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The emergence of antibiotic-resistant bacteria has intensified the need for novel therapeutic strategies targeting bacterial virulence rather than growth or survival. Bacterial virulence involves complex processes that enable pathogens to invade and survive within host cells. Chemical biology has become a powerful tool for dissecting these virulence mechanisms at the molecular level. This review highlights key chemical biology approaches for studying bacterial virulence, focusing on four areas: 1) regulation of virulence, where chemoproteomics has identified small molecule-protein interactions that modulate virulence gene expression; 2) identification of virulence proteins, using techniques like unnatural amino acid incorporation and activity-based protein profiling (ABPP) to uncover proteins involved in infection; 3) post-translational modifications of host proteins, where chemical probes have revealed how bacterial effectors alter host cell processes; and 4) effector-host protein interactions, with methods such as bifunctional unnatural amino acid incorporation facilitating the discovery of key host targets manipulated by bacterial effectors. Collectively, these chemical tools are providing new insights into pathogen-host interactions, offering potential therapeutic avenues that aim to disarm pathogens and combat antibiotic resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Advances in genomics, proteomics, and metabolomics have revealed associations between specific microbiota species in health and disease. However, the precise mechanism(s) of action for many microbiota species and molecules have not been fully elucidated, limiting the development of microbiota-based diagnostics and therapeutics. In this Review, we highlight innovative chemical and genetic approaches that are enabling the dissection of microbiota mechanisms and providing causation in health and disease. Although specific microbiota molecules and mechanisms have begun to emerge, new approaches are still needed to go beyond phenotypic associations and translate microbiota discoveries into actionable targets and therapeutic leads to prevent and treat diseases.

Objectives: This study was a retrospective study to investigate factors related to difficult tracheostomy decannulation, and to evaluate outcomes of tracheostomized neurosurgical patients., Methods: All consecutive tracheostomized neurosurgical patients in the Prince of Wales Hospital between 1st September 2016 and 31st August 2019 were reviewed retrospectively. Patients were grouped into easy decannulation and difficult decannulation groups using 3 months as cut-off time. Risk factors were analysed and outcomes were compared., Results: One hundred thirty-one patients were included. In univariate analyses, male gender, GCS less than or equal to 8 on admission, the presence of vocal cord palsy at 3 months, and pneumonia within 1-month post-tracheostomy were associated with difficult decannulation. In multivariable logistic regression for difficult decannulation, GCS on admission, the presence of vocal cord palsy at 3 months, and the presence of pneumonia within 1-month post-tracheostomy remained statistically significant. The easy decannulation group had a shorter length of in-patient stay, higher survival rate, and more favourable neurological outcome (GOS 4-5) than the difficult decannulation group at both 6 months and 1 year. The majority of easy decannulation group patients (54%) were discharged to home, while the majority of the difficult decannulation group (42%) of patients were discharged to the infirmary., Conclusion: GCS less than or equal to 8 on admission, the presence of vocal cord palsy, and the presence of pneumonia were associated with difficult tracheostomy decannulation in neurosurgical patients. Difficult decannulation is associated with a longer length of in-patient stay and poor neurological outcomes.

Enterococcus faecium is a microbiota species in humans that can modulate host immunity (Griffin and Hang, 2022), but has also acquired antibiotic resistance and is a major cause of hospital-associated infections (Van Tyne and Gilmore, 2014). Notably, diverse strains of E. faecium produce SagA, a highly conserved peptidoglycan hydrolase that is sufficient to promote intestinal immunity (Rangan et al., 2016; Pedicord et al., 2016; Kim et al., 2019) and immune checkpoint inhibitor antitumor activity (Griffin et al., 2021). 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, the 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., Competing Interests: SK, KF, XZ, PC, AM, TB, DG, DP No competing interests declared, HH has filed patent applications (PCT/US2016/028836, PCT/US2020/019038) for the commercial use of SagA-bacteria to improve intestinal immunity and checkpoint blockade immunotherapy, which has been licensed by Rise Therapeutics for probiotic development, (© 2024, Klupt, Fam, Zhang et al.)

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., Competing Interests: Declaration of interests B.R. received grand funding from Davolterra and Kanvas and honorarium from Merck, BMS and AstraZeneca. He is also the co-founder of Curebiota. C.K.B. is an employee and stockholder of AbbVie. M.B. sits on the scientific advisory board of Micronoma. A.B. was an employee of Genentech and is a holder of Roche stock. N.C. is CSO of MaaT Pharma. D.D. is a consultant for: ACM Bio, Ascendis Pharma, Clinical Care Options, Gerson Lehrman Group, Merck, Medical Learning Group, Xilio Therapeutics; CE Speakers’ Bureau: Castle Biosciences; and Intellectual Property: US Patent 63/124,231, “Compositions and Methods for Treating Cancer”, Dec 11, 2020 and US Patent 63/208,719, “Compositions and Methods For Responsiveness to Immune Checkpoint Inhibitors (ICI), Increasing Effectiveness of ICI and Treating Cancer”, June 9, 2021. L.D. sits on the scientific advisory board of EverImmune, holds patents covering the treatment of cancer and the therapeutic manipulation of the microbiota by LBP, and has been consulting for Ipsen and Sanofi. H.C.H. has filed a patent application (PCT/US2020/019038) for the commercial use of SagA-bacteria to improve checkpoint blockade immunotherapy, which is licensed by Rise Therapeutics to develop immunological-based biologics. M.I. is the founder and shareholder of Bio-Me. G.K. is a consultant for ReiThera; is on the Board of Directors of the Bristol Myers Squibb Foundation France; is a scientific co-founder of EverImmune Ltd., Osasuna Therapeutics, Samsara Therapeutics, and Therafast Bio; and is the inventor of patents covering therapeutic targeting of aging, cancer, cystic fibrosis, and metabolic disorders. G.K.’s brother, Romano Kroemer, was an employee of Sanofi and now consults for Boehringer-Ingelheim. F.M. reports honoraria from Therakos/Mallinckrodt, BMS, Sanofi, Jazz Pharmaceuticals, Gilead, Novartis, and Astellas, all outside the scope of this work. S.P. received travel reimbursement from CRISPR Therapeutics, and Ipsen. Z.R. is the founder of and a Scientific Advisor to Pangea Biomed. B.R. is the co-founder of Curebiota. G.D.S.-P. is an inventor on a US patent application (PCT/US2019/059647) submitted by The Regents of the University of California and licensed by Micronoma that covers methods of diagnosing and treating cancer using multi-domain microbial biomarkers in blood and cancer tissues; a founder of and reports stock interest in Micronoma; and has filed several additional US patent applications on cancer bacteriome and mycobiome diagnostics that are owned by The Regents of the University of California. R.F.S. reports consulting fees from Astellas, AstraZeneca, AVEO, BMS, EMD Serono, Editas, Exelixis, Gilead, Eisai, Janssen, Loxo, Lilly, Mirati, Pfizer, Silverback, and Seattle Genetics; research support (to institution) from Ascendis, ALX Oncology, Astellas, AstraZeneca, Bayer, BMS, CytomX, Eisai, Genentech/Roche, Gilead, Immunocore, Jounce, Loxo, Lilly, Merck, Moderna, Mirati, Novartis, Pfizer, Pionyr, Pyxis, Scholar Rock, QED Therapeutics; equity in AbbVie; and patents: Neoantigens in Cancer, PCT/US2020/031357. M.v.d.B. has received stock options from Seres Therapeutics, Notch Therapeutics, and Pluto Therapeutics; royalties from Wolters Kluwer; consultant for and honorarium from or participated in advisory boards for Seres Therapeutics, Rheos Medicines, Ceramedix, Pluto Therapeutics, Thymofox, Garuda, Novartis (Spouse), Synthekine (Spouse), BeiGene (Spouse), and Kite (Spouse); IP Licensing with Seres Therapeutics and Juno Therapeutics; and holds a fiduciary role on the Foundation Board of DKMS (a nonprofit organization). R.K.W. is a consultant for Takeda Pharmaceuticals. H.Z. sits on the scientific advisory board of EverImmune and is a consultant for MaaT Pharma. L. Zhao is a co-founder of Notitia Biotechnologies Company. J.M. received honoraria from Roche, BMS, and Merck. L. Zitvogel is a co-founder of EverImmune; the President of EverImmune scientific advisory board; holds patents covering the treatment of cancer and the therapeutic manipulation of the microbiota by LBP; has held research contracts with bioMérieux, Daiichi Sankyo, Glaxo Smith Kline, Incyte, Lytix, Kaleido, PiLeJe, Transgene, 9 Meters, Tusk Pharma, Merus, Roche, and Innovate Pharma; is on the scientific advisory board of Hookipa; and was on the Board of Directors of Transgene., (Copyright © 2023. Published by Elsevier Inc.)

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.

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., Competing Interests: The authors declare the following competing financial interest(s): T. T. and H. C. H. have filed a patent application for the commercial use of N-arylpyrazole NOD2 agonists for immunotherapy., (This journal is © The Royal Society of Chemistry.)

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 β-arrestin recruitment. Metabolomic and functional profiling also revealed specific amino acid decarboxylase-expressing microbiota species that produce aromatic monoamine agonists for GPRC5A-β-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., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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-1β (IL-1β) 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., Competing Interests: Declaration of interests K.C. has received research support from Pfizer, Takeda, Pacific Biosciences, Genentech, and Abbvie. K.C. has consulted for or received an honoraria from Puretech Health, Genentech, and Abbvie. K.C. is an inventor on U.S. patent 10,722,600 and provisional patent 62/935,035 and 63/157,225. H.C.H. has received research support from Rise Therapeutics and LISCure Biosciences. U.S. patents PCT/US16/28836 (H.C.H.) and PCT/US2020/019038 (H.C.H. and M.E.G.) were obtained for the commercial use of SagA-engineered bacteria. Rise Therapeutics has licensed these patents to develop SagA-probiotics as therapeutics. J.A. reports consultancy fees, honoraria, or advisory board fees from Abbvie, Adiso, Bristol Myers Squibb, Janssen, Pfizer, Fresnius, and BioFire Diagnostics., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: Declaration of interests M.E.G. and H.C.H. have filed a patent application (PCT/US2020/019038) for the commercial use of SagA-expressing bacteria to improve checkpoint blockade immunotherapy., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021 American Society for Microbiology.)

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., (© 2023 The Protein Society.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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.

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, Δ bapR 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.

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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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., Competing Interests: Declaration of Competing Interest M.E.G. and H.C.H. have filed a patent application (PCT/US2020/019038) to use SagA-expressing microbes as a therapeutic agent for cancer and infection., (Copyright © 2022. Published by Elsevier Inc.)

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.

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.

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., Competing Interests: Conflict of interest statement The authors declared that no conflict of interest exists., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests M.E.G. and H.C.H. have filed a patent application (PCT/US2020/019038) for the commercial use of SagA bacteria to improve checkpoint blockade immunotherapy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Protein S-fatty acylation or S-palmitoylation is a reversible and regulated lipid post-translational modification (PTM) in eukaryotes. Loss-of-function mutagenesis studies have suggested important roles for protein S-fatty acylation in many fundamental biological pathways in development, neurobiology, and immunity that are also associated with human diseases. However, the hydrophobicity and reversibility of this PTM have made site-specific gain-of-function studies more challenging to investigate. In this review, we summarize recent chemical biology approaches and methods that have enabled site-specific gain-of-function studies of protein S-fatty acylation and the investigation of the mechanisms and significance of this PTM in eukaryotic biology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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 .

Interferon-induced transmembrane proteins (IFITMs) are S -palmitoylated proteins in vertebrates that restrict a diverse range of viruses. S -palmitoylated IFITM3 in particular engages incoming virus particles, prevents their cytoplasmic entry, and accelerates their lysosomal clearance by host cells. However, how S -palmitoylation modulates the structure and biophysical characteristics of IFITM3 to promote its antiviral activity remains unclear. To investigate how site-specific S -palmitoylation controls IFITM3 antiviral activity, we employed computational, chemical, and biophysical approaches to demonstrate that site-specific lipidation of cysteine 72 enhances the antiviral activity of IFITM3 by modulating its conformation and interaction with lipid membranes. Collectively, our results demonstrate that site-specific S -palmitoylation of IFITM3 directly alters its biophysical properties and activity in cells to prevent virus infection.

S -palmitoylation is a reversible posttranslational lipid modification of proteins. It controls protein activity, stability, trafficking and protein-protein interactions. Recent global profiling of immune cells and targeted analysis have identified many S -palmitoylated immunity-associated proteins. Here, we review S -palmitoylated immune receptors and effectors, and their dynamic regulation at cellular membranes to generate specific and balanced immune responses. We also highlight how this understanding can drive therapeutic advances to pharmacologically modulate immune responses.

Increased heme levels, anemia, and desaturation occur during infection. We aimed to compare the levels of heme, heme oxygenase-1 (HO-1), ferritin, and bilirubin in coronavirus disease 2019 (COVID-19) patients at different saturation levels. Heme and HO-1 enzyme levels significantly increased in the low SpO 2 group, but further studies are required., Competing Interests: Declaration of competing interest The authors declare that they have no conflicting interests., (Copyright © 2020. Published by Elsevier B.V.)

Peptidoglycan is a vital component of the bacterial cell wall, and its dynamic remodeling by NlpC/p60 hydrolases is crucial for proper cell division and survival. Beyond these essential functions, we previously discovered that Enterococcus species express and secrete the NlpC/p60 hydrolase-secreted antigen A (SagA), whose catalytic activity can modulate host immune responses in animal models. However, the localization and peptidoglycan hydrolase activity of SagA in Enterococcus was still unclear. In this study, we show that SagA contributes to a triseptal structure in dividing cells of enterococci and localizes to sites of cell division through its N-terminal coiled-coil domain. Using molecular modeling and site-directed mutagenesis, we identify amino acid residues within the SagA-NlpC/p60 domain that are crucial for catalytic activity and potential substrate binding. Notably, these studies revealed that SagA may function via a catalytic Cys-His dyad instead of the predicted Cys-His-His triad, which is conserved in SagA orthologs from other Enterococcus species. Our results provide key additional insight into peptidoglycan remodeling in Enterococcus by SagA NlpC/p60 hydrolases.

Protein S-palmitoylation, or S-fatty-acylation, regulates many fundamental cellular processes in eukaryotes. Herein, we present a chemical fatty-acylation approach that involves site-specific incorporation of cycloalkyne-containing unnatural amino acids and subsequent bioorthogonal reactions with fatty-acyl tetrazines to install fatty-acylation mimics at target protein sites, allowing gain-of-function analysis of S-palmitoylation in live cells.

The characterization of specific metabolite-protein interactions is important in chemical biology and drug discovery. For example, nuclear receptors (NRs) are a family of ligand-activated transcription factors that regulate diverse physiological processes in animals and are key targets for therapeutic development. However, the identification and characterization of physiological ligands for many NRs remains challenging, because of limitations in domain-specific analysis of ligand binding in cells. To address these limitations, we developed a domain-specific covalent chemical reporter for peroxisome proliferator-activated receptors (PPARs) and demonstrated its utility to screen and characterize the potency of candidate NR ligands in live cells. These studies demonstrate targeted and domain-specific chemical reporters provide excellent tools to evaluate endogenous and exogenous (diet, microbiota, therapeutics) ligands of PPARs in mammalian cells, as well as additional protein targets for further investigation.

Interferon-induced transmembrane protein 3 (IFITM3) is a key interferon effector that broadly prevents infection by diverse viruses. However, the cellular factors that control IFITM3 homeostasis and antiviral activity have not been fully elucidated. Using site-specific photo-crosslinking and quantitative proteomic analysis, here we present the identification and functional characterization of VCP/p97 AAA-ATPase as a primary interaction partner of IFITM3. We show that IFITM3 ubiquitination at lysine 24 is crucial for VCP binding, trafficking, turnover, and engagement with incoming virus particles. Consistently, pharmacological inhibition of VCP/p97 ATPase activity leads to defective IFITM3 lysosomal sorting, turnover, and co-trafficking with virus particles. Our results showcase the utility of site-specific protein photo-crosslinking in mammalian cells and reveal VCP/p97 as a key cellular factor involved in IFITM3 trafficking and homeostasis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The discovery of specific microbiota metabolite mechanisms has begun to motivate new therapeutic approaches. Inspired by our mechanistic studies of microbiota-derived short chain fatty acid (SCFA) acylation of bacterial virulence factors, here we explored covalent protein acylation therapeutics as potential anti-infectives. For these studies, we focused on acetyl-salicylic acid, aspirin, and discovered that SCFA analogues such as butyryl-salicylic acid showed significantly improved anti-infective activity against Salmonella Typhimurium. Structure-activity studies showed that the ester functionality of butyryl-salicylic acid was crucial and associated with the acylation of key bacterial virulence factors and metabolic enzymes, which are important for Salmonella infection of host cells and bacterial growth. Beyond the Gram-negative bacterial pathogens, butyryl-salicylic acid also showed better antibacterial activity compared to aspirin against Clostridioides difficile , a clinically challenging Gram-positive bacterial pathogen. Notably, coadministration of butyryl-salicylic acid, but not aspirin, effectively attenuated Salmonella pathogenesis in vivo . This study highlights how the analysis of microbiota metabolite mechanisms may inspire the repurposing and development of new anti-infective agents.

Understanding the molecular mechanisms of endogenous and environmental metabolites is crucial for basic biology and drug discovery. With the genome, proteome, and metabolome of many organisms being readily available, researchers now have the opportunity to dissect how key metabolites regulate complex cellular pathways in vivo. Nonetheless, characterizing the specific and functional protein targets of key metabolites associated with specific cellular phenotypes remains a major challenge. Innovations in chemical biology are now poised to address this fundamental limitation in physiology and disease. In this review, we highlight recent advances in chemoproteomics for targeted and proteome-wide analysis of metabolite-protein interactions that have enabled the discovery of unpredicted metabolite-protein interactions and facilitated the development of new small molecule therapeutics., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The advances made in bioorthogonal chemistry and the development of chemical reporters have afforded new strategies to explore the targets and functions of specific metabolites in biology. These metabolite chemical reporters have been applied to diverse classes of bacteria including Gram-negative, Gram-positive, mycobacteria, and more complex microbiota communities. Herein we summarize the development and application of metabolite chemical reporters to study fundamental pathways in bacteria as well as microbiota mechanisms in health and disease., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Microbiota generates millimolar concentrations of short-chain fatty acids (SCFAs) that can modulate host metabolism, immunity and susceptibility to infection. Butyrate in particular can function as a carbon source and anti-inflammatory metabolite, but the mechanism by which it inhibits pathogen virulence has been elusive. Using chemical proteomics, we found that several virulence factors encoded by Salmonella pathogenicity island-1 (SPI-1) are acylated by SCFAs. Notably, a transcriptional regulator of SPI-1, HilA, was acylated on several key lysine residues. Subsequent incorporation of stable butyryl-lysine analogs using CRISPR-Cas9 gene editing and unnatural amino acid mutagenesis revealed that site-specific modification of HilA impacts its genomic occupancy, expression of SPI-1 genes and attenuates Salmonella enterica serovar Typhimurium invasion of epithelial cells, as well as dissemination in vivo. Moreover, a multiple-site HilA lysine acylation mutant strain of S. Typhimurium was resistant to butyrate inhibition ex vivo and microbiota attenuation in vivo. Our results suggest that prominent microbiota-derived metabolites may directly acylate virulence factors to inhibit microbial pathogenesis in vivo.

The NlpC/p60-family of peptidoglycan hydrolases are key enzymes that facilitate bacterial cell division and also modulate microbe-host interactions. These endopeptidases utilize conserved Cys-His residues in their active site and are expressed in most bacterial species as well as some eukaryotes. Here we describe methods for biochemical analysis of Enterococcus faecium SagA-NlpC/p60 peptidoglycan hydrolase activity (Kim et al., 2019; Rangan et al., 2016), which includes recombinant protein preparation and biochemical analysis using both gel-based and LC-MS profiling of peptidoglycan fragments. These protocols should also facilitate the biochemical analysis of other NlpC/p60 peptidoglycan hydrolases., Competing Interests: Conflict of interest statement The authors declared that no conflict of interest exists., (© 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: MEG and HCH are inventors on a patent filed by The Rockefeller University for the use of SagA towards the treatment of cancer and infection. Rise Therapeutics has licensed the patent on SagA for probiotic development., (© 2019 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

We discovered that Enterococcus faecium ( E. faecium ), a ubiquitous commensal bacterium, and its secreted peptidoglycan hydrolase (SagA) were sufficient to enhance intestinal barrier function and pathogen tolerance, but the precise biochemical mechanism was unknown. Here we show E. faecium has unique peptidoglycan composition and remodeling activity through SagA, which generates smaller muropeptides that more effectively activates nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in mammalian cells. Our structural and biochemical studies show that SagA is a NlpC/p60-endopeptidase that preferentially hydrolyzes crosslinked Lys-type peptidoglycan fragments. SagA secretion and NlpC/p60-endopeptidase activity was required for enhancing probiotic bacteria activity against Clostridium difficile pathogenesis in vivo . Our results demonstrate that the peptidoglycan composition and hydrolase activity of specific microbiota species can activate host immune pathways and enhance tolerance to pathogens., Competing Interests: BK, YW, CH, JE, JS, KR, DO, JK, VP, HH No competing interests declared, (© 2019, Kim et al.)

Cells require a constant supply of fatty acids to survive and proliferate. Fatty acids incorporate into membrane and storage glycerolipids through a series of endoplasmic reticulum (ER) enzymes, but how these enzymes are regulated is not well understood. Here, using a combination of CRISPR-based genetic screens and unbiased lipidomics, we identified calcineurin B homologous protein 1 (CHP1) as a major regulator of ER glycerolipid synthesis. Loss of CHP1 severely reduces fatty acid incorporation and storage in mammalian cells and invertebrates. Mechanistically, CHP1 binds and activates GPAT4, which catalyzes the initial rate-limiting step in glycerolipid synthesis. GPAT4 activity requires CHP1 to be N-myristoylated, forming a key molecular interface between the two proteins. Interestingly, upon CHP1 loss, the peroxisomal enzyme, GNPAT, partially compensates for the loss of ER lipid synthesis, enabling cell proliferation. Thus, our work identifies a conserved regulator of glycerolipid metabolism and reveals plasticity in lipid synthesis of proliferating cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) have emerged as important innate immune effectors that prevent diverse virus infections in vertebrates. However, the cellular mechanisms and live-cell imaging of these small membrane proteins have been challenging to evaluate during viral entry of mammalian cells. Using CRISPR-Cas9-mediated IFITM-mutant cell lines, we demonstrate that human IFITM1, IFITM2 and IFITM3 act cooperatively and function in a dose-dependent fashion in interferon-stimulated cells. Through site-specific fluorophore tagging and live-cell imaging studies, we show that IFITM3 is on endocytic vesicles that fuse with incoming virus particles and enhances the trafficking of this pathogenic cargo to lysosomes. IFITM3 trafficking is specific to restricted viruses, requires S-palmitoylation and is abrogated with loss-of-function mutants. The site-specific protein labeling and live-cell imaging approaches described here should facilitate the functional analysis of host factors involved in pathogen restriction as well as their mechanisms of regulation.

Protein fatty-acylation describes the covalent modification of protein with fatty acids during or after translation. Chemical proteomic profiling methods have provided new opportunities to explore protein fatty-acylation in microbial pathogens. Recent studies suggest that protein fatty-acylation is essential to survival and pathogenesis of eukaryotic pathogens such as parasites and fungi. Moreover, fatty-acylation in host cells can be exploited or manipulated by pathogenic bacteria. Herein, we first review the prevalent classes of fatty-acylation in microbial pathogens and the chemical proteomic profiling methods for their global analysis. We then summarize recent fatty-acylation profiling studies performed in eukaryotic pathogens and during bacterial infections, highlighting how they contribute to functional characterization of fatty-acylation under these contexts.

Protein S-fatty-acylation, the covalent addition of a long-chain fatty acid, predominantly palmitate (S-palmitoylation), to cysteine, is a highly dynamic and regulated process that controls protein function and localization of membrane-associated proteins in eukaryotes. The analysis of S-fatty acylated peptides by mass spectrometry remains challenging due to the hydrophobic and potentially labile thioester linkage of the S-fatty acylated peptides.Here we describe an optimized protocol for the global analysis of S-palmitoylated proteins based on the combination of an alkyne-tagged chemical reporter of palmitoylation, alk-16 with hydroxylamine-selective hydrolysis of thioester bonds. This protocol decreased the number of false positive proteins and was applied to identify S-fatty acylation sites, providing modification sites for 44 proteins out of the 106 S-fatty acylated proteins identified.

Shigella flexneri, an intracellular Gram-negative bacterium causative for shigellosis, employs a type III secretion system to deliver virulence effectors into host cells. One such effector, IcsB, is critical for S. flexneri intracellular survival and pathogenesis, but its mechanism of action is unknown. Here, we discover that IcsB is an 18-carbon fatty acyltransferase catalysing lysine N ε -fatty acylation. IcsB disrupted the actin cytoskeleton in eukaryotes, resulting from N ε -fatty acylation of RhoGTPases on lysine residues in their polybasic region. Chemical proteomic profiling identified about 60 additional targets modified by IcsB during infection, which were validated by biochemical assays. Most IcsB targets are membrane-associated proteins bearing a lysine-rich polybasic region, including members of the Ras, Rho and Rab families of small GTPases. IcsB also modifies SNARE proteins and other non-GTPase substrates, suggesting an extensive interplay between S. flexneri and host membrane trafficking. IcsB is localized on the Shigella-containing vacuole to fatty-acylate its targets. Knockout of CHMP5-one of the IcsB targets and a component of the ESCRT-III complex-specifically affected S. flexneri escape from host autophagy. The unique N ε -fatty acyltransferase activity of IcsB and its altering of the fatty acylation landscape of host membrane proteomes represent an unprecedented mechanism in bacterial pathogenesis.

S-Fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterizing the endogenously attached fatty acids are still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods. Notably, we directly identified the S-fatty-acylation sites of IFITM3, an important interferon-stimulated inhibitor of virus entry, and we further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid. The methods described here should facilitate the direct analysis of protein S-fatty-acylation sites and their endogenously attached fatty acids in diverse cell types and activation states important for mammalian physiology and diseases.

Interferon-induced transmembrane protein 3 (IFITM3) is a cellular endosome- and lysosome-localized protein that restricts numerous virus infections. IFITM3 is activated by palmitoylation, a lipid posttranslational modification. Palmitoylation of proteins is primarily mediated by zinc finger DHHC domain-containing palmitoyltransferases (ZDHHCs), but which members of this enzyme family can modify IFITM3 is not known. Here, we screened a library of human cell lines individually lacking ZDHHCs 1-24 and found that IFITM3 palmitoylation and its inhibition of influenza virus infection remained strong in the absence of any single ZDHHC, suggesting functional redundancy of these enzymes in the IFITM3-mediated antiviral response. In an overexpression screen with 23 mammalian ZDHHCs, we unexpectedly observed that more than half of the ZDHHCs were capable of increasing IFITM3 palmitoylation with ZDHHCs 3, 7, 15, and 20 having the greatest effect. Among these four enzymes, ZDHHC20 uniquely increased IFITM3 antiviral activity when both proteins were overexpressed. ZDHHC20 colocalized extensively with IFITM3 at lysosomes unlike ZDHHCs 3, 7, and 15, which showed a defined perinuclear localization pattern, suggesting that the location at which IFITM3 is palmitoylated may influence its activity. Unlike knock-out of individual ZDHHCs, siRNA-mediated knockdown of both ZDHHC3 and ZDHHC7 in ZDHHC20 knock-out cells decreased endogenous IFITM3 palmitoylation. Overall, our results demonstrate that multiple ZDHHCs can palmitoylate IFITM3 to ensure a robust antiviral response and that ZDHHC20 may serve as a particularly useful tool for understanding and enhancing IFITM3 activity., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The intestine is a highly complex ecosystem where many bacterial species interact with each other and host cells to influence animal physiology and susceptibility to pathogens. Genomic methods have provided a broad framework for understanding how alterations in microbial communities are associated with host physiology and infection, but the biochemical mechanisms of specific intestinal bacterial species are only emerging. In this review, we focus on recent studies that have characterized the biochemical mechanisms by which intestinal bacteria interact with other bacteria and host pathways to restrict pathogen infection. Understanding the biochemical mechanisms of intestinal microbiota function should provide new opportunities for therapeutic development towards a variety of infectious diseases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Increasing antibiotic resistance and beneficial effects of host microbiota has motivated the search for anti-infective agents that attenuate bacterial virulence rather than growth. For example, we discovered that specific flavonoids such as baicalein and quercetin from traditional medicinal plant extracts could attenuate Salmonella enterica serovar Typhimurium type III protein secretion and invasion of host cells. Here, we show epigallocatechin-3-gallate from green tea extracts also inhibits the activity of S. Typhimurium type III protein effectors and significantly reduces bacterial invasion into host cells. These results reveal additional dietary plant metabolites that can attenuate bacterial virulence and infection of host cells., (Copyright © 2017. Published by Elsevier Ltd.)