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Chemical tools for exploring metabolite-protein interactions

The emergence of microbial pathogens and adaptation of microbes to currently available drugs demands a better understanding of host-microbe interactions to prevent and treat infections. With the genetic blueprints of many microbes and animal hosts now available, new methods are needed to evaluate how key metabolites regulate host immunity and microbial virulence. To elucidate the mechanisms involved in host-microbe interactions, the Hang laboratory has developed robust chemical methods to image and profile the biochemical targets of metabolites in animal cells and microbes that are synthesized endogenously or derived from the environment (diet or microbiota). At the heart of this chemical approach is the design and synthesis of specific chemical reporters, metabolites bearing uniquely reactive groups, that can be chemically or enzymatically incorporated into biomolecules in vitro and in vivo and then selectively labeled with imaging or affinity purification reagents (Fig. 1). Using this strategy, a variety of chemical reporters based on important metabolites (nucleosides, amino acids, lipids and other cofactors) have been developed in the Hang laboratory for the sensitive detection and analysis of metabolite-protein modifications such as palmitoylation, myristoylation, prenylation, acetylation, AMPylation, ADP-ribosylation (reviewed in Grammel M & Hang HC Nat Chem Biol 2013) as well as those derived from environmental metabolites such as flavonoids from medicinal plants (Tsou LK et al JACS 2016).

Chemical reporters enable detection and enrichment of metabolite-modified proteins for target identification.

Figure 1. Chemical reporters enable detection and enrichment of metabolite-modified proteins for target identification.

Protein fatty-acylation in host immunity and microbial virulence

Metabolites from the diet, microbiota and cellular metabolism can significantly affect host susceptibility to infections. These active metabolites encompass a broad range of molecules ranging from fatty acids to bacterial cell well components. My laboratory has been especially focused on protein targets of long-chain fatty acids (LCFAs), since alterations in amounts and structure of LCFAs have profound effects on host immunity and pathogen susceptibility (Fig. 2A). For example, genetic defects in palmitic acid (C16:0) synthesis have been associated with impaired immune responses and resistance to infections. Conversely, high levels of palmitic acid from high-fat diets or adipocytes from obese animals have been suggested to activate immunity receptors and induce inflammation. These effects are specific to the structure of LCFAs, as unsaturated fatty acids such as oleic acid (C18:1) or pro-resolving lipid mediators such as resolvin and protectin facilitate the resolution of inflammatory responses and tissue repair (Fig. 2B). The balance of fatty acid levels and composition in animals is thus clearly very important in regulating appropriate immune responses, but the specific molecular targets and mechanisms by which LCFAs controls specific immune pathways are unclear.

Fatty acid control of host immunity.

 

Figure 2. Fatty acid control of host immunity. A) Long-chain fatty acids that modulate host immunity. B) LCFA chemical reporter for profiling S-fatty-acylated proteins. C) Summary of fatty-acylated proteins in activated macrophages.

Beyond essential functions in membrane biogenesis and metabolism, LCFAs can serve as ligands for specific receptors and also can be covalently attached to proteins (N-myristoylation, S-palmitoylation, Lys-fatty-acylation) and regulate their activity in membranes (Fig. 2B). While fatty acid binding to specific receptors can control some signaling pathways, these interactions are insufficient to explain the broad effects of LCFAs on inflammation and host susceptibility to infection. Indeed, chemical proteomic studies of immune cells in our laboratory have revealed many unpredicted fatty-acylated proteins involved in host immunity and pathogen susceptibility (Fig. 2C). These novel fatty-acylated proteins include IFITM-family proteins, which we first demonstrated are S-fatty-acylated on conserved Cys resides that are important for their membrane targeting and antiviral activity against influenza virus infection (Yount JS et al Nat Chem Biol 2010). Following our discovery of interferon-induced transmembrane protein 3 (IFITM3) S-fatty acylation-dependent antiviral activity, we are continuing to develop new approaches to characterize the precise mechanism of IFITM3 antiviral activity and its regulation by S-fatty-acylation that include 1) live cell imaging of IFITM3 during virus infection, 2) IFITM3-interactome studies and 3) in vitro reconstitution studies of recombinant and site-specifically lipidated IFITM3. These studies should help elucidate the precise mechanism of action for IFITM3 (Fig. 3), which is mutated in humans with increased morbidity and mortality to seasonal influenza A virus infections.

Working model for IFITM3 antiviral activity.

Figure 3. Working model for IFITM3 antiviral activity

To understand the scope of protein fatty-acylation in host immunity, we have also performed more quantitative proteomic analysis of fatty-acylated proteins in naïve and IFNg/LPS-activated macrophages (Fig. 2C) (Thinon E et al ChemBioChem 2016) and developed new S-fatty-acylation methods (Percher A et al PNAS 2016). These studies have revealed new fatty-acylated proteins with important roles in vesicle trafficking and host immunity, which are currently being explored in the laboratory. These studies highlight new targets of fatty acids and suggest many uncharacterized functions of protein fatty-acylation in mammals. To characterize non-covalent metabolite-protein interactions, we will employ bifunctional fatty acid reporters for photocrosslinking studies in living cells that we have recently described (Peng T et al JACS 2015), which should help reveal key targets of other immunologically active fatty acids. These studies should characterize the covalent and non-covalent protein targets of fatty acids and uncover how these essential metabolites chemically control host immunity and pathogen susceptibility.

Microbial pathogens have evolved sophisticated mechanisms to evade host defense mechanisms and cause disease in animals. The analysis of individual virulence factors has suggested that many pathogens alter or subvert metabolite targets or PTMs in host cells for infection. For example, our collaborative studies have demonstrated that secreted bacterial protein effectors/toxins co-opt host protein lipidation pathways to regulate the virulence of intracellular bacterial pathogen (Burnaevskiy N et al Mol Cell 2015, Hicks SW et al Cell Host Microbe 2011). However, the precise protein modifications and targets of these virulence factors during infection have been challenging to characterize biochemically. As bacterial pathogens secrete many enzymes with unknown biochemical targets and mechanisms of action, we are performing quantitative proteomics of infected-host cells using specific chemical reporters as a general strategy to discover alterations in host metabolite-targets during infection. These unbiased and quantitative biochemical analyses of pathogen-infected host cells are required to fully understand pathogen virulence mechanisms.

Discovery and development of anti-infectives

With dwindling supply of antibiotics and a growing appreciation for commensal microbes, new anti-infective strategies are needed to selectively target microbial pathogens without depleting the beneficial microbiota in humans. To develop new anti-infective approaches, the Hang laboratory is interested in understanding protective mechanisms of commensal bacteria and developing inhibitors of bacterial virulence pathways. While host-associated bacterial species have been shown to secrete metabolites and proteins for their beneficial affects, the mechanisms of action and targets of these commensal bacteria-derived molecules are not well understood and could lead to new anti-infective therapeutics. To identify new factors involved in host resistance or tolerance to pathogens, my laboratory has utilized Caenorhabditis elegans as a model system for exploring commensal bacteria and their secreted factors. We discovered that secreted antigen A (SagA) from Enterococcus faecium is sufficient to protect C. elegans against Salmonella pathogenesis by promoting pathogen tolerance. The NlpC/p60 peptidoglycan hydrolase activity of SagA is required and generated muramyl-peptide fragments that are sufficient to protect C. elegans against Salmonella pathogenesis in a tol-1-dependent manner. SagA can also be expressed and secreted to improve the protective activity of probiotics against Salmonella pathogenesis in mice. In collaboration with the Mucida Laboratory of Mucosal Immunology at Rockefeller, we showed that SagA-mediated protection mechanisms are conserved in mice and can be used to prevent enteric infections including C. difficile. These studies highlight how protective intestinal bacteria can modify microbial-associated molecular patterns to enhance pathogen tolerance and potentially be used enhance the activity of probiotics (Fig. 4).

. Model for E. faecium and SagA-protection against enteric pathogens in vivo.

Figure 4. Model for E. faecium and SagA-protection against enteric pathogens in vivo

To complement the mechanistic studies of commensal bacteria, the Hang laboratory is also developing chemical inhibitors of key microbial virulence pathways to limit infections. Specifically, the Hang laboratory is working on small molecule inhibitors of type III secretion systems (T3SSs), which are responsible for injecting bacterial toxins and effector proteins into host cells and are essential for the virulence of many Gram-negative bacterial pathogens. Towards this goal, the Hang laboratory has developed a high-throughput assay for type III protein secretion and discovered and currently characterizing specific small molecules from medicinal plants and synthetic chemical libraries that can antagonize T3SSs and inhibit bacterial infection. Our recent studies on the type III protein secretion system (T3SS) inhibitors from medicinal plants, which suggests specific plant metabolites (flavonoids) covalently label and inactivate T3SS substrates to attenuate bacterial virulence (Tsou LK et al JACS 2016). These T3SS inhibitors are not broadly bactericidal and provide new lead compounds for selectively targeting bacterial pathogens responsible for disease without killing protective commensal bacteria. These studies highlight our ongoing efforts to discover and characterize new infective approaches to combat infections.

Chemical tools for exploring metabolite-protein interactionsThe emergence of microbial pathogens and adaptation of microbes to currently available drugs demands a better understanding of host-microb