Microbial metabolites: cause or consequence in gastrointestinal disease?

Abstract

Systems biology studies have established that changes in gastrointestinal microbiome composition and function can adversely impact host physiology. Notable diseases synonymously associated with dysbiosis include inflammatory bowel diseases, cancer, metabolic disorders, and opportunistic and recurrent pathogen infections. However, there is a scarcity of mechanistic data that advances our understanding of taxonomic correlations with pathophysiological host-microbiome interactions. Generally, to survive a hostile gut environment, microbes are highly metabolically active and produce trans-kingdom signaling molecules to interact with competing microorganisms and the host. These specialized metabolites likely play important homeostatic roles, and identifying disease-specific taxa and their effector pathways can provide better strategies for diagnosis, treatment, and prevention, as well as the discovery of innovative therapeutics. The signaling role of microbial biotransformation products such as bile acids, short-chain fatty acids, polysaccharides, and dietary tryptophan is increasingly recognized, but little is known about the identity and function of metabolites that are synthesized by microbial biosynthetic gene clusters, including ribosomally synthesized and posttranslationally modified peptides (RiPPs), nonribosomal peptides (NRPs), polyketides (PKs), PK-NRP hybrids, and terpenes. Here we consider how bioactive natural products directly encoded by the human microbiome can contribute to the pathophysiology of gastrointestinal disease, cancer, autoimmune, antimicrobial-resistant bacterial and viral infections (including COVID-19). We also present strategies used to discover these compounds and the biological activities they exhibit, with consideration of therapeutic interventions that could emerge from understanding molecular causation in gut microbiome research.

INTRODUCTION

New sequencing technologies based on culture-free methods and metagenomics have revealed that humans are colonized by trillions of microorganisms collectively termed “microbiota.” In the gastrointestinal (GI) tract, the combined genetic information contained within the microbiota (known as the microbiome) greatly exceeds that of their host (1, 2). Host interactions with symbiotic microbiota have attracted great attention over the past two decades, resulting in tremendous growth of publications studying the human gut microbiome. The National Institutes of Health (NIH) Human Microbiome Project (HMP) and European Metagenomics of the Human Intestinal Tract (MetaHIT) consortia played a major role in rekindling interest in the gut microbiome by developing culture-independent 16S ribosomal RNA (16S rRNA) and whole shotgun metagenomic (WGS) sequencing platforms to characterize bacterial community structure and composition in different parts of the human body (1, 3). In addition to bacteria, microbiota is also composed of archaea, fungi, protists, and viruses that inhabit different body sites, including the oral cavity, gut, lung, skin, and genitalia (4). In the GI tract, the majority of bacteria belong to phyla Firmicutes and Bacteroidetes, which represent over 90% of the phylogeny identified in this organ (3, 5). However, a substantial difference in microbiome composition is evident among healthy individuals (3, 5), and this diversity is especially significant among infants (6).

Since the host and its associated microbiome have coevolved, transkingdom interactions between eukaryotes and prokaryotes (human-microbiome interactions) exert great impact on the fitness and well-being of the host (7). It is now widely recognized that the microbiome can contribute to human health and disease, which has led some physician-scientists to see this symbiotic entity as a new “human organ” (8). Others have described humans as “holobionts” composed of microbial and human cells (9). In fact, it is now universally accepted that the gut microbiome acts as an accessory organ that plays a critical role in the digestion of food and production of essential vitamins, host immunity, and defense against pathogens (10–13). With state-of-the-art metagenomic, bioinformatic, and statistical tools being readily available, these methods are widely applied to study the diversity and composition of the gut microbiome in healthy versus disease states. These holistic studies have paved the way to understanding what constitutes a healthy microbiome profile among individuals as opposed to the disturbed or altered microbiome “dysbiosis,” which has been correlated to several diseases including diabetes (14), inflammatory bowel diseases (IBD) (15), cancer (16), asthma (17), obesity (14), pathogen colonization with organisms such as Clostridioides difficile (18), and neurodegenerative diseases including Parkinson’s and Alzheimer’s disease (19). Clear correlations established changes in microbiome structure and composition with several diseases, including the abundance, presence, or absence of specific microbial members linked to susceptibility to autoimmune disease or pathogenic invasion including COVID-19 or H1N1 influenza (20–22).

Despite the significant progress made in microbiome research, mechanisms that define host-microbiome interactions impacting human health are still poorly understood. Furthermore, there is still a lack of studies that move from simple associations or correlations between changes in microbiome composition with disease states to more reductionist questions that explore microbe-produced molecules, their functions, and mechanisms in host-microbiome interactions. However, bridging this knowledge gap is of utmost importance to exploit the power of the microbiome in disease prevention, diagnosis, and treatment. Since microbes interact with their host and the environment by producing specialized metabolites (also known as natural products), it is likely that these molecules are key players in modulating microbiome composition and/or phenotypic host responses. Recent bioinformatic investigations of bacterial genomes from human subjects disclosed that the microbiome genomes encode several small-molecule biosynthetic gene clusters (BGCs) with little information about their structures or biological functions (23, 24). In this review, we consider bioactive secondary metabolites produced by members of the microbiome that could cause disease or promote health in cancer, autoimmune, and drug resistance-associated diseases. We describe mechanisms and potential therapeutic interventions that could emanate from a better understanding of causation in microbiome-based research.

BIOSYNTHETIC GENE CLASSES AND SECONDARY METABOLITES FROM THE HUMAN MICROBIOME

The human body is colonized by microbial cells living inside and outside of our body sites with a total mass of ∼0.2 kg (25). Sender et al. (25) estimated the weight of the microbiota to be ∼0.2 kg based on microbial and human cells count and calculations revising past estimates. Our GI tract harbors ∼500–1,000 microbial species at any given time (26). The sum of these microbes contains genes that significantly exceed the human genome, offering greater genetic variety and unique biological capability that is lacking in the host genome (26). Some of these capabilities are offered by the presence of biosynthetic gene clusters (BGCs) or metabolic gene clusters (MGCs), which are genetic elements containing two or more genes encoding the biosynthetic machinery to produce primary and specialized metabolites (27, 28). These metabolites are of potentially great importance and can provide a deeper understanding of host-microbiome interactions because they are often produced in the gut at physiological concentrations and can access the bloodstream to influence human homeostatic function locally or at remote sites (29). Although few studies are reported, some pioneering work has helped to shed light on the diversity and classes of primary and secondary metabolites synthesized by the gut microbiome (23, 24, 30–33).

DIVERSITY AND DISTRIBUTION OF BIOSYNTHETIC GENE CLUSTERS IN THE HUMAN MICROBIOME

Recently, an interdisciplinary study combining techniques from chemistry, molecular biology, bioinformatics, and omics technologies revealed the main families of BGCs that encode natural products in thousands of bacterial isolates from different sites of the human body (23). More than 2,000 reference genomes constituting the human microbiome were comprehensively analyzed identifying over 14,000 BGCs belonging to diverse classes of metabolites including polysaccharides, nonribosomal peptides (NRPs), ribosomally encoded and posttranslationally modified peptides (RiPPs), polyketides (PKs), NRPs-independent siderophores, terpenes, and hybrid molecules. The 14,000 global BGCs were matched with metagenomic data obtained from 752 samples of the Human Microbiome Project to detect more than 3,000 BGCs representative of the small molecules encoded in the genome of the microbiota of healthy humans (23, 34).

The molecules and biological functions resulting from these BGCs are largely unknown, highlighting the knowledge gap in understanding their roles in mediating host-microbe interactions. The distribution of these 3,000 BGCs varies significantly across body sites with the gut and oral habitats having by far the greater diversity, showing their richness in the gut microbiome compared with other parts of the body (Fig. 1). For instance, a normal gut or oral cavity sample contains on average ∼500–1,000 BGCs, whereas the number in skin, airway, or genital samples is less diverse containing ∼30–100 BGCs (23). Saccharides are the most abundant BGCs in the microbiome, being especially enriched in gut and oral samples. In addition to saccharides are RiPPs, NRPs, and PKs, which occur in all the body sites. The majority of BGCs described above are encoded in the genomes of human bacterial phyla belonging to Bacteroides, Corynebacterium, Ruminococcus, Rothia, and Parabacteroides. These findings regarding the diversity and classes of BGCs in the human microbiome are in alignment with a recent bioinformatics study focusing on BGCs from the oral microbiome in health or disease (30). In this report, saccharides, RiPPs, NRPs, PKs, NRP/PK hybrids, and terpenes represented the main BGCs of the oral microbiome in oral health or in disease association with caries or periodontitis. Interestingly, ∼50% of the 4,915 BGCs in their study remains unknown reflecting the great potential to discover novel function in the human microbiome.

Figure 1.

Average distribution of biosynthetic gene clusters (BGCs) present in samples from a typical healthy individual from the Human Microbiome Project (23, 34).

SECONDARY METABOLITES FROM THE HUMAN MICROBIOME

To limit the focus of this section of the review, we summarize endogenous specialized secondary metabolites synthesized by members of the microbiota. We highlight genetically encoded bacterial secondary metabolites and consider in the next sections how these molecules or genes can potentially be accessed or used for better mechanistic understanding or microbial therapeutic interventions. Literature describing other important biologically active metabolites is reviewed elsewhere, including reports on short-chain fatty acids (SCFAs) (35, 36), bile acids (29, 37, 38), polysaccharides (39, 40), and tryptophan (or amino acid)-derived metabolites (41–43). Our focus is centered on secondary metabolites genetically encoded by the human microbiome, namely, ribosomally synthesized and posttranslationally modified peptides (RiPPs), nonribosomal peptide synthetases (NRPSs), polyketide synthases (PKs), PKS/NRPS hybrids, and terpene biosynthetic gene clusters (Fig. 2) (3, 23, 24, 30–32, 44–47).

Figure 2.

Examples of secondary metabolites isolated from the human microbiome and their biosynthetic origins.

RIBOSOMALLY SYNTHESIZED AND POSTTRANSLATIONALLY MODIFIED PEPTIDES

RiPPs are a major class of secondary metabolites produced by the human microbiome. They are common in bacteria and their occurrence is agnostic to bacterial sources (human, soil, plant, etc.). To produce RiPPs, a precursor peptide frequently containing ∼20–110 amino acids and possessing a signal and a leader sequence is synthesized by ribosomal enzymes. Diversity of RiPPs is mediated by posttranslational modification (PTM) of the precursor core peptide using the leader sequence as the recognition site. The PTM is achieved by decorating and modifying enzymes and usually involves a final cleavage step that releases the end product outside of the cell (48–52). RiPPs have been predicted using bioinformatics from the genomes of bacterial phyla of the GI tract including Firmicutes, Bacteroidetes, Actinobacteria, Fusobacteria, Synergistetes, and Proteobacteria (23, 47). The detailed biosynthetic mechanisms of RiPPs are described elsewhere (53, 54). RiPPs produced by the human microbiome can be subdivided into several subclasses that include lantipeptides (or lanthipeptides) (55), sactipeptides (48, 56), thiopeptides (23), and microcins or thiazole/oxazole-modified microcins (TOMMs) (57).

Lanthipeptides

Lanthipeptides (for lanthionine-containing peptides) are RiPPs containing thioether amino acids lanthionine (Lan) or methyllanthionine (MeLan). Two steps are required for the integration of Lan and MeLan during the biosynthesis of lanthipeptides. Serine/threonine moieties in the precursor peptide undergo dehydration to yield 2,3-didehydroalanine (Dha)/(Z)-2,3-didehydrobutyrine (Dhb), followed by the formation of thioether bridges by the Michael-type addition of Cysteine residues onto the dehydrated Dha and Dhb (53, 54). Lanthipeptides exhibiting antimicrobial properties are known as lantibiotics. Bioinformatic studies have revealed the presence of biosynthetic gene clusters encoding lanthipeptides in Firmicutes and Actinobacteria from different parts of the body (23, 30). Few lanthipeptides have been described from human-associated microorganisms as most were isolated from pathogenic bacteria or rare commensals. Some examples of lanthipeptides include mutacins (e.g., mutacin 1140), which are produced by Streptococcus mutans commonly inhabiting the human oral cavity (58, 59). Further examples include the lantibiotics BPSCSK characterized from the human-associated bacterium Blautia producta (55) and nisin O discovered from the human gut bacterium Blautia obeum A2-162 (60).

Sactipeptides

Sactipeptides (sulfur to α-carbon cross-linked peptides) belong to a new and growing class of RiPPs with important biological activities including antimicrobial functions. Key features in their molecular structures are intramolecular bridges between sulfur of cysteine moieties and α-carbon belonging to other amino acid residues (53, 54, 61). Only a small number of sactipeptides are reported and the majority are characterized in Bacillus species (61). Biosynthetic gene clusters of sactipeptides are also identified in the human microbiome and the secondary metabolites ruminococcin C (isolated from Ruminococcus gnavus) and streptosactin (isolated from Streptococcus spp.) represent examples of sactipeptides encoded by human-associated commensal bacteria (48, 56).

Thiopeptides

Thiopeptides are RiPPs characterized by a macrocycle backbone comprising several thiazole moieties and sometimes feature several dehydrated amino acids. Small molecules of this category have attracted wide scientific attention for decades owing to their antibiotic properties. A key characteristic of thiopeptides is the presence of a six-membered nitrogen-containing ring in their chemical structures (54). Thiopeptide-encoded biosynthetic gene clusters are predicted to be widely distributed in different sites of the human body and are found in members of the human microbiota, including Lactobacillus gasseri, Cutibacterium acnes, Enterococcus faecalis, Streptococcus downei, and S. sobrinus (23). Some of these biosynthetic gene clusters were investigated experimentally to discover small molecule thiopeptides, including cutimycin and lactocillin, which are produced by Cutibacterium acnes and Lactobacillus gasseri, respectively (23, 62).

Microcins and Thiazole/Oxazole-Modified Microcins

Microcins represent a subset of ribosomal peptides produced by members of Enterobacteriaceae (especially Escherichia coli) and display interesting biological properties. They are formed by a biosynthetic pathway that involves substrate transformation by complex posttranslational modifying enzymes. Thiazole/oxazole-modified microcins (TOMMs) are biosynthetically comparable with microcins but account for a larger group of small molecules produced by both Gram-positive and Gram-negative bacteria. Some well-known microcins or TOMMs from the human microbiota include microcins L and J25 produced by E. coli (63, 64) and clostridiolysin S produced by Clostridium botulinum (65). The biosynthesis and functions of microcins and microcin-related molecules have been reviewed elsewhere (53, 57).

NONRIBOSOMAL PEPTIDES

Besides saccharides and RiPPs, nonribosomal peptides (NRPs) represent a major class of natural products genetically encoded by the human microbiome (23, 30). NRPs encompass a large family of secondary metabolites of bacterial or fungal origin produced by complex, modular, and multidomain catalytic pipelines governed by enzymes known as nonribosomal peptide synthetases (NRPSs). These compounds exhibit medicinally relevant activities and several NRPs are currently approved for use as therapeutics, including antibiotics (e.g., penicillins and cephalosporins) and immunosuppressants (e.g., cyclosporine A) (66, 67). Unlike RiPPs which are ribosomally synthesized and modified posttranslationally, the biosynthesis of NRPs occurs independently of the ribosome or messenger RNA and represents a natural alternative process to the formation of peptide bonds, i.e., generation is opposite to typical protein synthesis (68). Furthermore, the structural complexity and diversity of NRPs are enhanced by cyclization, branched structures, or incorporation of nonproteinogenic amino acid residues bearing decorations such as N-methyl, N-formyl, hydroxyl groups, carboxylic acids, sugar moieties, or even occurring in D-isomeric forms (66, 69). These characteristics clearly distinguish nonribosomal peptides from their ribosomal counterparts. Several authors have reviewed the biosynthetic mechanisms of NRPs (68, 70–72).

Genomic and metabolomic investigations of NRPs from the human microbiome started only recently and the vast majority of BGCs predicted to encode NRPs have not yet been verified experimentally and the structure and function of these secondary metabolites remain largely unknown. These BGCs were identified in several microbial phyla of the human microbiome, including Actinobacteria, Proteobacteria, Bacteroidetes, and Firmicutes (23, 30, 31). The list of isolated and fully characterized NRPs from members of the human microbiota is short and includes humimycins discovered from Rhodococcus species genome sequence (44, 45), lugdunin isolated from Staphylococcus lugdunensis (73), and pyrazinones from Staphylococcus aureus (74).

POLYKETIDES AND PK-NRP HYBRIDS

Another large group of chemically diverse and biologically active small molecules of natural origin is polyketides (PKs). Polyketide-type secondary metabolites are characterized by different sources including plants, animals, and microorganisms, and several representatives of this group like erythromycin (antibiotic) (75), anthracycline (anticancer) (76), ivermectin (anthelmintic) (77), lovastatin (cholesterol-lowering agent) (78), and amphotericin (antifungal) (79) are prescribed as drugs. This remarkable group of compounds is synthesized by large and multifunctional enzymes known as polyketide synthases (PKSs). PKSs can be divided into three types, namely, type I PKSs, which are proteins comprised of repeated functional domains working together to elongate a carbon chain, occurring in both bacteria and fungi. Type II PKSs are primarily present in bacteria and are comprised of monofunctional peptides synthetizing diverse aromatic compounds. Type III PKSs are ketosynthase homodimers that are mainly present in plants and harbor coenzyme A as an attachment site for chain elongation. The nature, diversity, and functions of PKS domains (e.g., ketosynthase, acyl carrier protein, acyltransferase, thioesterase, and optional domains), and detailed biosynthetic mechanisms leading to polyketides have been thoroughly reported.

Although different in their sequences, PKS modules share similarities that can easily be recognized using bioinformatic mining of bacterial genomes (23, 24, 79–81). BGCs encoding polyketide-type molecules are identified within the human microbiome in bacterial phyla including Actinobacteria, Proteobacteria, Bacteroidetes, and Firmicutes. Noteworthy, most polyketide BGCs found within human microbial genomes are mixed with NRP assembly lines to form PK-NRP hybrids with the potential to generate more complex chemical scaffolds (23, 24, 30, 82–84). Despite the biosynthetic capacity of human-associated bacteria to produce PK/PK-NRP hybrids as inferred from recent metagenomic studies, only a handful of these small molecules have actually been isolated and characterized from human microbiota. This highlights a potential wealth of metabolites with unknown function and possible medicinal application left for discovery. Some examples of previously described compounds include reutericyclins and mutanocyclin, which are antimicrobial tetramic acid secondary metabolites synthesized by a cascade of PK-NRP enzymes in Lactobacillus reuteri and S. mutans (85, 86). Further studies disclosed mutanobactins A-D, which are antifungal and macrocyclic lipopeptides produced by PK-NRP assembly lines harbored in the genome of S. mutans (84, 87–91). Mutanofactin-697 is a PK-NRP hybrid exhibiting an intriguing novel chemical scaffold recently discovered from the chemically diverse bacterial strain S. mutans to potentiate adhesion and biofilm formation (92). Additional PK examples include wexrubicin, a type II PKS encoded aromatic compound that was generated through cloning of the BGC from the gut bacterium Blautia wexlerae DSM 19850 into the heterologous host Bacillus subtilis-168-sfp, and metamycins A-D obtained by heterologous expression of DNA sequences from the human metagenome in host Streptomyces albus (24).

Several well-studied PKs and PK-NRPs are found in microbe-associated pathogenicity. An example is colibactin, a specialized metabolite the presence of which is associated with colorectal cancer (93–97). Colibactin is produced by certain bacterial strains belonging to Enterobacteriaceae (e.g., E. coli and Klebsiella pneumoniae) harboring the 54-Kb gene clusters clb or pks. Clb⁺ E. coli strains synthetizing colibactin are generally present in the human colon, damage host DNA, facilitate tumor formation in experimental animal models, and are more abundant in patients with colorectal cancer than in healthy individuals (94, 95). The structural elucidation of colibactin, a long-standing chemistry challenge that remained for almost a decade because of colibactin instability and small quantities, was recently solved (97).

TERPENES

Terpenes represent the largest and most diverse class of specialized metabolites characterized to date. They comprise more than 70,000 small molecules distributed into more than 400 compound families primarily identified from plants and fungi (98, 99). Owing to their structural complexity, diversity, and functionality (e.g., defense metabolites, fragrance, taste, pheromones, vitamins, etc.), terpenes exhibit a wealth of biological activities and many among them (e.g., the anticancer drug taxol and antimalarial drug artemisinin) are currently on the front lines of clinical studies or are prescribed as drugs against several diseases (98–102). The biosynthesis of terpenes involves isoprene units [C₅: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are fused together to generate elongated (C₁₀, C₁₅, C₂₀, C₃₀)] and branched hydrocarbon chains in prenyl transferase enzyme-catalyzed reactions. These acyclic and linear precursors can undergo cyclization under complex mechanisms governed by enzymes called terpene cyclases (or terpene synthases) providing (poly)cyclic terpene backbones (103, 104). The basic (poly)cyclic terpenoid can be modified by oxidizing enzymes and the addition of moieties including sugars, amino acids, and fatty acids to provide further structural diversity and complexity (98).

Historically, bacteria were previously thought not to represent a major source of terpenes as only a fraction of compounds could be traced back to bacterial sources. However, recent genome mining and bioinformatic investigations of terpene BGCs in bacteria revealed genome-encoded information for the synthesis of thousands of terpenes with unknown structures and functions (98, 99). A variety of these bacterially derived terpene BGCs was identified from human metagenome samples and belong to Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes (23, 30, 98, 99). Nevertheless, terpenes have rarely been experimentally characterized from human-associated bacteria or indeed from any bacterial source. The majority of terpenoids identified from the microbiota are in fact not directly encoded by microbiome genomes, but rather represent microbial biotransformations of metabolites from dietary products or bile acid derivatives synthesized in the liver (29, 38, 105–107). One example of a genuinely microbiota-produced terpene is staphyloxanthin, an orange-red triterpene carotenoid-type compound discovered from S. aureus. Staphyloxanthin is one of the colored pigment metabolites found in S. aureus, which provided the bacterium with its nomenclature (108, 109).

BIOLOGICAL ACTIVITIES, FUNCTIONS, AND MECHANISMS OF ACTION OF MICROBIOME-DERIVED SECONDARY METABOLITES

Antimicrobials and Metabolites Associated with Colonization Resistance

Antimicrobial resistance (AMR) poses a serious public health threat that has gradually increased over the years with a high mortality rate estimated to reach 10 million deaths globally by 2050, with a worldwide financial burden of $100 trillion (110). For example, drug-resistant pathogens including vancomycin-resistant enterococcus (VRE), methicillin-resistant S. aureus (MRSA), and epidemic C. difficile are responsible for thousands of deaths yearly in the United States with an associated healthcare cost in billions of dollars (74, 110). There is an urgent need for new therapeutic molecules to tackle these unmet medical needs. Some of these pathogens, for example, S. aureus, S. mutans, or C. difficile, are often present in the human microbiome, where these microbes are in competition with commensal bacteria, expanding rapidly when there is disruption of the microbiota, caused by antibiotics or immunosuppression in patients (111). Although it is known that the gut microbiota plays an important role in protecting the host against invading pathogens in the GI tract, the molecular mediators of these interactions are largely unknown and can provide new functional metabolites or lead compounds for precision antimicrobial therapy. Several recent studies have begun to investigate the human microbiome for colonization resistance and functional antimicrobial compounds (Table 1) (23, 31, 44, 45, 48, 55, 56, 58–60, 62, 64, 65, 73, 86, 88, 90).

Table 1.

Examples of compounds with biological activity synthesized by human bacteria

Compound Or Extract	Compound Class	Bacterial Source	Literature
Antimicrobial agents
Mutanobactin A	PK-NRP hybrid	S. mutans	(83)
Lugdunin	NRP	S. lugdunensis	(69)
Humimycin A	NRP	Rhodococcus erythropolis	(41)
Streptosactin	RiPP	Streptococcus thermophiles	(44)
1-ethoxycarbonyl-β-carboline	Alkaloid	Lactobacillus species	(107)
Anticancer or cancer-promoting agents
Hemolysin BL	Protein	Bacillus toyonensis	(109)
Polysaccharide A	Saccharide	Bacteroides fragilis	(110)
Colibactin (DNA damaging)	PK-NRP hybrid	S. aureus	(90)
Immunomodulatory agents
Pyro-tryptophan	Dipeptide	Lactobacillus plantarum	(111)
Mutanamide	PK-NRP hybrid	S. mutans	(83)
Commendamide	Lipid	Bacteroides vulgatus	(42)
Antiviral agents
Supernactants of L. gasseri and L. fermentum (anti-HIV)	Uncharacterized extracts	Lactobacilli	(112)
Polysaccharide A	Saccharide	B. fragilis	(113)

HIV, human immunodeficiency virus; NRP, nonribosomal peptides; RiPP, ribosomally synthesized and posttranslationally modified peptides; PK, polyketide.

Mutanobactin A is a hybrid PKS-NRPS compound characterized by coculture experiments in which the antimicrobial activity of mutanobactin-competent S. mutans was compared with a mutanobactin deletion mutant strain against the fungus C. albicans. It was observed that S. mutans producing mutanobactin A was able to maintain C. albicans in its yeast morphological state, whereas in the presence of S. mutans lacking the mutanobactin gene, C. albicans was able to form mycelia, which represent the invasive state of the fungus (88). Another recent study determined that the reutericyclin family of PKS-NRPSs is used by S. mutans to inhibit the growth of neighboring oral commensal bacteria before colonizing the host environment and causing disease (86).

Lugdunin was found to exhibit significant antibacterial activity against several Gram-positive pathogenic bacteria including MRSA, VRE, and glycopeptide-intermediate resistant S. aureus with minimum inhibitory concentrations (MICs) ranging from 1.5 to 12 μg/mL. This compound is produced by oral S. lugdunensis strains and causes bacterial cells to stop incorporating DNA, RNA, protein, or cell-wall precursors, suggesting an antimicrobial mechanism involving rapid collapse of the bacterial energy machinery (73). Individuals colonized by S. lugdunensis carry a reduced load of S. aureus, indicating that lugdunin or lugdunin-producing human bacteria could be important in inhibiting colonization by infectious Staphylococcus (73).

Humimycins are potent antibiotics discovered from sequence mining of BGCs and chemical synthesis of predicted molecules encoded in Rhodococcus species, especially Rhodococcus erythropolis, which is widely distributed across oral and nasal cavities in humans. Humimycin A exhibited antibiotic activity against methicillin-resistant S. aureus (MRSA) strains with MIC values ranging from 8 to 128 μg/mL. The humimycin antibiotic acts by inhibiting lipid II flippase and potentiating β-lactam activity against MRSA (45). Claesen et al. (62) recently functionally characterized a BGC from C. acnes, a commensal bacterium harbored by the human skin and is widely spread across subjects and skin parts, identifying a thiopeptide antibiotic named cutimycin. This antibiotic showed activity against MRSA and Staphylococcus epidermidis strains with MICs ranging from 0.2 to 0.8 μM, and reduced the ability of Staphylococcus species to colonize skin hair follicles (62). Further examples of thiopeptide antibiotics from the human microbiome include lactocillin, a compound identified by mining encoded BCGs in the genome of Lactobacillus gasseri, a bacterium commonly found in the vagina microbiome. The encoded lactocillin molecule demonstrated activity against a wide range of pathogenic bacteria (e.g., Staphylococus aureus and Enterococcus faecalis) and was inactive against several vaginal commensals, suggesting a protective role of this compound in the urogenital tract by preventing colonization by harmful bacteria (23).

In further recent studies, the two sactipeptides, ruminococcin C and streptosactin, were described as antibiotics derived from the human microbiome (48, 56). Ruminococcin C was isolated from the gut commensal R. gnavus and exhibited anticlostridial activity against Clostridium perfringens. Streptosactin, found in a strain of Streptococcus thermophilus, was described as the first example of a fratricidal compound from a member of the human microbiome. It completely inhibited the growth of S. thermophilus LMD-9 and S. thermophilus LMG 18311 at 1 μM (48). Ribosomally synthesized antimicrobial natural products produced by bacteria are generally referred to as bacteriocins, which include lantibiotics and microcins. Detailed reviews about bacteriocins from the human microbiome have been published (49, 57, 110, 112).

MICROBIOME-DERIVED ANTICANCER OR CANCER-PROMOTING MOLECULES

Dysbiosis is associated with several cancers, including cervical (113), pancreatic (114), colorectal (115), oral (116), and gastric cancer (117). It is proposed that the microbiome plays a role in tumorigenesis by inducing inflammation, DNA damage, and programmed cell death, or by adversely influencing the host immune and/or tumor response to treatment (114). The potential contribution of specific bacterial members of the microbiome in the pathogenesis of cancers through proinflammatory metabolic interactions with human organs or DNA damaging effects has been reported (118–120). It is becoming evident that the collective metabolism products of the intestinal microbiota (or of specific bacterial members of the microbiota) strongly influence host immunity against cancer, as well as in the development of pathology or susceptibility to disease (119, 121). The anticancer roles and mechanisms of action of metabolites derived from the transformation of dietary products and host metabolites by microbiota have been reviewed elsewhere (121–124). These metabolites are not directly synthesized by bacteria and include short-chain fatty acids (SCFA), indole derivatives, polyamines, and bile acid derivatives (121, 123, 124). For instance, SCFAs produced by the healthy human bacterium E. coli KUB-36 strain exhibited anticancer and anti-inflammatory activities by affecting the inflammatory reactions in lipopolysaccharide-induced THP-1 macrophage cells and by suppressing the proinflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α whil promoting the expression of anti-inflammatory cytokine IL-10 (123). Some proteins (e.g., hemolysin BL reported from Bacillus toyonensis) and polysaccharides (e.g., polysaccharide A from Bacteroides fragilis) directly synthesized by specific members of the microbiome exhibited beneficial effects against cancers by modulating the immune response (125, 126).

Despite growing evidence that the microbiome can protect against or contribute to the development of cancer, most natural products or secondary metabolites (small molecules covered in this review) directly synthesized by the microbiota were not screened for antitumor activity. This can be explained by the functional roles of microbial metabolites in the defense against microbial invasion, for own-species survival, and competition with other species. Mechanistically, these metabolites have the potential to shape microbiome diversity of bacterial communities harbored by different individuals (49, 112), as well as signals to the host. Nevertheless, few studies have elucidated the roles of bacterially produced secondary metabolites from the human microbiome, especially genotoxins (e.g., colibactin), in causing cancer or indeed in shaping microbiome community structure or diversity. Lactobacillus casei strains possess activity against colonic cancer cells (93, 96). The small molecule responsible for the antitumoral activity of L. casei was identified to be ferrichrome, a siderophore nonribosomal peptide with tumor-suppressive activity. Ferrichrome application induced programmed cell death (apoptosis), involving the activation of c-jun NH₂-terminal kinase as a protective mechanism (127).

IMMUNOMODULATORY MOLECULES

Active bidirectional interactions between the gut microbiome and immune system are essential in maintaining host homeostasis. However, changes in microbiome composition or perturbation of the immune system can lead to autoimmune diseases and metabolic disorders, including inflammatory bowel disease (IBD), type-1 diabetes, and rheumatic arthritis (22). Microbiota-derived immunomodulatory molecules are reported to be implicated in the signal interplay between microbes and the immune system. In this section, we consider natural products produced by the microbiota that exhibit immunomodulatory activity, especially those implicated in the protection against or in the development of autoimmune diseases.

Lactobacillus plantarum is found in the intestinal microbiota and is known for its anti-inflammatory therapeutic benefits and is used as a microbial component in several probiotic preparations (128). L. plantarum is reported to play a role in the prevention or attenuation of IBD and this role is proposed to be mediated by the secretion of immunomodulatory agents (129). Four pyroglutamic (pyro) dipeptides including pyrophenylalanine, pyro-leucine, pyro-isoleucine, and pyro-tryptophan were identified from L. plantarum and associated with its immunomodulatory activity (128). Among them, pyrophenylalanine and pyro-tryptophan were evaluated for immunomodulatory activity in vivo in mice. These two small molecules were able to decrease the secretion of proinflammatory cytokine interferon (IFN)-γ, a key humoral regulator in the maintenance of homeostasis.

The natural product N-acyl-3-hydroxypalmitoyl-glycine trivially named commendamide is a metabolite produced by the human-associated bacterium Bacteroides vulgatus. Commendamide was found to activate G protein-coupled receptors (GPCRs), especially GPCR G2A/GPR132, which plays a signaling role in autoimmune diseases and atherosclerosis (46). Further studies with S. mutans revealed that mutanobactins A and B and mutanamide displayed immunomodulatory activity. In particular, mutanobactin B significantly enhanced the production of IL-6 and IL-12 pro-inflammatory cytokines but reduced the production of MCP-1, G-CSF, and TNF-α in an assay using lipopolysaccharide (LPS)-stimulated macrophages (84). The immunomodulatory activity of microbiota-derived proteins and polysaccharides or microbiota-derived biotransformed molecules including SCFAs has been recently reviewed (130).

ANTIVIRAL MOLECULES

A rapidly growing field in microbiome-based research is the study of host-microbe interactions in the presence of viral infections, including severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2, the viral cause of coronavirus disease 2019 (COVID-19)] (131), influenza virus (132), human immunodeficiency virus (HIV) (133), and human papillomavirus (HPV) (133). Viruses are biological agents that require host cell protein biosynthetic assembly to replicate. They usually penetrate the host through the GI tract, skin, nasal, or genitalia, organs that are all colonized by host microbiota (134). In interactions reported between the microbiota or microbiota products and pathogenic viruses, the microbiota can promote viral infection or inhibit it. Some members of the microbiota can inhibit viral infection by preventing the adhesion of viruses or by activating antiviral immune defenses (132, 135, 136). Conversely, in other cases, the microbiota could help viral agents escape host immune defenses by presenting excess lipopolysaccharide (LPS) and activating immunotolerance or by facilitating adhesion of virus to host cells (134, 136). In the former scenario preventing viral infection, host microbiota-produced metabolites are likely mediators of the observed antiviral effects.

Lactobacilli were reported to protect mice against H1N1 influenza infection (132), or inhibit HIV and herpes simplex virus (HSV) infection (133). The neutral pH supernatants obtained from the liquid culture of vaginal L. gasseri and L. fermentum were not toxic and demonstrated inhibitory activity against HIV replication. Supernatant from coculture of L. gasseri, L. rhamnosus, and L. crispatus inhibited HSV replication (133). Although in these studies the authors did not investigate the chemical composition of the supernatants, they postulated that small molecules secreted by Lactobacilli are most likely responsible for the virucidal activity by blocking the attachment of viruses to cells or by simply killing them. This proposed mechanism of action is different from the hypothesis that Lactobacilli neutralizes viral agents by lowering the environmental pH (133). Even though the investigation of antiviral small molecules from human-associated bacteria is in its infancy, natural products extracted from soil and marine bacteria demonstrated antiviral activity (137, 138).

Recent studies exploring the relationship between COVID-19 symptoms and the human microbiota reported differences between the microbiota of healthy human subjects and patients with COVID-19 (139–142). The microbiota can determine the severity and susceptibility to COVID-19, as well as the response to the treatment or vaccination against the disease. In most of these interactions, the underlying molecular mechanisms are poorly understood, and it is highly feasible that medications, e.g., antibiotic use, represent a major confounder in appropriately deciphering protective mechanisms in these studies. In a study using fecal samples, it was observed that out of 34 investigated bacterial phyla, Firmicutes and Bacteroidetes represented the major bacterial phyla where the abundance varied significantly between healthy individuals and subjects infected with COVID-19 (139). The occurrence of changed bacterial members in the Firmicutes, including bacterial genera where Ruminococcus, Butyrivibrio, Lachnoclostridium, and Dorea, showed significantly reduced relative abundance in subjects with COVID-19. On the other hand, the relative abundance of Bacteroidetes members, including Bacteroides coprophilus, Bacteroides coprocola, and Bacteroides graminisolvens, was augmented in patients infected with COVID-19 (139). Moreover, microbiota-related harmful small molecules were examined in fecal samples, where phenylacetylglutamine, salsolinol, and uric acid were more abundant in patients with COVID-19 (139). A separate microbiome study performed on nasopharyngeal samples from healthy individuals and individuals with COVID-19 revealed that commensal microbes including Gemella morbillorum, Gemella haemolysans, and Leptotrichia hofstadii were notably decreased in patients with COVID-19 while the abundance of bacteria such as Prevotella histicola, Streptococcus sanguinis, and Veillonella dispar was higher (141). Comparative metabolomic investigations of sera (blood, urine, nasopharyngeal swabs) obtained from the same subjects indicated that chlorogenic acid methyl ester was significantly reduced in patients infected with COVID-19 compared with healthy subjects, and this might hint toward an anti-SARS-CoV-2 small molecule (141).

Another line of study of the human microbiota and viral infection resides in the virus-microbiome-immune interplay. For example, the microbiome-immune cross talk can induce the production of type I interferons (IFN-Is), acting to defend against viral infections (131). IFN-Is have an important role in antiviral immune responses by mediating antiviral mechanisms in cells or inducing apoptosis in compromised tissues. The reduction of IFN-Is production following viral infection correlated with susceptibility or severity of disease (131, 135, 143). However, there is a lack of knowledge about which immune cells and small molecules are produced by specific taxa or how these molecules modulate antiviral immune responses. Stefan et al. (135) reported that glycolipids found on the membrane of specific taxa can activate the production of IFN-Is. Taking polysaccharide A isolated from B. fragilis, they demonstrated that IFN-Is production by dendritic cells was mediated through TLR4-TRIF signaling, and the antiviral potency of this bacterial-derived molecule against vesicular stomatitis virus or influenza virus depended on the expression of IFN-Is (135). Elsewhere it was reported that desaminotyrosine, a bacterially produced metabolite following degradation of dietary flavonoids induces protection against influenza virus in mice via expression of IFN-Is. C. orbiscindens, a member of the gut microbiota, degrades flavonoids to yield desaminotyrosine (144). The microbiota can also transform host secreted molecules, such as bile acids to mediate antiviral immune responses. In fact, the gut bacterium C. scindens or its biotransformed metabolite deoxycholic acid can protect mice against chikungunya virus infection via the plasmacytoid dendritic cells (pDCs)-IFN signaling axis resulting in restriction of virus propagation and potential resistance to virus transmission (143).

Overall, the literature lacks reports of protective effects of secondary metabolites synthesized de novo by members of the microbiota that exhibit antiviral activity. The role of SCFAs on viral infections has been reviewed elsewhere (145).

RESEARCH STRATEGIES USED TO DETERMINE MOLECULAR CAUSALITY AND EFFECTOR MECHANISMS OF THE HUMAN MICROBIOME

Three main strategies are generally used to identify and characterize biologically active small molecules generated by the human microbiome, namely, 1) phenotypic screenings based on bioactivity-guided fractionation of functional small molecules, 2) comparative untargeted metabolomics, and 3) gene-to-molecule discovery (genome mining) or combinations of the above.

BIOACTIVITY-GUIDED FRACTIONATION

Bioactivity-guided fractionation has been the method of choice for the separation of natural products with biological activity from plants and microorganisms for several decades and many drugs in clinical use were discovered by this technique (146). In this approach, an active fraction is obtained by extracting a bacterial culture with an organic solvent or a mixture of solvents, and then evaluated for biological activity (e.g., in phenotypic assays based on cells). When an extract exhibits activity, it is fractionated based on the physicochemical properties or polarity of molecules it contains to reduce the complexity of the extract and to enrich the concentration of bioactive molecule(s) in the fraction(s) by eliminating antagonistic effects or undesirable/inactive molecules (Fig. 3). Each fraction is tested for biological activity and the molecule(s) present in the most active fraction are purified by chromatography, chemically characterized by spectroscopy, and studied for more detailed biological activity and mechanism of action. The strength of bioactivity-guided fractionation is that it is driven by the biological relevance of the isolated compounds and when successful can directly hint toward mechanistic insights.

Figure 3.

Bioactivity-guided fractionation: example for antimicrobial compounds discovery.

Bioactivity-guided fractionation was used to characterize an inflammatory molecule from the extract of R. gnavus, a human gut bacterium that has been linked to Crohn’s disease (147). The active molecule, a polysaccharide derivative, induced the expression of proinflammatory agents such as TNFα produced by dendritic cells and provided one possible mechanism for the association between R. gnavus and inflammatory disease (147). Another example based on bioactivity-guided fractionation is the identification of the antitumor molecule hemolysin BL from a fraction obtained from the supernatant of B. toyonensis BV-17, a member of the healthy human gut microbiome (125). In this study, the genus Bacillus was associated with healthy subjects and was absent in patients with colorectal cancer as revealed by 16S rRNA comparative metagenomics of the microbiome in both groups. Hemolysin BL inhibited tumor growth in mice, providing a possible molecular explanation for the susceptibility to colorectal cancer and a potential therapeutic lead that can be further developed (125). Recently, 1-ethoxycarbonyl-β-carboline produced by Lactobacillus species was identified by bioactivity-guided fractionation as a bioactive compound against the opportunistic pathogen C. albicans (148). This molecule hinders C. albicans filamentation.

COMPARATIVE METABOLOMICS

Metabolomics is a powerful strategy to simultaneously examine several molecules in biological samples. It is based on analytical techniques (e.g., LC-MS and NMR) and most of the time combined with bioinformatics to rapidly compare differential metabolite compositions of samples or to analyze large data sets (105, 146, 149). Databases are used to rapidly identify or annotate the metabolites or compound classes correlating with the observed phenotype or activity without the need for tedious compound isolation and the novo structural elucidation. Metabolomics can be used to obtain information about the chemical composition of microbial extracts and fractions, thus facilitating extraction strategies or prioritizing fractions for isolation and characterization when exploring new functional molecules (128). This method can also be applied to compare the metabolite profiles (metabolome) of different samples (e.g., extracted from feces, urine, sera, nasal swabs) from healthy and disease subjects to determine molecules present or absent in study samples (Fig. 4), thus providing candidate molecular mechanisms and disease biomarkers (141). In general, targeted metabolomics focusing on a set of known metabolites (e.g., SCFAs, bile acid derivatives) and using available standard compounds has been successful in linking the abundance of a metabolite or set of metabolites to a particular phenotype. However, untargeted metabolomics considering unknown and more complex specialized metabolites (e.g., terpenes, RiPPs, NRPs) directly synthesized by microbes is still in its infancy.

Figure 4.

Comparative metabolomics for biomarker discovery in microbiome research.

Metabolomics has been used in a variety of experimental investigations. Zvanych et al. (84) used metabolomics to explore the products of NRP BGCs in S. mutans UA159. They applied comparative metabolomics and synthetic biology using wild-type S. mutans UA159 containing NRPs and mutant strains with deleted NRP genes to identify more than 50 molecules, of which some were structurally elucidated and evaluated for their immunomodulatory properties (84). Franzosa et al. (150) performed untargeted metabolomics and metagenomics analysis of stool samples from patients with IBD and without IBD to identify species, compounds, and compound classes that are upregulated in IBD, namely, sphingolipids and bile acids, and downregulated metabolites including triacylglycerols and tetrapyrroles (150). The taxonomic and metabolomic associations provided an understanding of the microbiome-metabolome dysbiosis in IBD. Feng et al. (151) used metabolomics to understand the link between serum molecules and the colonic microbiome in chronic kidney disease (151). Detailed reviews about the use of metabolomics in the human microbiome have been published elsewhere (149, 152, 153).

GENE-TO-MOLECULE DISCOVERY OR (META)GENOMIC MINING

Gene-to-molecule discovery encompasses a set of techniques using metagenomics or genomic sequences to identify and predict the biosynthetic products of genes that are responsible for the production of specialized metabolites (27, 82, 154). With readily available bacterial genome sequences since the early 2000s, these techniques have been applied in the field of natural product identification to tackle problems arising from the inability to culture many gut bacteria or due to silencing of biosynthetic gene clusters under culture conditions. General concepts have been extensively reviewed elsewhere (82, 146, 154, 155). Generally, computational approaches (e.g., anti-SMASH) are used to identify BGCs or family of BGCs, followed by the heterologous expression of the identified gene in a genetically tractable and easy-to-culture organism (e.g., E. coli), and extraction and chemical characterization of the produced metabolite using analytical approaches (HPLC, MS, NMR, etc.) (28, 80, 81). In the context of the human microbiota where a vast number of bacteria has been sequenced, genome mining has been applied to microbiome sequences to identify new functional molecules (156). Metamycins A-D (24), commendamide (46), and lactocillin (23) are some examples of bioactive natural products discovered from primary sequences of human microbiota members by combining bioinformatics, (meta)genomic mining, synthetic biology, and heterologous expression of BGCs and analytical chemistry (Fig. 5).

Figure 5.

Mining and characterizing BGC products through heterologous expression or chemical synthesis. BCG, biosynthetic gene cluster.

Genetic manipulation and heterologous expression of BGCs are not trivial since the experimentalist needs to manipulate large genes and identify or create a suitable host that fulfills all of the requirements to produce the targeted metabolites in culture (146). To bypass the need for microbial culture and heterologous expression, Chu et al. (45) developed an approach (synthetic-bioinformatics natural products, syn-BNPs) in which the biosynthetic products of bioinformatically predicted gene clusters are chemically synthesized (45). Humimycins are examples of antibiotic NRP natural products discovered from the human microbiome using syn-BNPs (31, 45).

Overall, the three main strategies described in this section combined with physiologically relevant biological assays can help to expand the chemical space of the human microbiome, thereby providing molecular and mechanistic information with potential therapeutic significance.

PHARMACOLOGICAL SIGNIFICANCE AND THERAPEUTIC INNOVATIONS

With findings in the past decade shedding new light on the role of the microbiome impacting human health, this has become a “new organ” for targeting innovative therapeutic interventions with a promising future market (8, 157, 158). The recent decade has seen rapid growth of microbiome-based projects in academia and biotech companies focusing mainly on gut bacteria. The presence or absence of defined members of the microbiome can be associated with health or disease states (26, 34). This hypothesis is the main driven force of microbiome-centered therapies, of which probiotics and fecal microbiota transplantation (FMT) to treat recurrent C. difficile infection (CDI) are front-line microbial interventions that promote a healthy gut microbiota (159).

Probiotics are preparations containing live microbes (natural or engineered) that are claimed to have health benefits on the host when administered. FMT is the process of delivering stool from a healthy donor into the GI tract of a patient. Only FMT restores the gut microbiota, which is used as a treatment for recurrent C. difficile infection (CDI). FMT is considered a drug in the United States and is clinically used under a special policy of the Food and Drug Administration (FDA) without full approval (159). To date, probiotics are mainly considered and are sold as dietary supplements. Several companies including Rebioitx, Seres Therapeutics, Vedanta Biosciences, and NuBiyota are focusing on the development of probiotics or microbial-based drugs to treat diseases ranging from recurrent CDI to cancer, IBD, and obesity (160). Among microbiome-based drugs, SER-109 from Seres is the most advanced microbiome-based therapeutic candidate, being recently cleared for phase III clinical trials against recurrent CDI (160). SER-109 is a cocktail of purified Firmicutes and has been designated by the FDA as a breakthrough therapy. Other therapeutic approaches targeting the microbiome include prebiotics (food components or substances that promote the activity and growth of beneficial microbes), compounds that can modulate pathways in host-microbiome interactions, and next-generation narrow-spectrum antibiotics as previously reviewed (161–166). Recently, a microbiota-targeted dietary supplement intervention for children with malnutrition was conducted and the results demonstrated the benefit of this approach on growth stunted children over the duration of the clinical study (167). This study highlights a clinical example of applying a microbial therapeutic approach to human disease, in this case, malnutrition and failure to thrive.

Although microbiome-based drugs (e.g., probiotics/live biotherapeutics and FMT) represent a new therapeutic avenue, the challenges to their development and approval include a lack of strong and validated data about their quality (Fig. 6), safety, and efficacy; the lack of a proper characterization of the “healthy microbiome;” the poor understanding of their mechanism of action; and difficulty in meeting government safety regulations (168–170). For instance, there is continuing discussion about the long-term risks of FMT. Adverse side effects and mortality caused by transferred pathogens and weight gain are reported as health concerns associated with the FMT strategy (171). Furthermore, the definition of a whole bacteria phylum as “healthy” can be misleading as single members at the strain level and within the same genus can contribute to homeostasis or disease progression. A good example is Prevotella, which has been classified both as a beneficial bacterium (P. histicola) or a detrimental bacterium (P. copri) in rheumatoid arthritis (169). Thus, strategies arising from a better understanding of molecular mechanisms, especially the mode of action of bacterial-derived small molecules are needed. This includes identifying highly purified microbial strain(s)-producing specific molecules that target known members of the microbiota or specific host-microbiome signaling pathways without affecting homeostatic balance. All of these approaches could achieve greater therapeutic efficacy and further the promise of precision-based microbial therapy.

Figure 6.

Advantages and disadvantages of major therapeutic innovations targeting the microbiome including FMT, prebiotics, probiotics, and small molecules. FMT, fecal microbiota transplantation.

Some examples of small molecules (e.g., polysaccharide A) in clinical studies and targeting the microbiome were recently reviewed (163). Trimethylamine-N-oxide (TMAO), a microbe-dependent compound originating from trimethylamine (TMA) metabolism, promotes cardiovascular disease (CVD, thrombosis potential). The gene catalyzing the synthesis of TMA in the microbiota was identified as CutC/D, which is a mechanism-based target for the inhibition of CVD potential (164, 169). Using this mechanism-directed strategy, the Hazen’s laboratory developed small molecules (including the natural product 3,3-dimethyl-1-butanol structurally related to choline) targeting TMAO/TMA-producing proteins (CutC/D) as microbiota-directed nontoxic drug inhibitors of thrombosis potential (164). Progress in the understanding of such molecular mechanisms in the microbiome field will provide an opportunity for more precise microbiome-based therapeutic interventions.

SUMMARY AND PERSPECTIVES

Changes in the composition of the human gut microbiota have been linked to several clinical diseases. However, the molecular mechanisms underlying these host-microbiome interactions largely remain unknown. Since microbes are metabolically active and interact with their environment using small molecules, microbial molecules play a significant role in the mechanism of host-microbe interactions. The role of microbe biotransformation products (e.g., bile acid derivatives and SCFAs) has been increasingly recognized, but little is known about the identity and function of compounds directly synthesized by the microbiome, or indeed whether diet can change the de novo synthesis of such compounds by directly regulating their synthesis or through modulation of microbiome community structure. In this review, we mainly describe BGCs and respective metabolites directly encoded by the human microbiome. We also present strategies used to discover these compounds and the biological activities they exhibit and their roles in disease or health processes as well as different therapeutic innovations targeting the microbiome. Although great progress has been achieved in analyzing and understanding the microbiome structure and composition, translation of these results into clinical practice to combat disease will be facilitated by a better understanding of the mechanisms of action, which are mainly driven by small molecules or biologicals. Although most microbiome-based projects still focus on associations and consortia of bacteria, the field is now slowly shifting toward molecular causation. Future directions in the field should also include more systematic approaches not only limited to bacteria and their mechanisms of interactions, but considering all components of the microbiome (bacteria, fungi, virus archaea, and protists) that impact health, the host, and the environment.

GRANTS

This work was supported by the National Institutes of Health Grants National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) P30-DK56338 and R01DK130517, National Institute of Allergy and Infectious Diseases (NIAID) U01-AI24290 and P01-AI152999, and National Institute of Nursing Research (NINR) R01-NR013497.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.A.F. prepared figures; S.A.F. drafted manuscript; T.S. edited and revised manuscript; S.A.F. and T.S. approved final version of manuscript.