutant, restored Δ sagA growth, production of active muropeptides and NOD2 activation. SagA is therefore essential for E. faecium growth, stress resistance and activation of host immunity.

Melanoma and microbiota: Current understanding and future directions.

Over the last decade, the composition of the gut microbiota has been found to correlate with the outcomes of cancer patients treated with immunotherapy. Accumulating evidence points to the various mechanisms by which intestinal bacteria act on distal tumors and how to harness this complex ecosystem to circumvent primary resistance to immune checkpoint inhibitors. Here, we review the state of the microbiota field in the context of melanoma, the recent breakthroughs in defining microbial modes of action, and how to modulate the microbiota to enhance response to cancer immunotherapy. The host-microbe interaction may be deciphered by the use of "omics" technologies, and will guide patient stratification and the development of microbiota-centered interventions. Efforts needed to advance the field and current gaps of knowledge are also discussed.

Discovery and Characterization of IFITM S-Palmitoylation.

Interferon-induced transmembrane proteins (IFITM1, 2 and 3) are important host antiviral defense factors. They are active against viruses like the influenza A virus (IAV), dengue virus (DENV), Ebola virus (EBOV), Zika virus (ZIKV) and severe acute respiratory syndrome coronavirus (SARS-CoV). In this review, we focus on IFITM3 S-palmitoylation, a reversible lipid modification, and describe its role in modulating IFITM3 antiviral activity. Our laboratory discovered S-palmitoylation of IFITMs using chemical proteomics and demonstrated the importance of highly conserved fatty acid-modified Cys residues in IFITM3 antiviral activity. Further studies showed that site-specific S-palmitoylation at Cys72 is important for IFITM3 trafficking to restricted viruses (IAV and EBOV) and membrane-sterol interactions. Thus, site-specific lipid modification of IFITM3 directly regulates its antiviral activity, cellular trafficking, and membrane-lipid interactions.

Small molecule modulators of immune pattern recognition receptors.

Pattern recognition receptors (PRRs) represent a re-emerging class of therapeutic targets for vaccine adjuvants, inflammatory diseases and cancer. In this review article, we summarize exciting developments in discovery and characterization of small molecule PRR modulators, focusing on Toll-like receptors (TLRs), NOD-like receptors (NLRs) and the cGAS-STING pathway. We also highlight PRRs that are currently lacking small molecule modulators and opportunities for chemical biology and therapeutic discovery.

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.

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.

Functional analysis of protein post-translational modifications using genetic codon expansion.

Post-translational modifications (PTMs) of proteins not only exponentially increase the diversity of proteoforms, but also contribute to dynamically modulating the localization, stability, activity, and interaction of proteins. Understanding the biological consequences and functions of specific PTMs has been challenging for many reasons, including the dynamic nature of many PTMs and the technical limitations to access homogenously modified proteins. The genetic code expansion technology has emerged to provide unique approaches for studying PTMs. Through site-specific incorporation of unnatural amino acids (UAAs) bearing PTMs or their mimics into proteins, genetic code expansion allows the generation of homogenous proteins with site-specific modifications and atomic resolution both in vitro and in vivo. With this technology, various PTMs and mimics have been precisely introduced into proteins. In this review, we summarize the UAAs and approaches that have been recently developed to site-specifically install PTMs and their mimics into proteins for functional studies of PTMs.

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.

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.

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.

Lytic bacteriophages facilitate antibiotic sensitization of Enterococcus faecium.

Enterococcus faecium, a commensal of the human intestine, has emerged as a hospital-adapted, multi-drug resistant (MDR) pathogen. Bacteriophages (phages), natural predators of bacteria, have regained attention as therapeutics to stem the rise of MDR bacteria. Despite their potential to curtail MDR E. faecium infections, the molecular events governing E. faecium-phage interactions remain largely unknown. Such interactions are important to delineate because phage selective pressure imposed on E. faecium will undoubtedly result in phage resistance phenotypes that could threaten the efficacy of phage therapy. In an effort to understand the emergence of phage resistance in E. faecium, three newly isolated lytic phages were used to demonstrate that E. faecium phage resistance is conferred through an array of cell wall-associated molecules, including secreted antigen A (SagA), enterococcal polysaccharide antigen (Epa), wall teichoic acids, capsule, and an arginine-aspartate-aspartate (RDD) protein of unknown function. We find that capsule and Epa are important for robust phage adsorption and that phage resistance mutations in sagA, epaR, and epaX enhance E. faecium susceptibility to ceftriaxone, an antibiotic normally ineffective due to its low affinity for enterococcal penicillin binding proteins. Consistent with these findings, we provide evidence that phages potently synergize with cell wall (ceftriaxone and ampicillin) and membrane-acting (daptomycin) antimicrobials to slow or completely inhibit the growth of E. faecium Our work demonstrates that the evolution of phage resistance comes with fitness defects resulting in drug sensitization and that lytic phages could serve as effective antimicrobials for the treatment of E. faecium infections.

Anti-infective bile acids bind and inactivate a Salmonella virulence regulator.

Bile acids are prominent host and microbiota metabolites that modulate host immunity and microbial pathogenesis. However, the mechanisms by which bile acids suppress microbial virulence are not clear. To identify the direct protein targets of bile acids in bacterial pathogens, we performed activity-guided chemical proteomic studies. In Salmonella enterica serovar Typhimurium, chenodeoxycholic acid (CDCA) most effectively inhibited the expression of virulence genes and invasion of epithelial cells and interacted with many proteins. Notably, we discovered that CDCA can directly bind and inhibit the function of HilD, an important transcriptional regulator of S. Typhimurium virulence and pathogenesis. Our characterization of bile acid-resistant HilD mutants in vitro and in S. Typhimurium infection models suggests that HilD is one of the key protein targets of anti-infective bile acids. This study highlights the utility of chemical proteomics to identify the direct protein targets of microbiota metabolites for mechanistic studies in bacterial pathogens.

Chemical proteomic analysis of bile acid-protein targets in Enterococcus faecium.

Bile acids are important gut microbiota metabolites that regulate both host and microbial functions. To identify the direct protein targets of bile acids in Enterococcus, we synthesized and validated the activity of a lithocholic acid (LCA) photoaffinity reporter, x-alk-LCA-3. Chemical proteomics of x-alk-LCA-3 in E. faecium Com15 reveals many candidate LCA-interacting proteins, which are involved in cell well synthesis, transcriptional regulation and metabolism. To validate the utility of bile acid photoaffinity labeling, we characterized a putative bile salt hydrolase (BSH) crosslinked by x-alk-LCA-3, and demonstrated that this BSH was effective in converting taurolithocholic acid (TLCA) to LCA in E. faecium and in vitro. Chemical proteomics should afford new opportunities to characterize bile acid-protein targets and mechanisms of action in the future.

Identification of a Bile Acid-Binding Transcription Factor in Clostridioides difficile Using Chemical Proteomics.

Clostridioides difficile is a Gram-positive anaerobic bacterium that is the leading cause of hospital-acquired gastroenteritis in the US. In the gut milieu, C. difficile encounters microbiota-derived, growth-inhibiting bile acids that are thought to be a significant mechanism of colonization resistance. While the levels of certain bile acids in the gut correlate with susceptibility to C. difficile infection, their molecular targets in C. difficile remain unknown. In this study, we sought to use chemical proteomics to identify bile acid-interacting proteins in C. difficile. Using photoaffinity bile acid probes and chemical proteomics, we identified a previously uncharacterized MerR family protein, CD3583 (now BapR), as a putative bile acid-sensing transcription regulator. Our data indicate that BapR specifically binds to and is stabilized by lithocholic acid (LCA) in C. difficile. Although loss of BapR did not affect C. difficile's sensitivity to LCA, ΔbapR cells elongated more in the presence of LCA compared to wild-type cells. Transcriptomics revealed that BapR regulates several gene clusters, with the expression of the mdeA-cd3573 locus being specifically de-repressed in the presence of LCA in a BapR-dependent manner. Electrophoretic mobility shift assays revealed that BapR directly binds to the mdeA promoter region. Because mdeA is involved in amino acid-related sulfur metabolism and the mdeA-cd3573 locus encodes putative transporters, we propose that BapR senses a gastrointestinal tract-specific small molecule, LCA, as an environmental cue for metabolic adaptation.

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.

S-Palmitoylation and Sterol Interactions Mediate Antiviral Specificity of IFITMs.

Interferon-induced transmembrane proteins (IFITM1, 2, and 3) are important antiviral proteins that are active against many viruses, including influenza A virus (IAV), dengue virus (DENV), Ebola virus (EBOV), Zika virus (ZIKV), and severe acute respiratory syndrome coronavirus (SARS-CoV). IFITM proteins exhibit specificity in activity, but their distinct mechanisms of action and regulation are unclear. Since S-palmitoylation and cholesterol homeostasis are crucial for viral infections, we investigated IFITM interactions with cholesterol by photoaffinity cross-linking in mammalian cells along with molecular dynamic simulations and nuclear magnetic resonance analysis in vitro. These studies suggest that cholesterol can directly interact with S-palmitoylated IFITMs in cells and alter the conformation of IFITMs in membrane bilayers. Notably, we discovered that the S-palmitoylation levels regulate differential IFITM protein interactions with cholesterol in mammalian cells and specificity of antiviral activity toward IAV, SARS-CoV-2, and EBOV. Our studies suggest that modulation of IFITM S-palmitoylation levels and cholesterol interaction influence host susceptibility to different viruses.

Membrane targeting enhances muramyl dipeptide binding to NOD2 and Arf6-GTPase in mammalian cells.

To further understand the mechanisms of muramyl dipeptide (MDP) sensing by NOD2, we evaluated key properties involved in the formation of the Arf6-MDP-NOD2 complex in mammalian cells. We found that the conserved Arf aromatic triad is crucial for binding to MDP-NOD2. Mutation of Arf6 N-myristoylation and NOD2 S-palmitoylation also abrogated the formation of the Arf6-MDP-NOD2 complex. Notably, lipid-modified MDP (L18-MDP) increased Arf6-NOD2 assembly. Our results indicate recruitment of Arf6 may explain enhanced activity of lipidated MDP analogues and membrane targeting may be important in developing next-generation NOD2 agonists.

Chemical proteomics for identifying short-chain fatty acid modified proteins in Salmonella.

Microbiota-metabolized small molecules play important roles to regulate host immunity and pathogen virulence. Specifically, microbiota generates millimolar concentration of short-chain fatty acid (SCFA) that can directly inhibit Salmonella virulence. Here, we describe chemical proteomic methods to identify SCFA-modified proteins in Salmonella using free fatty acids as well as their salicylic acid derivatives. In addition, we include CRISPR-Cas9 gene editing protocols for epitope-tagging of specific proteins to validate SCFA-modification in Salmonella. These protocols should facilitate the discovery and functional analysis of SCFA-modified proteins in Salmonella microbiology and pathogenesis.

Chemoproteomic Analysis of Microbiota Metabolite-Protein Targets and Mechanisms.

The microbiota have emerged as an important factor in host physiology, disease, and response to therapy. These diverse microbes (bacteria, virus, fungi, and protists) encode unique functions and metabolites that regulate intraspecies and interspecies interactions. While the mechanisms of some microbiota species and metabolites have been elucidated, the diversity and abundance of different microbiota species and their associated pathways suggest many more metabolites and mechanisms of action remain to be discovered. In this Perspective, we highlight how the advances in chemical proteomics have provided new opportunities to elucidate the molecular targets of specific microbiota metabolites and reveal new mechanisms of action. The continued development of specific microbiota metabolite reporters and more precise proteomic methods should reveal new microbiota mechanisms of action, therapeutic targets, and biomarkers for a variety of human diseases.