Microbiota-host Transgenomic Metabolism, Bioactive Molecules from

Perspective pubs.acs.org/jmc

Microbiota−Host Transgenomic Metabolism, Bioactive Molecules from the Inside Miniperspective Silvia Turroni,† Patrizia Brigidi,† Andrea Cavalli,†,‡ and Marco Candela*,† †

Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy Compunet, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genoa, Italy

‡

S Supporting Information *

ABSTRACT: Molecular factors from the gut microbiota provide the host with the right metabolic, immunological, and neurological components to support health and wellbeing. However, certain circumstances can rupture the mutualistic pact with our intestinal counterpart, pushing the gut microbiome toward a dysbiotic layout, where microbiomederived molecules may contribute to a disease state. We are now beginning to understand the microbiota−host co-regulated pathways underlying these processes, paving the way for a new era of rational piloting of the gut microbiome functions, through the design of a new generation of microbiome-targeting drugs. Microbiota-derived metabolites are emerging as promising starting hit compounds to modulate human targets, hence triggering certain pharmacological responses. In conclusion, drug discovery targeting the gut microbiota as well as the characterization of microbiota-derived metabolites can represent innovative medicinal chemistry possibilities toward the identification of novel drug candidates, targets, and more in general innovative ways for the treatment of unmet medical needs.

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HUMAN GUT MICROBIOTA We live in a planet dominated by microbial life, where human beings are just an island surrounded by a sea of microorganisms.1 It is thus unavoidable that our body hosts different microbial communities, formed through a series of exposures to environmental factors, a process that begins at our birth.2 Among the microbial communities populating our body, the one inhabiting the gastrointestinal (GI) tract (known as the gut microbiota (GM)) is the most stable, dense, diversified, and individually specific.3 Separated from the external environment by the gastric barrier and continuously fed by the host diet, the GM has found the best niche to establish a close association with the host, paving the way for a unique coevolution process in which human beings and their hosted gut microbial communities coexist in an admirable mutualistic scenario.4 Our symbiont gut microbial community is numerically enormous; with more than 100 trillion cells and 5 million nonredundant genes (up to nearly 500 times the human genome),5 the human GM is probably the most densely populated bacterial ecosystem on our planet.6 To date, thousands of different bacterial species have been detected in the human GM meta-community. However, the great majority belong to only 6 of the hundreds of bacterial phyla populating our planet: Firmicutes and Bacteroidetes, which together represent approximately 90% of the community, and Actinobacteria, Proteobacteria, Fusobacteria and Verrucomicrobia, as subdominant phyla.4,7 The GM provides the human host with essential physiological features that we have not evolved by ourselves.8 Indeed, our © 2017 American Chemical Society

symbiont bacterial ecosystem is a strategic component of our immune system, playing a key role in its development and functionality. In parallel, the GM is an important regulator of the human metabolic homeostasis, with an essential role in our nutrition and metabolism. We are currently witnessing increasing awareness of the importance of our gut microbial counterpart as an integral component of our central nervous system (CNS), with an ultimate capacity to modulate our behavior.9 The extraordinary importance of the GM as an inherent part of the human physiology is also highlighted by the number of chronic diseases involving the rupture of the mutualistic relationship with our intestinal symbionts.8 Detectable in terms of dysbiosis, such a rupture of the host−GM homeostatic equilibrium is the result of the establishment of an unbalanced GM compositional and functional layout, which can support and/or consolidate the disease state.8 To date, imbalances of the intestinal microbial community have been associated with a plethora of gastroenterological disorders, as well as neurological, respiratory, metabolic, hepatic, and cardiovascular illness.10 GM metabolites and GM-derived bioactive molecules represent the functional connection between the GM and the physiology of the human holobiont (Table 1).11−13 Indeed, the GM can be viewed as a bioreactor producing an extraordinarily diverse molecular repertoire, through the anaerobic fermentation of dietary components or the biotransformation of endogenous compounds generated by the host or other community members. Received: February 14, 2017 Published: July 26, 2017 47

DOI: 10.1021/acs.jmedchem.7b00244 J. Med. Chem. 2018, 61, 47−61

Journal of Medicinal Chemistry

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Table 1. Principal Gut Microbiome Bioactive Compounds with a Major Role in Human Physiology and Pathophysiologya

a

When available, for each gut microbiome-derived bioactive compound, the substrate, the metabolic activity involved, and the specific role in human biology are reported. In red are the metabolites with a link to disease. 48



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Figure 1. The diet from the gut microbiome to the host axis. Gut microbiome is at the connection between diet and host physiology, establishing a “diet from the microbiome to the host axis” strategic for human biology. The bioconversion of dietary components by intestinal symbionts results in the production of a wide and diverse range of bioactive small molecules, available to the host, that regulate several aspects of human biology critical to our metabolic, immunological, and neurological homeostasis. In particular, the catabolism of indigestible polysaccharides by gut microbes leads to the production of short-chain fatty acids as fermentation end-products, mainly acetate, propionate, and butyrate. These microbial waste products represent, for the host, an important energy source, strategic metabolic regulators, and homeostatic modulators of immune function. On the other side, intestinal microbes can convert dietary inputs to bioactive compounds, such as vitamins (e.g., folic acid and riboflavin), indoles, neurotransmitters (e.g., GABA, DA, noradrenaline, and 5HT), and phytoestrogens (e.g., urolithins and equol), which play a major role in host nutrition, immune functionality, central nervous system function, and cancer protection, respectively.

factor, GM variations over time can have an adaptive or maladaptive nature, the first important to improve the host adaptive response to environmental and endogenous changes, the second driving disease-associated dysbioses.15 Past studies on the human GM highlighted the vast strain-level compositional diversity of this bacterial ecosystem, as well as the correspondingly high degree of genetic complexity.22 However, we are now beginning to appreciate another dimension of the GM complexity: the enormous biochemical diversity of this ecosystem, resulting in an astonishing potential to produce a vast range of bioactive molecules that are not represented in the human metabolome.23,24 We are beginning to understand the importance of this vast molecular repertoire for the human physiology, entering a new era in GM research, where descriptive studies give way to more mechanistic ones. In this scenario, we provide here a critical review of the main GM-delivered bioactive molecules (see Figure 1 and Supporting Information, Figure S1), describing their biological role in human health and disease. Subsequently, we discuss how this unique repertoire of small organic molecules may help identify innovative targets for drug discovery and may represent starting hits for medicinal chemistry campaigns toward the identification of GM-derived drug candidates for the treatment of a wide range of pathological

Though produced locally, molecular signals from the GM can reach extra-intestinal organs, establishing a system-level connection between the GM and the immune, endocrine, metabolic, and nervous apparatus. Indeed, a vast range of bacterial small molecules, produced in the colon at physiologically relevant concentrations (mM order),14 are absorbed by colonocytes and released into the bloodstream, reaching the liver through the portal vein. From the liver, these molecular actors are then released systemically to the peripheral venous system, in some cases after further chemical modification by the host. As a bacterial ecosystem, the GM is an intrinsically dynamic entity.15 For instance, its metabolic products, as well as its compositional and functional layouts, respond to the variation of several environmental and endogenous factors, such as diet, geography, and drug intake, as well as host inflammation and age.4,16−19 Dietary deviations are probably the main driver of the GM variability over time. Diet is indeed a potent modulator of the GM compositional structure, with ultimate repercussions on its metabolic potential.16 Moreover, by providing the GM with substrates for the biosynthesis of a multitude of small molecules, 20,21 the host diet controls the overall GM metabolome, performing a key role in defining the GM-mediated impact on the human biology. Independent of the triggering 49



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potent activators of GPCR43, with EC50 values ranging between 250 and 500 μM. Similarly, GPCR41 is activated in decreasing order of affinity by propionate, butyrate, and acetate, with the EC50 for propionate in the range of 12−274 μM. Since the average SCFA concentration in the colon lumen may range between 10 and 100 mM, it is reasonable to assume that the GPCRs are constantly saturated by the GM-produced SCFAs, and subtle variations in their concentration should not affect signaling.12 Moreover, butyrate and propionate have been reported, in rats, to activate intestinal gluconeogenesis (IGN) through two complementary mechanisms: (i) butyrate triggers IGN gene expression by increasing the cAMP concentration in colonocytes, and (ii) propionate activates IGN by regulating gene expression through a gut−brain neural circuit involving GPCR41 and itself can be converted into glucose by IGN.34 This last propionate-dependent mechanism of IGN is particularly strategic, providing the host with several metabolic benefits that ultimately regulate body weight, modulating the host glucose control by increased insulin sensitivity and glucose tolerance. Finally, butyrate also represents an important energy source for the host, providing 6−10% of the total daily energy requirement in individuals consuming a typical British diet.35 By functioning as a source of oxidative energy for intestinal epithelial cells (IECs), it is also fundamental to maintaining anaerobiosis in the gut lumen.36 Thus, in addition to its major contribution to the host energetics, butyrate is also of great importance in containing the aerobic expansion of potential pathogens (e.g., Enterobacteriaceae) in the gut. Outside the gut, SCFAs are potent modulators of the endocrine function of the white adipose tissue (WAT). In particular, acetate has been reported to promote antilipolytic activity in WAT through GPCR43 signaling, improving glucose and lipid metabolism.37 In parallel, by suppressing the intestinal transcription of the angiopoietin-like protein-4 (Angptl4), a circulating lipoprotein lipase inhibitor also known as fastinginduced adipose factor (Fiaf), SCFAs may result in increased lipid incorporation in adipocytes, as shown in mouse models.38 Finally, through the activation of GPCR41 and GPCR43, SCFAs control the production of the anorexigenic hormone leptin in adipocytes, as specifically demonstrated in mouse adipocyte cell lines and mouse adipose tissue in primary cultures.39,40 Mainly secreted by WAT, leptin plays a central role in the human basal metabolism, informing the brain of the whole-body nutritional status and regulating glucose homeostasis, insulin and GLP-1 secretion, and appetite.41 SCFAs are potent immune regulators. The mammalian immune system, especially the adaptive immunity, has evolved in parallel to the GM acquisition, probably in response to the need to maintain control over the vast array of microbial cells inhabiting our gut.42 On the other hand, our microbial counterpart has become an active and, in some ways, essential component of the host immune apparatus, being strategic for training the immune system during infancy as well as for its functional tuning for the rest of our lives.15,43 In this scenario, microbial SCFAs play a primary role.44 They are capable of finely calibrating several aspects of our immune biology, putting the GM at the connection point between diet and the host immune apparatus.45,46 Exerting their action locally and systemically, on both innate and adaptive components of our immune system, SCFAs are potent anti-inflammatory agents, which provide immunosuppressive action that is important to maintaining immune homeostasis throughout the body.11,12,47,48 As immune modulators, they act primarily through two convergent

conditions. In parallel, drug therapies aimed at inhibiting GMactivated signaling pathways relevant for disease pathogenesis can be implemented. With our work, we aim to consolidate a new perspective, in which a more rational understanding of the GM contribution to the human physiology will be translated into innovative GM-based precision medicine interventions.

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SHORT-CHAIN FATTY ACIDS, MICROBIOTA METABOLITES WITH A PLURIPOTENT ROLE IN HUMAN PHYSIOLOGY As end-products of the GM fermentation of dietary fiber, the short-chain fatty acids (SCFAs) acetate, propionate, and butyrate play a pluripotent role in human biology.25 SCFA concentration varies along the gut length, showing a higher value in the proximal colon (70−140 mM), which progressively declines toward the distal colon (20−40 mM).14 This gradient is the result of the progressive absorption of SCFAs by the colonocytes through the Na+-coupled monocarboxylate transporter SLC5A8. Absorbed SCFAs are then released into the bloodstream and reach the liver through the portal vein. From there, they can diffuse systemically to the peripheral venous system, where they reach concentrations in the μM range. As a preferred energy source for colonocytes, butyrate is mostly consumed locally, while propionate is metabolized in the liver. Thus, these two SCFAs occur at low concentration in the periphery. Conversely, acetate abounds in the peripheral circulation. Able to cross the blood−brain barrier (BBB), it can be considered the most “systemic” of the SCFAs.26 However, despite their low peripheral concentration, propionate and butyrate still retain the potential to control distant organs by activating hormonal and nervous systems.12 SCFAs are involved in all the main physiological functionalities of the human host, such as metabolic regulation, immune function, and the activity of the CNS. At the molecular level, they act primarily through the activation of the “metabolite sensing” G-protein-coupled receptors (GPCRs),27 which are expressed by several cell types throughout the body (including colonocytes but also immune cells, endocrine cells, and adipocytes).28 Remarkably, GPCRs represent the most important protein family from the drug discovery standpoint, as several currently available medicines target specifically receptors belonging to this family. Therefore, new chemical knowledge coming from GMderived SCFAs and from their mechanisms of interaction with targets can be key for identifying novel chemical scaffolds toward next generation binders of GPCRs. Another mechanism of action for SCFAs involves the inhibition of a further, quite important family of target proteins, histone deacetylases (HDACs).11,29 Via these mechanisms, SCFAs control, for example, several aspects of our metabolic homeostasis. SCFAs can indeed play an important role as signaling molecules, controlling the expression and secretion of the major appetite and glucose regulatory peptides, peptide PYY, and the incretin hormone glucagon-like peptide-1 (GLP-1), through enteroendocrine L-cells, as repeatedly demonstrated in mouse models.30−32 These enteroendocrine peptides are important regulators of the host metabolism: the first inhibits the gut motility, increasing energy harvest from the diet, and the second is an important antihyperglycemic hormone, with a substantial impact on pancreatic function and insulin release, in addition to playing a role in the central regulation of food intake by mediating satiety. SCFAs trigger the secretion of PYY and GLP-1 through the activation of GPCR41 and GPCR43, respectively, both of which are expressed on the Lcell surface.33 Specifically, acetate and propionate are the most 50



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shown to exert direct and indirect action on several components of our neuronal apparatus, such as the enteric nervous system (ENS), afferent nerves of the peripheral nervous system (PNS) (e.g., vagal sensory neurons, spinal sensory neurons, and intrinsic primary afferent neurons) and the CNS.28 For instance, by direct signaling to GPCR41, butyrate has been reported to modulate the activity of the ENS in mouse models.33 Moreover, butyrate can act on PNS neurons, which also express GPCR41, as shown in mice and mouse neuroblastoma cells.61,62 In particular, in the PNS, the activation of GPCR41 by SCFAs induces sympathetic activation via noradrenaline release, leading to increased energy expenditure and heart rate.61 Furthermore, butyrate and propionate have been reported to promote Tph1-mediated 5hydroxytryptamine (5HT, serotonin) biosynthesis by colonic enterochromaffin cells in mice.63 Contributing to both the colon and serum pool, gut-derived 5HT represents more than 90% of the total body amount and, in addition to its traditional role as neurotransmitter, may regulate diverse functions, including enteric motor function, secretory reflex, platelet aggregation, bone development, and cardiac function. Finally, in addition to being crucial to the strengthening of BBB integrity, through increased expression of the TJ protein occludin,64 SCFAs themselves can cross this barrier and modulate CNS neuronal activity by GPCR43 activation or HDAC inhibition, as demonstrated in rodent models.60,65 For instance, after crossing the BBB, acetate induces anorectic signals involving the glutamate−glutamine transcellular cycle and γ-amino butyric acid (GABA) production in the hypothalamic arcuate nucleus, reducing appetite via a central homeostatic mechanism.26 A comment is finally required on the wide variety of mechanisms of action SCFAs exert in the human body. Actually, SCFAs are very small molecules with sizes more similar to fragment-like rather than to lead-like compounds. Also because of their high concentration, such fragments are able to modulate a wide range of biological targets, thus triggering several responses of physiological and therapeutic relevance. Additionally, the simultaneous modulation of different targets could eventually be responsible for multitarget mechanisms of action, which are inherently related to the small size and promiscuous character of fragment-like molecules,66 and can be at the basis of their high efficacy in vivo. Furthermore, the many targets modulated by SCFAs may require structural biology and computational investigations to identify the binding mode of these molecules at their biological counterpart, thus helping the design and synthesis of SCFAs-based compounds for pharmaceutical research.

mechanisms: HDAC inhibition, epigenetically modulating the immune system function, and signaling through GPCR41, GPCR43, and GPCR109A, which are expressed in immune cells and IECs.27 First, the SCFA action is of primary importance in fortifying the innate immunity of the intestinal mucosa.11 Indeed, SCFAs have been reported to reinforce the IEC barrier, increase mucus production by goblet cells, and strengthen the tight junctions (TJs). Moreover, by supporting the production of the inflammasome-related cytokine interleukin (IL)-18 by IECs, as demonstrated in mouse models,49 SCFAs may contribute to the maintenance of epithelial integrity. Finally, by signaling to GPCR43 and GPCR109A, SCFAs have been reported in mice to control the activation process of the inflammasome,50 an essential component to maintaining immune homeostasis in the gut. The overall anti-inflammatory activity of SCFAs in the intestine also stems from their ability to directly modulate the pattern of cytokine production by IECs and immune cells in the lamina propria, such as dendritic cells (DCs) and the effectors, macrophages, and monocytes. Indeed, SCFAs reduce the expression of several proinflammatory cytokines, such as tumor necrosis factor (TNF) α, IL-6, and interferon (IFN) γ, while enhancing the production of the anti-inflammatory cytokine IL10 by DCs, as shown in human-derived cultures.51−53 These activities generally involve the activation of GPCR109A and GPCR41 by butyrate and propionate, respectively, but HDAC inhibition may also contribute to this modulatory process. For instance, by inactivating the nuclear factor κB (NF-kB) in peripheral blood mononuclear cells, macrophages, and DCs, HDAC inhibition may result in the down-regulation of the production of TNF-α.54,55 The SCFAs act on the local T cell populations, further consolidating the tolerance layout of the intestinal immune apparatus. In particular, through DC conditioning, SCFAs, mainly propionate and butyrate, have been reported, in mice, to enhance the naive CD4+ T cell polarization toward the antiinflammatory Foxp3-expressing (Treg) population, reducing the load of proinflammatory Th1 and Th17 cells.44,47,56 Further, SCFAs can regulate the DC chemokine production pattern. By reducing the expression of several proinflammatory chemokines, SCFAs may also mold the cell traffic within secondary lymphoid tissues, ultimately affecting the leukocyte recruitment process.57 Another local SCFA function that is strategic for maintaining intestinal homeostasis is the promotion of mucosal IgA secretion by B cells, as demonstrated in mice.47,58 Indeed, secreted into the intestinal lumen, these immunoglobulins bind to microbes, dietary components, and luminal antigens, preventing their direct interaction with the host. Circulating SCFAs can influence the biology of the immune system throughout our whole body. In particular, propionate has been reported to drive myelopoiesis in the bone marrow, where it can directly modulate the DC and macrophage biology.48 Moreover, in mouse models propionate has been shown to have the potential to shape the immunological environment in the airways by enhancing the hematopoiesis of DC precursors with an impaired ability to promote Th2 cell effector function.59 Finally, through GPCR43 binding, SCFAs have been reported in mice to be involved in the maturation and function of microglia,60 the resident macrophages of the brain, acting as the key players in the immune defense in the CNS. This function is particularly relevant for the SCFA-mediated prevention of neuroinflammation and associated diseases of the aging brain.9 A new and emerging role of SCFAs is their contribution to the functionality of the host nervous system. SCFAs have been

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BEYOND SCFAS, GUT-MICROBIOME-DERIVED SMALL MOLECULES MODULATE PHARMACOLOGICALLY RELEVANT TARGETS As an inside bioreactor fueled by dietary macronutrients, symbiont bacterial cells can produce numerous small molecules other than SCFAs through primary and secondary metabolic pathways.20,67 Like SCFAs, these small molecules, which can be either bacterial metabolites or structural components, can diffuse throughout the body, affecting organs either directly or by hormonal and neuronal signaling.68 First, intestinal microbes represent an important endogenous source of vitamins.69 To fulfill their metabolic needs, some gut microorganisms can produce menaquinone, folate, cobalamin, and riboflavin, which, from the host perspective, act as vitamins K2, B9, B12, and B2, respectively. These vitamins are known to 51



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be involved in several biological functions, from blood coagulation to bone metabolism and insulin sensitivity.70 Another class of GM-derived compounds with extraordinary biological importance for the human host are neurotransmitters. The GM can control the concentration of several neurotransmitters, including catecholamines and their derivatives, amino acids, and indolic compounds, by both direct and indirect action.28 For instance, enteric symbionts can produce GABA,71 the chief inhibitory neurotransmitter in the mammalian nervous system, with numerous physiological impacts along the GI tract, as it is implicated in intestinal motility, gastric acid secretion, gastric emptying, and perception of visceral pain.72 Analogously, some intestinal bacteria have been reported to be sources of adrenaline, noradrenaline, dopamine (DA), and 5HT.73 As mentioned previously, the production of 5HT in the gut by tryptophan hydroxylase has been shown to contribute significantly to its plasma and brain levels, with potential implications in several physiological functions, from intestinal immunity to visceral sensitivity and behavior. Finally, GM components have been reported to be able to synthesize and release another major neurotransmitter, nitric oxide (NO), by bacterial NO synthese,74 which might enhance host longevity and stress resistance in nonmammalian organisms.75 Further pharmacological and physiological studies, particularly at a molecular level, will be needed to more in depth investigate this mechanism in humans and more generally in mammals. Through the action of tryptophan hydroxylases and decarboxylases, or tryptophanases, the GM can produce a range of tryptophan metabolites in addition to 5HT, with an important role in modulating the host immune and metabolic homeostasis.63,76,77 In particular, tryptophan is metabolized by intestinal microbes to several aryl hydrocarbon receptor (AhR) agonists, such as indole, indole acetic acid, indole propionic acid, indole-3-acetaldehyde, and tryptamine.78,79 AhR is a ligandinducible transcription factor, expressed by immune cells and IECs, whose activation is essential to governing metabolic and immunological functions.28,80 Local AhR activation by GM metabolites can indeed engage gene expression for the modulation of important immune homeostatic factors, such as the production of IL-22 (a crucial cytokine for regulating epithelial cell repair), the transcription of antimicrobial peptides, the activity of Treg, Th17, and innate lymphoid cells, and the reinforcement of TJs, preventing permeability.11,27,48,68,70 Moreover, indole has been reported to specifically trigger GLP-1 secretion from mouse enteroendocrine L cells, regulating the host metabolic layout.81 Finally, further strengthening the strategic importance of these metabolites in the context of the GM−host mutualism, nicotinic acid, another microbe-dependent tryptophan metabolite, possesses well-known anti-inflammatory properties.82 On the other hand, p-cresol and p-cresol sulfate, which are generated from the GM metabolism of the other two aromatic amino acids, tyrosine and phenylalanine, have been associated with poor cardiovascular outcomes and chronic kidney disease, the latter as a result of the accumulation of toxic metabolites in the kidney.83,84 Another noteworthy GM-produced metabolite is lactate. Principally produced by the milk-fermenting GM ecosystem of breast-fed infants, bacterial lactate can exert important metabolic and regulatory effects,12 being an energy source and acting as an immune modulator, HDAC inhibitor, and signaling molecule. In particular, lactate is a natural ligand for GPCR81, inhibiting the cAMP-mediated intracellular signaling events governing lipolysis, as shown in an ex vivo assay employing mouse fat pads.85

By means of constitutive or inducible forms of amino acid decarboxylases, gut microbes can produce polyamines.86 These molecules are essential for several host functions, mostly linked to cell growth, survival, and proliferation; however, if produced at dysregulated levels, they can have detrimental effects on our health. For instance, at low levels, they have been associated with cell growth defects, while at higher concentrations, they can promote carcinogenesis.11,87 Conversely, a balanced production of polyamines, namely, spermine and spermidine, is critical to maintain IEC turnover and enhance the integrity of the IEC barrier. Polyamines can also stimulate mucin and IgA production, as demonstrated in rodents,88 playing a central role in the regulation of host immunity. In particular, spermine can inhibit macrophage polarization toward an inflammatory phenotype, decreasing the production of TNF-α and IL-6.89 Polyamines have also been reported in rats to mold the adaptive immunity, being important in accelerating the maturation of intraepithelial CD8+ T cells and lamina propria CD4+ T cells.90 Among other GM-derived metabolites, we would like to mention that intestinal microbes are potentially capable of producing a range of fatty acids with longer chain lengths than SCFAs, with different impacts on the host health. For instance, GM-produced long-chain fatty acids can modulate Th17 gene expression, forcing the balance between homeostatic and potentially pathogenic Th17 cells toward the latter, as shown in murine T cell cultures.91 In contrast, some GM members can conjugate ω-6 fatty acids to produce conjugated linoleic acid,92 which has been reported to have anti-inflammatory properties and increase insulin sensitivity, reducing adiposity, atherosclerosis, and carcinogenesis. These effects are likely related to its action on the peroxisome proliferator-activated receptors (PPARs) γ and α, cyclooxygenases, and lipoxygenases.93 Finally, yet importantly, protein fermentation by the GM can result in the production of branched-chain fatty acids (BCFAs), such as isobutyrate, 2-methylbutyrate, and isovalerate. Mainly originating from the branched-chain amino acids valine, leucine, and isoleucine, BCFAs have been associated with insulin resistance, as demonstrated in a groundbreaking study on mice transplanted with the GM from twins discordant for obesity.94 Up to this point, we have critically reviewed the importance of bacterial metabolites as GM signaling molecules. However, the gut microbes can also communicate with the host through their structural components, known as microbe-associated molecular patterns (MAMPs). MAMPs are bacterial cell components, such as lipopolysaccharide (LPS), peptidoglycan, flagellin, lipoteichoic acid, double-stranded RNA and DNA.95 Showing a specific variance for each GM group, MAMPs are recognized by host pattern recognition receptors (PRRs), which are expressed on the surface of IECs and immune cells. The main PRRs are Tolllike receptors (TLRs), NOD-like receptors, and RIG-I-like receptors. PRR activation by MAMPs results in the production of IL-18, which is involved in the regulation of the antimicrobial cell response.96 PRR signaling also regulates the circadian clock within IECs and adjusts the secretion of epithelial cell-derived metabolic hormones, such as glucocorticoids, as shown in mouse cells.97 Interestingly, TLRs are also expressed in the dorsal root ganglia, suggesting that GM-associated MAMPs might directly target enteric neurons to control local functions.98 For example, TLR2, which specifically recognizes the peptidoglycan, is expressed by ENS neurons, suggesting the potential of this bacterial structural component to regulate neurological processes.99 Notably, the myeloid differentiation primary response gene 88 (MyD88) is a central adaptor for the majority 52



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toward a full exploitation of the GM as a source of bioactive small organic compounds for pharmaceutical purposes.

of TLRs, which use it to activate NF-kB, and thus it plays a major role at the interface between the GM and the host. The presence of MyD88 in virtually all cell types suggests that during evolution, physiology and immunity have coevolved to shape the GM-host interaction.28 The final class of GM-produced bioactive compounds we want to mention is represented by small peptides with a primary role in bacteria−bacteria interaction processes, which in turn can affect the host biology. For instance, bacteria can communicate with one another through hormone-like signals that modulate gene expression in a process known as quorum sensing.100 This process can control several bacterial phenotypes, such as adhesion, biofilm formation, production of pathogenicity factors (e.g., toxins), and virulence.101,102 Among the bacterial quorum sensing mediators, the γ-butyrolactone derivative autoinducer 2 (AI-2) is the most “universal”, being involved in interspecies signaling and mediating interaction even among different genera. Produced by certain GM components, AI-2 levels have been reported in mice to influence the abundance of the major GM phyla,103 the balance of which is known to influence the overall GM metabolome layout, with potentially important repercussions on human physiology and health. For instance, AI-2 production by the GM member Ruminococcus obeum can hamper intestinal colonization by Vibrio cholerae, repressing the biosynthesis of several V. cholerae colonization factors.103,104 The hypothesis that bacterial quorum sensing mediators can modify even mammalian cell signal transduction has recently been advanced, opening the possibility of an interkingdom signaling process.100 Among other GM-derived bioactive proteins, we cannot fail to mention bacteriocins. As high potential peptide toxins, produced by some GM components, these molecules can exert potent and specific inhibition activity toward allochthonous microorganisms,105 protecting the host from colonization by pathogens and opportunistic pathogens. Very recently, a new family of GM-produced peptidic metabolites, i.e., dipeptide aldehydes, has been discovered. In particular, one of these compounds, Phe-Phe-H, has been demonstrated to exhibit potent and selective protease inhibitor activity against human cathepsins, mainly cathepsin L, revealing a possible role for lysosomal proteases in the GM−host interaction.106 This study represents a very inspiring example of small molecules derived from the GM, which may be useful as modulators of host targets with eventual pharmacological effects. Indeed, from drug discovery and medicinal chemistry perspectives, one may envision a scenario in which GM-derived metabolites can be utilized to identify new classes of small organic molecules with a desired pharmacological profile. Subsequent chemical campaigns can eventually optimize starting hit compounds derived from the GM toward more potent and selective modulators of certain classes of pharmacological targets. Additionally, experimental protocols such as that reported by Guo et al.106 can also be key from the target identification and validation standpoint, as GMderived metabolites can bind to undisclosed proteins, which may represent innovative targets for drug discovery. At this step, computational methods can play a twofold role: on the one side, they can help optimize GM-derived small organic molecules toward the discovery of leadlike and eventually druglike compounds; on the other side, reverse in silico screening can help fish potential targets, once a metabolite derived from the GM has been identified and chemically characterized. Tight interdisciplinary projects combining genomics and bioanalytics with computational biology and bioinformatics will be pivotal

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GUT MICROBIOME METABOLISM OF XENOBIOTICS AND DRUGS The GM has also the potential to metabolize numerous xenobiotics found in our diet, including natural products and chemical additives. This metabolism results in the production of a series of small bioactive molecules with a wide range of biological effects, from health-promoting to toxic.19,107 For instance, dietary plant polyphenols, such as isoflavones, lignans, and ellagitannins, are bioactivated by intestinal microbes to form substances collectively known as phytoestrogens, which, by binding to the estrogen receptors α and β, exert important protective effects against breast and prostate cancers.108−112 Specifically, isoflavones, such as daidzein, and lignans are activated by the GM to form equol and enterolignans, respectively, whereas fruit-derived ellagitannins are metabolized to urolithins, which possess well-known antioxidant, antiinflammatory, and anticancer activities.111 Analogously, plant glucosinolates are converted by the GM myrosinase to isothiocyanates,113 bioactive compounds that induce the production of host cytoprotective proteins by activating the Keap1-Nrf2-ARE pathway.114 Nrf2-regulated genes are involved in the biosynthesis of antioxidant proteins, drug-metabolizing enzymes, drug efflux pumps, and heat shock proteins, limiting oxidative stress and xenobiotic toxicity.115 In contrast, the GMdependent bioconversion of some dietary molecules may exert a detrimental impact on human health, breaking the mutualistic pact with the host. For instance, the GM can convert cyclamate, present in some noncaloric artificial sweeteners, to cyclohexylamine, which exhibits toxicity in animals and has been associated with bladder tumors.116,117 It is now a matter of fact that the GM has the potential to interfere with the first- and/or second-pass metabolism of several pharmaceuticals.19,118 In this scenario, the possible relevance of the personal GM layout in determining treatment response is emerging. For instance, the therapeutic efficacy of several azo bond-containing prodrugs (e.g., the antibacterial prodrug prontosil) requires bioactivation by microbial azoreductases in the gut, liberating the biologically active compound (triaminobenzene and sulfanilamide).119 The rate of microbial azo reduction may depend on the individual GM complement, with an ultimate impact on drug efficacy.120 On the other hand, gut microorganisms can also metabolize the downstream metabolites of azo reduction. For instance, 5-aminosalic acid, the bioactive component of the anti-inflammatory drug sulfasalazine, can be inactivated by GM arylamine Nacetyltransferases. The activity of these enzymes can vary up to 10 times between individuals, depending on the GM layout.121 Further, as a result of the GM-mediated reduction of the α,βunsaturated lactone ring, about 10% of the patients excrete high levels of the inactive metabolite dihydrodigoxin of digoxin, a drug generally utilized for the treatment of cardiac arrhythmia and heart failure.122,123 Finally, the interindividual variation in statin efficacy, with up to 30% of patients failing to reach lipid-lowering targets, is associated with the potential of the individual GM profile to metabolize primary bile acids.124 Indeed, primary bile acids may compete for the same intestinal transporters of statin, lowering its absorption. An emerging field of research is now exploring the interaction between the GM and cancer treatment. In particular, studies carried out in mice and patients have shown that the GM can act 53



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compound and augmentation of its genotoxicity, as shown in rats.131 Another noteworthy GM−host co-metabolic interaction involves the bile acid metabolism. It is indeed known that intestinal microbes are capable of deconjugating primary bile acids by means of bile salt hydrolase (BSH), preventing their uptake from the small intestine.13 The deconjugated bile acids are then further metabolized by gut microbes into secondary bile acids by means of their 7-dehydroxylation activity. In particular, the secondary bile acid lithocholic acid (LCA) is formed from chenodeoxycholic acid (CDA), and deoxycholic acid (DCA) is formed from cholic acid (CA). Another major microbial biotransformation of bile acids is the generation of oxo (or keto) bile acids by the oxidation of hydroxyl groups at ring position 3, 7, or 12.132 This reaction is catalyzed by bacterial hydroxysteroid dehydrogenases (HSDHs) and can ultimately result in bile acid epimerization.133 The GM metabolic activity on bile acids thus leads to increased diversity of the bile acid pool and, in general, facilitates their fecal secretion by enhancing hydrophobicity. However, a minor portion of secondary bile acids can be absorbed and reach the enterohepatic circulation, where they may function as signaling molecules.13 Indeed, in addition to their traditional role in dietary fat absorption, bile acids bind to cellular receptors, such as the GPCR TGR5 and the nuclear farnesoid X receptor (FXR).134,135 In particular, FXR tightly regulates the production of bile acids by negative feedback inhibition. The most potent FXR ligand is CDA, followed by CA, DCA, and LCA. By changing the bile acid pool in favor of secondary bile acids, intestinal microbes can thus affect both the metabolism and biosynthesis of bile acids in an FXR-dependent manner. Since balanced FXR signaling leads to improved glucose metabolism and body weight reduction,136 a homeostatic GM− host co-metabolism of bile acids is fundamental to our metabolic health, while its deregulation can induce adipose tissue inflammation and increased hepatic expression of genes involved in lipid uptake, as shown in mice.137 In contrast to FXR, TGR5 predominantly recognizes the secondary bile acids LCA and DCA. Ubiquitously expressed, TGR5 plays an important role as a host metabolic regulator, favoring the increased release of GLP-1, thereby improving liver and pancreatic function and controlling glucose homeostasis and energy expenditure in the brown adipose tissue, as demonstrated in mice and cell cultures.138 On the other hand, the GM−host co-metabolism of dietary choline, phosphatidylcholine and carnitine, abundant in seafood, cheese, eggs, and red meat, represents a clear example of unhealthy alliance between the GI microbial communities and the host. Intestinal microbes can produce high levels of trimethylamine (TMA) from choline through the action of TMA lyases. Once absorbed from the gut, TMA circulates to the liver, where it is oxidized to TMA N-oxide (TMAO) by host enzymes of the flavin monooxygenase family. TMAO is a metabolite that can affect the cholesterol metabolism by inhibiting hepatic bile acid synthesis, and it has recently been identified as a novel and independent risk factor for atherosclerosis and cardiovascular disease.68,139,140

as a potent adjuvant for anticancer immunotherapies, specifically immune checkpoint inhibitors.125,126 In particular, in mice reared under germ-free conditions or treated with antibiotics, the immune checkpoint inhibitors lost their therapeutic efficacy. Conversely, specific GM components, such as Bif idobacterium and Bacteroides, can promote the tumor immune surveillance processes, increasing the efficacy of anti-PD-1 monoclonal antibodies (or its ligands, PD-L1 and PD-L2). Finally, we should mention the antidiabetic drug metformin, which has recently been shown to alter the GM and to enhance the growth of mucin-degrading bacteria in diet-induced obese mice.127 This study has shown that this antidiabetic drug is able to select specific gut bacteria and that this mechanism could be at the basis of the eventual therapeutic effect of metformin in humans. In addition to its antidiabetic effect, it has been reported that this drug is also able to induce life-span in rodents and to alter bacterial folate and methionine metabolism.128 Further studies will be needed to investigate similar mechanisms in mammals and humans. In summary, all these studies have shown that there exists a biunivocal link between drugs and the GM. On the one side, the GM can metabolize drugs altering their absorption, metabolism and eventually impacting on their bioavailability and pharmacokinetics. With the GM being profoundly different in diverse patients, the future of personalized medicine should take into account the metabolic role of the GM in defining proper therapeutic options, as the GM can impact on DM/PK profile of drugs. On the other side, drugs can alter gut commensal community, opening up innovative ways for rational drug discovery that specifically targets the GM, with the final objective to increase the community of “good” bacteria over those that can have a negative impact on human health. In this context, next generation sequencing for genomics and meta-genomics analyses will play a key role in assessing the microbiological profile of new molecules targeting the GM.

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GUT MICROBIOME−HOST TRANSGENOMIC METABOLISM In some circumstances, the GM and host metabolisms integrate into a microbiome−host co-metabolic network, which may have extraordinary importance for the host biology. For instance, gut bacteria can affect the host detoxification pathway of glucuronidation. Through the action of uridine diphosphate (UDP)-glucuronosyltransferases in the liver, glucuronic acid is added to several substrates, including not only drugs and dietary compounds but also endogenously produced compounds, such as hormones and bile acids. This biotransformation interferes with the substrate’s biological activity and, by increasing its molecular weight and solubility, functions to favor its elimination through the urine by renal secretion or through the stools by biliary secretion. Possessing an intrinsic β-glucuronidase activity, intestinal microbes can interrupt this host detoxification process, liberating the parent aglycone from the glucuronide moiety, thus reactivating drugs and/or favoring the reabsorption of hormones and bile acids.19,129 Bacterial β-glucuronidases have also been shown to contribute to the association between the intake of heterocyclic amines and the risk of colorectal cancer (CRC). Indeed, these compounds, formed during the charring of meat, are normally detoxified by hepatic glucuronidation and the subsequent secretion of glucuronidated compounds in the intestinal lumen through the bile.130 At this point, the bacterial β-glucuronidases can intervene, favoring the release of the conjugate group, with consequent reactivation of the toxic

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CONCLUSIONS AND PERSPECTIVES In a mutualistic scenario, the GM is configured to provide the host with a vast range of bioactive molecules, which, complementing the human physiology, can confer on us the right metabolic, immunological, and neurological components to support health and well-being. Our increasing comprehension of the molecular actors and mechanisms involved is allowing us to 54



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unravel, for the first time, the GM imprint on human physiology. Bioactive molecules from symbiont gut microbes can cause the host metabolism to increase the energy harvest from the diet, favor energy storage, and induce postprandial satiety. In parallel, GM-derived molecules may shape our immune function, boosting the innate immune functionality on the one hand and favoring the immunological tolerance of the adaptive immune system on the other. Importantly, we are now beginning to see the potential of GM metabolites to control the most complex aspect of the human biology, the nervous system functionality. Even if the relevance of this functional imprint is not completely clear, we are now aware that GM-derived neuropeptides and fermentation products can regulate several aspects of the PNS and CNS functionality, such as gastric motility, visceral pain perception, appetite, and behavior. In particular, experiments conducted in mice have provided glimpses of how the GM can influence the host behavioral sphere.141 For instance, by producing important neurotransmitters, such as GABA and 5HT, and/or the SCFA butyrate, intestinal microbes can reduce anxiety and depression. This effect can be mediated either by direct action on the respective receptors or by epigenetic modulation. Further, by providing DA, intestinal microbes can activate the neuroimmunological circuitry, with a possible ultimate impact on the host psychomotricity. Finally, recent research carried out in rodents supports the importance of metabolites from the mother GM to drive a healthy trajectory of the offspring developmental program.9 Indeed, microbial signals in the early life, and probably even in the fetal environment, mold the rate of neurogenesis in the hippocampus, with an ultimate effect on the offspring behavioral attitudes. Interestingly, when these processes are perturbed, e.g., by antibiotic administration during pregnancy and/or in germ-free mice, behavioral deficits in the offspring are induced. All these studies clearly point to GM metabolites as a unique repertoire of small molecules and mechanisms of action, which could be investigated in depth from the computational and medicinal chemistry standpoint to launch drug discovery campaigns based on novel targets and hit compounds. We can also envision that certain target proteins in humans have been evolved to specifically bind small molecules derived from GM metabolism, as the genomics of host and GM have been modified throughout the evolution possibly in a concerted manner. We can therefore here ask the very provocative question: is there any orphan target in the human body able to univocally bind a metabolite specifically produced by the GM? In some circumstances, environmental (e.g., diet, infection, drug intake) and endogenous factors (e.g., inflammation and aging) can force the individual GM to deviate from its mutualistic configuration. This process results in functional and metabolic dysbioses, where the pattern of GM-produced biomolecules influences the host physiology toward a nonhealthy configuration, which can eventually establish a disease state. Fortunately, some steps toward the mechanistic comprehension of these processes have been achieved. For instance, it is now well established that excess consumption of animal protein can result in GM metabolites with a detrimental impact on the host health,142 such as cresolic compounds and TMA, both associated with poor cardiovascular outcomes, and BCFAs, which can trigger insulin resistance. Analogously, obese people consuming a high-fat, high-sugar diet show a dysregulated production of SCFAs, which could exacerbate the obesity phenotype by providing extra energy intake from the diet.20 High-fat diets can also support the increase of “bile-loving” GM components, such

as Bilophila wadsworthia. By producing an excess of H2S in the gut, these microorganisms can lead to increased permeabilization of the gut epithelium, supporting intestinal and systemic inflammation,143 which is the basis of many acute and chronic diseases of modern Western society. In light of these findings, it is thus tempting to speculate that the GM−host mutualistic pact was “signed” during the Paleolithic age, when human populations followed a hunter−gatherer lifestyle, consuming an essentially fiber-based diet with a salutary intake of game meat and no animal fat.144 However, we must point out that despite the many advances we have witnessed in recent years, the human GM still harbors a large amount of uncharacterized functional diversity, suggesting that only the tip of the iceberg has been explored. Indeed, of over 3000 biosynthetic gene clusters identified to date, the vast majority are predicted to encode for small molecules of unknown structure and function.24 This metabolic dark matter needs to be unraveled to further extend our comprehension of the GM contribution to the human physiology in health and disease. Another strategic frontier will be the dissection of the molecular mechanisms linking the GM metabolic diversity with the efficacy and toxicity of drugs. It is known that the GM can influence the metabolism and thus the pharmacokinetics of dozens of pharmaceuticals.19 However, the mechanisms involved have not always been described, and the impact of individual GM functional variation on the subjective response to pharmaceuticals needs to be better explored. On the other side, approaches of rational drug design could be specifically implemented to remove or modify chemical and functional groups known to be subjected to microbial metabolism in the gut, allowing minimizing the GM impact on the first-pass drug metabolism. Research in this direction will allow the implementation of more precise drug utilization, where the individual GM functional layout will be considered to define the best intervention strategy. In conclusion, it is now urgent to implement innovative strategies to predict and investigate the biological function of GM molecules in health and disease, as well as to reveal key molecules resulting from co-regulated microbiota−-host pathways as potential therapeutic targets. Such new strategies must synergize different and complementary approaches, such as functional -omics and systems medicine, with classical in silico drug discovery, target fishing and quantum biomolecular modeling. This approach will permit a deeper and more comprehensive understanding of the regulation of the human holobiont physiology, allowing the implementation of precision intervention strategies for specific steering of the GM contribution to the human health. Recent pioneer research has provided examples in this direction. For instance, being aware of the key role of gut microorganisms in the TMAO production from dietary choline, Wang et al. implemented a new target inhibition approach to inhibit the GM pathways responsible for TMA generation.145 On the basis of a structural analogue of choline, 3,3-dimethyl-1butanol, the authors provide a new potential therapy to “drug the microbiome” for the prevention/treatment of atherosclerosis. Analogously, the hypothesis of inhibiting GM-associated metabolites as a therapeutic approach to block signaling pathways associated with disease pathogenesis has been advanced, as in the case of type 2 diabetes associated metabolic inflammation.146,147 On the other hand, in a very recent publication, Guo et al. combined bioinformatics, synthetic biology, chemistry, and heterologous gene expression to expand our knowledge on the GM metabolic potential, providing 55



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evidence supporting the metabolic adaptation of certain GM components to survive in the intracellular niche.106 Indeed, the authors discovered and characterized the biological function of a new family of nonribosomal peptide synthetase gene clusters conserved in the GM. Coding for cell-permeable protease inhibitors (dipeptide aldehydes) specifically acting on the host cysteine protease system, these gene clusters provide some GM symbionts with the ability to occupy and emerge from a niche in the phagolysosome, providing evidence of a new (and unexpected) dimension of the biology of the GM−host interplay. From a medicinal chemistry perspective, an in depth understanding of molecular mechanisms and atomistic interactions between GM-derived metabolites and human targets can provide key information for chemical campaigns toward rational GMbased drug discovery. To this objective and in light of the very high diversity of commensal bacteria, interdisciplinary projects should be soon undertaken to properly detangle microbiota− metabolite−human relationships at a molecular level. The progressive understanding of the molecular processes governing the GM−host interaction in health and disease will enable novel therapeutic approaches based on metabolites secreted, modulated, or degraded by the microbiome.147 Dietary inputs, probiotics, or GM-targeting drugs can be designed to regulate precise GM metabolic functions for the production of specific metabolites. These interventions should be finely calibrated to the individual GM layout for a rational modulation of the ecosystem to provide the host with an adequate biomolecular layout to support individual metabolic, immune, and CNS health.

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Patrizia Brigidi is Professor of Chemistry and Biotechnology of Fermentations, and Delegate of the Rector for European Research at the University of Bologna, Italy. She graduated specializing in Chemistry and Pharmaceutical Technology (University of Bologna) in 1980. She was Associate Professor of Fermentation Chemistry and Industrial Microbiology (University of Catania) from 1991 to 1994. She was Director of the Institute of Advanced Studies (University of Bologna) from 2012 to 2015. She is currently responsible for the scientific activities of the National Agrifood Cluster, Area “Nutrition and Health”, and coordinator of the expert group of the University of Bologna working on actions bridged to Horizon2020 (KIC, JPI) and related activities. She leads a number of European and national projects focused on the intestinal microbiome for healthy aging and disease prevention. Andrea Cavalli is Professor of Medicinal Chemistry at the University of Bologna and Research Director at the Italian Institute of Technology, where he is also Deputy Director for Computation and Data Science. His research interests are in the field of computational drug discovery. In particular, he has developed and applied computational approaches to accelerate the discovery of druglike compounds in several therapeutic areas, including cancer, neurodegenerative diseases, and neglected tropical diseases. Prof. Cavalli is the author of more than 200 publications in high-ranked journals, is an inventor of several PCT international patents, and has delivered numerous invited lectures and seminars. In 2003, he was awarded the “Farmindustria Prize for Pharmaceutical Research”. He is cofounder of the high-tech startup company, BiKi Technologies. Marco Candela is Associate Professor of Chemistry and Biotechnology of Fermentations at the Department of Pharmacy and Biotechnology, University of Bologna, Italy. He is a senior PI of the “Microbial Ecology of Health” unit with research activities focused on the gut microbial ecology. He graduated specializing in Biological Sciences (University of Bologna) in 1999. He obtained a European Ph.D. in Biomedical Sciences (University of Bologna) in 2004. In 2003, he worked at the Institute of Microbiology and Biotechnology, University of Ulm, Germany, with Prof. B. Eikmanns as part of his Ph.D. program. He was postdoctoral research fellow at the Department of Pharmacy and Biotechnology (University of Bologna) from 2004 to 2014. He is scientific founder and member of the SpinOff company Wellmicro S.r.l.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00244. Figure S1 showing chemical structures of the main GMdelivered bioactive molecules (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +39-051-2099727. Fax: +39-051-2099734. E-mail: [email protected].

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ABBREVIATIONS USED

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REFERENCES

AhR, aryl hydrocarbon receptor; AI-2, autoinducer 2; BBB, blood−brain barrier; BCFA, branched-chain fatty acid; CA, cholic acid; CDA, chenodeoxycholic acid; CNS, central nervous system; CRC, colorectal cancer; DC, dendritic cell; DCA, deoxycholic acid; ENS, enteric nervous system; FXR, farnesoid x receptor; GM, gut microbiota; IEC, intestinal epithelial cell; IFN, interferon; IGN, intestinal gluconeogenesis; IL, interleukin; LCA, lithocholic acid; MAMP, microbe-associated molecular pattern; MyD88, myeloid differentiation primary response gene 88; PNS, peripheral nervous system; PRR, pattern recognition receptor; SCFA, short-chain fatty acid; TJ, tight junction; TMA, trimethylamine; TMAO, trimethylamine N-oxide; WAT, white adipose tissue

ORCID

Andrea Cavalli: 0000-0002-6370-1176 Marco Candela: 0000-0001-7420-790X Notes

The authors declare no competing financial interest. Biographies Silvia Turroni is Adjunct Professor and a senior postdoctoral research fellow at the Department of Pharmacy and Biotechnology, University of Bologna, Italy, where she works on the microbial ecology of the human gastrointestinal tract and characterization of probiotic strains. She graduated specializing in Pharmaceutical Biotechnology from the University of Bologna in 2004. She obtained a Ph.D. in Applied Biocatalysis and Industrial Microbiology at the University of Bologna in 2008. Since 2004, she has been Assistant Lecturer at the School of Pharmacy and Biotechnology, University of Bologna. She is cofounder and member of the SpinOff compay Wellmicro S.r.l., started in 2015, whose mission is to provide a service for the characterization of the gut microbiome in terms of compositional and functional structure and impact on human health.

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