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Our laboratory is broadly interested in the molecular mechanisms by which chemical signals modulate host-microbe interactions in infection and immunity (Fig. 1). These chemical signals (metabolites) may be derived from host metabolism (endogenous metabolites) or the environment (diet, microbiota, therapeutics) and have been challenging to mechanistically elucidate. To dissect the mechanisms that govern host-microbe interactions, my laboratory has 1) developed chemical methods to characterize metabolite-protein interactions and 2) employed key animal models to discover new protective factors from specific microbiota species and elucidated their mechanisms of action (Fig. 1). These studies have revealed unpredicted metabolite-protein functions in host immunity and microbial pathogenesis as well as novel microbiota protective factors, which have afforded new therapeutic leads and biomarkers for microbial infections and cancer immunotherapy. Our major discoveries as well as ongoing and future directions are summarized below.

Figure 1. Chemical dissection of specific metabolites and microbiota species has revealed key mechanisms of action and afforded new protective factors for therapeutic and biomarker development towards microbial infections and cancer immunotherapy.

Chemical dissection of metabolite-protein targets. To characterize direct metabolite-protein modifications, my laboratory developed chemical reporters and robust proteomic methods to evaluate covalent metabolite-protein modifications (Fig. 2A), such as fatty-acylation, acetylation, prenylation, AMPylation and ADP-ribosylation as well as non-covalent metabolite-protein interactions using photoaffinity reporters (Fig. 2B). Notably, these chemical methods have enabled my laboratory and in collaboration with other groups to discover novel metabolite modifications and regulation in eukaryotic cell biology, host immunity and microbial pathogenesis. These studies showcase how our chemical proteomic methods can be used to characterize unpredicted metabolite targets and regulatory mechanisms in microbes and host cells.

Figure 2. Chemical reporters for characterizing metabolite-protein interactions. A) Functionalization of metabolites with small chemical tags such as terminal alkynes (alk) facilitates the analysis of covalent or photocrosslinked metabolite-protein interactions in vitroand in vivo. Addition of small photocrosslinkers such as diazirinesenable photocrosslinking of non-covalent metabolite interactions. Subsequent bioorthogonal labeling with azide-modified reagents facilitates the fluorescent detection, affinity enrichment and direct identification of metabolite-protein interactions. B) Chemical reporters of primary metabolites and their protein modifications. C) Representative chemical reporters of dietary medicinal plant metabolites and microbiota metabolites; muramyl-dipeptide (MDP), secondary bile acids and indole acetic acid.

Mechanistic analysis of dietary and microbiota metabolite-protein targets. Building upon our expertise in chemical biology, my laboratory has used chemical proteomics, site-specific protein labeling and CRISPR-Cas9 gene editing to elucidate the mechanisms of dietary and microbiota-metabolites. For example, we have developed chemical reporters of medicinal plant metabolites (Fig. 2C) as well as short-chain fatty acids (Fig. 2B) (Zhang Z et al Nat Chem Biol in revision) and discovered that these reactive metabolites can covalently modify and inactivate virulence factors in microbial pathogens to attenuate infection. Moreover, our recent studies of peptidoglycan metabolite protein targets using photoaffinity reporters (Fig. 2C) revealed that muramyl-dipeptide (MDP) can directly bind to the intracellular innate immune receptor NOD2 in cells for the first time and also interact with the small GTPase Arf6, which may be a key cellular adapter for the formation of the active MDP-NOD2 complex in mammalian cells (Wang Y-C et al ACS Chem Biol 2019). Beyond this work, recent in vivo and metabolomics studies have suggested that specific microbiota species can generate other metabolites such as secondary bile acids, aromatic amino acid derivatives (Fig. 2C) and others, but their protein targets and mechanisms of action in microbial pathogens and host cells remain to be determined. To further understand the functions of dietary and microbiota metabolites as well as small molecule therapeutics, my laboratory will continue synthesizing and applying chemical reporters to biochemically characterize the direct metabolite-protein targets and elucidate their mechanism(s) of action in microbes and host cells. In addition, our discover and characterization of metabolite targets has motivated my laboratory to develop synthetic derivatives and explore other small molecules as new therapeutic candidates for modulating infection and host immunity.

Discovery of specific microbiota protective mechanisms. The microbiota (diverse bacteria, fungi and viruses) are now recognized as major factors in human physiology, disease and therapy, but still challenging to mechanistically elucidate. To explore the protective mechanisms of specific microbiota species, we employed the roundworm Caenorhabditis elegans as an animal model to investigate microbiota modulation of microbial infections (Fig. 3A). Using C. elegans, we discovered that Enterococcus faecium could improve the host intestinal barrier and protect against Salmonella Typhimurium pathogenesis (Rangan K et al Science 2016). Enterococci are ubiquitous Gram-positive bacteria that have been recovered from the environment, food, diverse animals and humans. Amongst the >40 species of Enterococci identified to date, E. faecium (Efm) and E. faecalis (Efs) are the most prominent in humans. While some pathogenic strains of Enterococci can acquire virulence factors, antibiotic resistance and cause nosocomial infections, commensal strains of Efm have been reported to protect animals from diverse pathogens and developed as probiotics for humans, livestock and pets by unknown mechanism(s). Our mechanistic studies revealed that Efm secreted antigen A (SagA), a unique secreted NlpC/p60-family peptidoglycan hydrolase that is conserved in several species of Enterococci was sufficient to protect C. elegans against S. Typhimurium pathogenesis. Efm and SagA-mediated protection did not affect S. Typhimurium colonization or replication in vivo,but required the Tol-1 signaling (C. elegans ortholog of toll-like receptor in mammals), suggesting the activation of host immunity and improved intestinal barrier are the key mechanisms of action.

Figure 3. Analysis of specific microbiota species in C. elegans and mice revealed protective activity and mechanisms of SagA-Enterococci. A) Efm, SagA and MurNAc treatment was sufficient to prevent S. Typhimurium pathogenesis in C. elegans via Tol-1 signaling. B) SagA-Enterococci generates small muropeptides that enhance host immunity via NOD2 and protects against intestinal pathogens.

Mechanistic analysis of specific microbiota protective mechanisms. Our subsequent studies in mouse models showed that SagA-expressing Enterococci can enhance expression of antimicrobial peptides and mucins to improve the intestinal barrier function and protect against S. Typhimurium as well as Clostridium difficile infection (Pedicord V et al Science Immunology 2016) (Fig. 3B). The protective activity of Efm required innate immune sensing factors (MyD88 - adaptor for TLR signaling and NOD2 - innate immune sensor of peptidoglycan) as well as RegIIIg (a key antimicrobial peptide that also regulates microbiota-intestinal barrier segregation). Additional mechanistic studies from my laboratory also demonstrated that Efmcan directly activate human NOD2, but not NOD1, consistent with NOD2 recognition of small non-crosslinked Lys-type muropeptides that are more abundant in Efm compared to non-protective Efs. To further characterize the biochemical activity SagA protection, we determined the X-ray crystal structure of the SagA-NlpC/p60 hydrolase domain and further demonstrated that it cleaves crosslinked peptidoglycan fragments into smaller muropeptides such as GlcNAc-muramyl dipeptide (GlcNAc-MDP), which can be processed into MDP and activate NOD2 in mammalian cells (Fig. 3B). Notably, SagA can be expressed in probiotic bacteria such as Lactobacillus plantarum and confer protection against S. Typhimurium and C. difficile infection in vivo, which requires SagA secretion as well as hydrolase activity (Kim B et al Elife 2019). These studies provide a key mechanism by which Efm and SagA can modulate host immunity and protect against intestinal pathogen infections (Fig. 3B). To further elucidate the functions of specific microbiota species in human health, my laboratory will continue to employ key animal models for mechanistic studies and identification of novel protective factors.

RESEARCH SUMMARYOur laboratory is broadly interested in the molecular mechanisms by which chemical signals modulate host-microbe interactions in infection and immunity (Fig. 1). These chemical signa