Prebiotics

Prebiotics are selectively fermented ingredients that trigger specific changes in the microbiome composition and activity to promote host health (Gibson et al., Reference Gibson, Probert, Loo, Rastall and Roberfroid2004). Safely administering live microbes and establishing their colonisation in the gut is difficult and faces regulatory hurdles, making small molecule interventions more attractive (Cully, Reference Cully2019). Small molecules, especially endogenous metabolites can accumulate to high concentrations with negligible toxicity, remain stable in the systemic circulation, and obey the principles of pharmacokinetics. The major prebiotics are human milk oligosaccharides (HMOs), inulins (1 in Figure 1B), fructose oligosaccharides (FOS), xylooligosaccharides (XOS), mannan oligosaccharides (MOS), and galactooligosaccharides, which are polymers/oligomers of glucose, fructose, mannose, fucose, galactose, sialic acid, xylose, uronic acid, and arabinofuranose units linked together with β2, β3, and β4 linkages (Enam and Mansell, Reference Enam and Mansell2019).

Developments in chemical synthesis are bringing the goal of complex carbohydrate assembly closer. Difficulties arise mainly from (1) the need to selectively protect and deprotect monosaccharides and (2) regioselectivity and stereoselectivity. Improved glycosylation strategies have been reported, which enables glycosyl donors to react in a specific order, yielding a single oligosaccharide product (Vohra et al., Reference Vohra, Vasan, Venot and Boons2008). Automated glycan assembly (AGA) currently enables access to a maximum length of 100, while convergent block coupling of 30- and 31-mer oligosaccharide fragments made by AGA was used to make a multiple-branched 151-mer polymannoside (Joseph et al., Reference Joseph, Pardo-Vargas and Seeberger2020).

Enzymatic and chemoenzymatic processes offer better regioselectivity and stereoselectivity, along with fewer steps in the synthesis, which makes them faster and more cost effective (Li et al., Reference Li, McArthur and Chen2019). For example, the HMO 2′-fucosyllactose (2′FL) has been synthesised in engineered Escherichia coli strains (Baumgärtner et al., Reference Baumgärtner, Seitz, Sprenger and Albermann2013). One-pot multi-enzyme synthesis has been reported which employs glycosyltransferases to synthesise sialyl- and fucosyl-derivatives (Chen et al., Reference Chen, Zhang, Xue, Liu, Li, Chen, Wang, Wang and Cao2015). Sialylated HMOs with high regioselectivity and stereoselectivity have been synthesised using a chemoenzymatic strategy, whereby automated solid-phase synthesis of the glycan backbone was followed by α-(2,3)-sialyltransferase treatment (Fair et al., Reference Fair, Hahm and Seeberger2015). Interest in sustainable chemical feedstocks has led to method development for the conversion of lignocellulose biomass into valuable prebiotics such as XOS (Poletto et al., Reference Poletto, Pereira, Monteiro, Pereira, Bordignon and de Oliveira2020).

Prebiotics can have synergistic interactions with approved drugs. Konjac MOS from the plant Amorphophallus konjac are prebiotics containing β-D-mannose and β-D-glucose residues linked by one to four linkages (Liu et al., Reference Liu, Xu, Zhang, Zhou, Lyu, Zhao and Ding2015). The combined administration of the drug metformin and konjac MOS mitigates insulin resistance and glucose tolerance, while also improving islet and hepatic tissue function (Zheng et al., Reference Zheng, Li, Zhang, Jiang, Luo, Lu, Xu and Shi2018). The beneficial effects were correlated with the reduced abundance of the Rikenellaceae family and the Clostridiales order, with an increased relative abundance of Bifidobacterium pseudolongum, Akkermansia muciniphila, and OTU05945 of family S24–7 (Zheng et al., Reference Zheng, Li, Zhang, Jiang, Luo, Lu, Xu and Shi2018). Further studies focussing on prebiotic-drug interactions could lead to more targeted application of prebiotics in combination with approved drugs to mitigate the impact of specific diseases.

Stimulants of CR

CR is a mechanism by which the gut microbiota protects itself against the incursion and establishment of largely harmful microorganisms. This protection can be accomplished by several routes, such as antimicrobial secretion, nutrient limitation, and stimulation of gut barrier integrity and the action of bacteriophages (Ducarmon et al., Reference Ducarmon, Zwittink, Hornung, van Schaik, Young and Kuijper2019). Disturbances to the gut resulting from the use of antibiotics, other drugs, or inflammation can reduce CR, allowing enteric pathogens such as C. difficile, Salmonella enterica serovar Typhimurium, E. coli, Shigella flexneri, Campylobacter jejuni, Vibrio cholerae, Yersinia enterocolitica, and Listeria monocytogenes, to colonise the niches vacated by microbiome disruption (Sorbara and Pamer, Reference Sorbara and Pamer2019). Both endogenous molecules such as SCFA and tryptophan metabolites produced by the GM and exogenous synthetic small molecules can restore CR function. Synthetic molecules are beginning to be used in efforts to stimulate CR following disturbances to the GM, for example, after antibiotic administration. For example, vancomycin-resistant enterococci (VRE) flourish when CR is compromised following antibiotic treatment. A synthetic molecule, resiquimod or R848 (2 in Figure 1B), binds to a Toll-like receptor 7 that stimulates innate immune defences, leading to the restoration of CR against VRE by triggering the expression of the antimicrobial peptide Reg3γ (Abt et al., Reference Abt, Buffie, Sušac, Becattini, Carter, Leiner, Keith, Artis, Osborne and Pamer2016). R848 can be taken orally and induces the secretion of interleukins IL-23 and IL-22.

Bacterial conjugation inhibitors

Antibiotic resistance is spread by several mechanisms, including horizontal gene transfer mediated by plasmids. Analysis of Bacteroidetes strains sharing the intestinal niches of specific individual humans, demonstrated the extensive occurrence of horizontal gene transfer among those strains. In this case, the genetic elements exchanged coded for orphan DNA methylases, fimbriae synthesis proteins, novel metabolic enzymes, antibiotics, and proposed type VI secretion systems (T6SS) (Coyne et al., Reference Coyne, Zitomersky, McGuire, Earl and Comstock2014). More recent studies have recorded extensive plasmid exchange in the gut environment using CRISPR-Cas spacer acquisition analysis in an E. coli strain (Munck et al., Reference Munck, Sheth, Freedberg and Wang2020). Unlike earlier studies that relied on phenotypic markers or post-transfer replication to detect mobile genetic elements, the spacer acquisition analysis reveals plasmid transfer in real time, and the results showed that the IncX plasmid type was most frequently transferred (Munck et al., Reference Munck, Sheth, Freedberg and Wang2020). Therefore, inhibiting bacterial conjugation in a bacteria-dense environment could enable the host to mitigate antibiotic-resistant infections. In general, resident bacteria in the healthy GM may be able to suppress the evolution of antibiotic resistance in vivo. However, the wide distribution of plasmid-borne resistance in the environment is well known and exposure to them might be common. Moreover, gut inflammation boosts plasmid transfer between pathogenic and commensal Enterobacteriaceae (Stecher et al., Reference Stecher, Denzler, Maier, Bernet, Sanders, Pickard, Barthel, Westendorf, Krogfelt and Walker2012). Therefore, inhibiting plasmid transfer in the gut is expected to promote host health, and conjugation inhibitors (COINs) are unlikely to disturb the GM composition, unlike conventional antibiotics. We describe a few known COINS, but some need to be further specifically tested in the gut environment.

Early studies to identify COINs unearthed many unspecific molecules that affected DNA replication or growth (Cabezón et al., Reference Cabezón, de la Cruz and Arechaga2017). Plant phenolics seems to be a good source of COINS and have yielded two molecules that specifically inhibited bacterial conjugation, namely rottlerin and 8-cinnamoyl-5,7-dihydroxy-2,2,6-trimethylchromene (Oyedemi et al., Reference Oyedemi, Shinde, Shinde, Kakalou, Stapleton and Gibbons2016). Screening of a library of over 12,000 NPs (NatChem library) based on high-throughput whole-cell-based assays enabled the discrimination between true COINS and false “hits” which merely affected cell growth, leading to the discovery of the COIN dehydrocrepnynic acid (DHCA) (Fernandez-Lopez et al., Reference Fernandez-Lopez, Machón, Longshaw, Martin, Molin, Zechner, Espinosa, Lanka and de la Cruz2005). DHCA belongs to the chemical family of unsaturated fatty acids (UFAs), which is generally a good source of COINS. DHCA is derived from a tropical seed, and its supply is limited. However, it was used as the starting point for the synthesis of other COINS, particularly 2-hexadecynoic acid (2-HDA) and other 2-alkynoic fatty acids (2-AFAs), which specifically inhibited the transfer of a range of plasmids, including the common and highly infective IncF, in various bacteria (Getino et al., Reference Getino, Sanabria-Ríos, Fernández-López, Campos-Gómez, Sánchez-López, Fernández, Carballeira and de la Cruz2015). 2-HAD was later reported to prevent bacterial conjugation in the mouse gut (Palencia-Gándara et al., Reference Palencia-Gándara, Getino, Moyano, Redondo, Fernández-López, González-Zorn and de la Cruz2021). A series of UFA NPs called tanzawaic acids were discovered (tanzawaic acid B or TZA-B is depicted as (3 in Figure 1B); they mainly inhibited conjugation by the IncW- and IncFII-based plasmids. Other plasmids classified under the IncFI, IncI, IncL/M, IncX, and IncH incompatibility groups were less affected, whereas the IncN and IncP plasmids were unaffected (Getino et al., Reference Getino, Fernández-López, Palencia-Gándara, Campos-Gómez, Sánchez-López, Martínez, Fernández and de la Cruz2016).

Conjugation is driven by the type 4 secretion system, whose architecture is conserved in most bacteria, and contains the pilus, the core channel complex, the inner membrane platform, and the ATPases that provide energy for substrate transport and pilus biogenesis (Cabezón et al., Reference Cabezón, Ripoll-Rozada, Peña, de la Cruz and Arechaga2015). Nicking the DNA to relax the plasmid, DNA transfer to the secretion channel, the transfer of pilin molecules during pilus biogenesis, and pilus biogenesis are performed by four distinct ATP-ase enzymes, among which carboxylic acid COINS were shown to target the last step (TrwD protein). Based on structural and computational data, the UFAs and AFAs were suggested to bind at the end of the N-terminal domain as well as at the beginning of the linker region that connects the N-terminal and C-terminal domains, likely hindering the swapping movements of the domains needed for the catalytic cycle (Ripoll-Rozada et al., Reference Ripoll-Rozada, García-Cazorla, Getino, Machón, Sanabria-Ríos, de la Cruz, Cabezón and Arechaga2016).

Molecular decoys

These molecules bind enteric pathogens and stimulate their elimination from the gastrointestinal tract. This binding is thought to “fool” pathogens by mimicking the receptors used by them to attach to the gut epithelia in the lower gastrointestinal tract. The global enteric diseases is substantial and cases may number in the hundreds of millions annually. HMOs act as soluble decoys for receptors and block the binding of enteric pathogens. Rotavirus infection is prevented most effectively by the HMO 2′FL, although several other HMOs also have similar inhibitory effects (Laucirica et al., Reference Laucirica, Triantis, Schoemaker, Estes and Ramani2017). C. jejuni infects the mammalian gut and causes diarrhoea and sometimes also motor neuron paralysis. The infection is initiated by the bacterium binding to the fucosylated intestinal H(O) antigen (Fuc alpha 1, 2Gal beta 1, 4GlcNAc). However, FOS in human milk can act as decoys by binding to the pathogen, thereby preventing infection (Ruiz-Palacios et al., Reference Ruiz-Palacios, Cervantes, Ramos, Chavez-Munguia and Newburg2003).

Uropathogenic E. coli (UPEC) uses the extracellular appendages called Type 1 pili to colonise the intestine by binding a mannosylated host receptor; the Type 1 pili are also essential for colonisation and infection in the bladder. Mannosides (4 in Figure 1B) are small-molecule drugs bearing mannose group(s) that act as decoys by mimicking the mannosylated receptor and can clear both bladder and intestinal UPEC upon oral administration in mouse models, leaving the GM largely intact (Spaulding et al., Reference Spaulding, Klein, Schreiber, Janetka and Hultgren2018). The decoy approach has been further extended to combat cholera, and in this case, nanotechnology is also employed. The V. cholerae toxin binds to the host receptor monosialotetrahexosylganglioside (GM1), and coating GM1 on the surface of polymeric nanoparticles was enough to reduce cyclic-AMP production in epithelia and fluid responses to live V. cholerae in both cell cultures and a mouse infection model (Das et al., Reference Das, Angsantikul, Le, Bao, Miyamoto, Gao, Zhang and Eckmann2018). The modulation of disease via molecular mimicry extends to non-sugar molecules, such as metalloenzymes allows for the manipulation of the gut chemical environment using synthetic catalysts. A metalloporphyrin mimic of the enzyme superoxide dismutase could reduce lipid peroxidation levels and thereby shielded epithelial cells from damage in rats injected with the common antigen bacterial lipopolysaccharide (LPS) (Das et al., Reference Das, Angsantikul, Le, Bao, Miyamoto, Gao, Zhang and Eckmann2018).

Fluorophores

The most common tools for probing the GM are fluorophores, which may be attached to different types of other chemical entities. Commensal anaerobic bacteria, including Bacteroides fragilis when fed azide-labelled sugars, which subsequently conjugated with alkyne-fluorophores via click chemistry, facilitate the imaging of bacteria in live mice (Geva-Zatorsky et al., Reference Geva-Zatorsky, Alvarez, Hudak, Reading, Erturk-Hasdemir, Dasgupta, von Andrian and Kasper2015). Three different bacterial surface molecules from the GM, which interact with the host immune system, namely LPS, CPS, and PGN, can be tracked (Hatzenpichler et al., Reference Hatzenpichler, Scheller, Tavormina, Babin, Tirrell and Orphan2014), helping to dissect host–microbe interactions. Azide-bearing amino acids when fed to complex gut microbial communities showed that newly synthesised proteins could be visualised in situ (Hatzenpichler et al., Reference Hatzenpichler, Scheller, Tavormina, Babin, Tirrell and Orphan2014). Two D-amino acid-based fluorescent probes, TADA and Cy5ADA (5,6 in Figure 2), which get incorporated into bacterial PGN, have been instrumental in enabling live monitoring of GM growth and division patterns in mice (Lin et al., Reference Lin, Wu, Song, Du, Gao, Song, Wang and Yang2020). Probes based on D-amino acids are also being used to track the viabilities of bacteria in faecal transplants by using sequential tagging (Wang et al., Reference Wang, Lin, Du, Song, Peng, Chen and Yang2019). In this approach, the bacteria are treated with a probe before the transplantation and then the recipient mice are fed a second probe following the transplantation. Therefore, the bacteria surviving the process show the emission for both probes, enabling the identification of viable bacteria in the transplant (Wang et al., Reference Wang, Lin, Du, Song, Peng, Chen and Yang2019).

Figure 2.

Examples of chemical probes used to interrogate the GM – D-amino acid-based fluorescent probes = TADA (5) and Cy5ADA (6); a multifunctional probe showing different parts shaded in distinct colours = amine directed probe based on sulpho-N-hydroxysuccinimide (7); photoactive unnatural amino acid probes = DiZPK (8) and ACPK (9); a cysteine-targeted probe = Biotin-Gly-CMK (10); bioluminescent bile acid-luciferin conjugates for bile salt hydrolase (BSH) activity = series of compounds with H or OH at the positions R1 and R2 (11).

Multifunctional selective probes

Direct extraction from human faecal samples and release under mild conditions is possible using multifunctional chemoselective probes (Garg et al., Reference Garg, Conway, Ballet, Correia, Olsson, Vujasinovic, Löhr and Globisch2018), allowing for the analysis of femtomole levels of metabolites with enhanced sensitivity. Probe 7 in Figure 2 is anchored at one end to magnetic beads, linked by a spacer to a novel p-nitrocinnamyloxycarbonyl biorthogonal cleavage site, while the reactive site features an amine-selective sulpho-N-hydroxysuccinimide “warhead,” which reacts with metabolic amines (Garg et al., Reference Garg, Conway, Ballet, Correia, Olsson, Vujasinovic, Löhr and Globisch2018). Since 2011, it has been possible to monitor enteric pathogens via the incorporation of the photoactive unnatural amino acids DiZPK and ACPK (8, 9 in Figure 2) into specific pathogen proteins, which react to form cross links revealing the interactions between the modified protein and its client proteins (Lin et al., Reference Lin, Zhang, Xu, Li, Chen, Li, Hao and Chen2011). This approach is enabling the direct identification of proteins involved in pathogenesis and acid-stress defence mechanisms, which is quite challenging to perform with conventional methods.

Simple reactive probes

Sphinganines are bioactive components of foods, but the GM also modifies them. The use of alkyne-tagged sphinganines allows for the identification sphinganine-utilising GM strains based on labelling followed by a cell sorting workflow (Lee et al., Reference Lee, Le and Johnson2021). The subsequent sequencing of the sorted bacteria revealed that this metabolism is nearly exclusively performed by members of the Bacteroides (Lee et al., Reference Lee, Le and Johnson2021). An activity-based probe, Biotin-Gly-CMK (10 in Figure 2), has been used to differentiate between mice models harbouring “normal” human GM and “inflammatory bowel disease” (IBD) affected human GM, whereby a novel cysteine-reactive probe tagged several proteases and hydrolases in the IBD model, but not in the healthy controls (Mayers et al., Reference Mayers, Moon, Stupp, Su and Wolan2017).

An elegant recent study by Nie et al. using a click chemistry strategy isolated and identified a previously unknown BA 3-succinylated cholic acid correlated with reduced progression of metabolic dysfunction-associated steatohepatitis in humans (Nie et al., Reference Nie, Luo, Wang, Ding, Jia, Zhao and Li2024). Using this discovery, the authors were able to characterise an annotated β-lactamase in the GM member Bacteroides uniformis as the enzyme catalysing the 3-succinylation of CA (Mayers et al., Reference Mayers, Moon, Stupp, Su and Wolan2017).

Bioluminescent probes

Luciferin-based bioluminescent probes (11 in Figure 2) have been employed to detect bile salt hydrolase (BSH) activity in a wide variety of sample environments including purified enzymes, bacterial cells, faecal slurries as well as non-invasive imaging in mice and humans (Khodakivskyi et al., Reference Khodakivskyi, Lauber, Yevtodiyenko, Bazhin, Bruce, Ringel-Kulka, Ringel, Bétrisey, Torres and Hu2021). BSH activity releases luciferin from the conjugated BA, which can be further assayed using luciferase. These BA-luciferin probes were useful in demonstrating the stimulatory effect of prebiotics on BSH activity and as diagnostic tests that non-invasively detect the clinical IBD status in human patients (Khodakivskyi et al., Reference Khodakivskyi, Lauber, Yevtodiyenko, Bazhin, Bruce, Ringel-Kulka, Ringel, Bétrisey, Torres and Hu2021).

Choline metabolism

A “chemically guided functional profiling” could be a strategy to uncover the presence of novel enzymes in the GM and subsequently, to modulate their function to achieve therapeutic effects. The conversion of choline into trimethylamine (TMA) by anaerobic gut bacteria is correlated with disease conditions in humans, and more specifically, the production of TMA in both isolated bacteria and complex communities can be inhibited by betaine aldehyde (12 in Figure 3) (Orman et al., Reference Orman, Bodea, Funk, Campo, Bollenbach, Drennan and Balskus2019). The identified target is GM choline TMA-lyase (CutC) and this opens up the scope for the development of other inhibitors.

Figure 3.

Specific enzyme inhibition can be a strategy to selectively manipulate the gut microbiome, and some inhibitors of gut bacterial enzymes are shown. 12 = betaine aldehyde, inhibits choline TMA-lyase (CutC); 13 = fluoromethyl ketone suicide inhibitor of bile salt hydrolase (BSH); 14, 15 = piperazine-containing β-glucuronidase inhibitors; 16 = acarbose, inhibits starch and pullulan utilisation; and 17 = M4284 mannoside, inhibits FimH in uropathogenic E. coli.

Bile salt metabolism

Bile salts have major effects on the physiology and virulence of C. difficile. When patients are restored to a C. difficile-resistant state, it is observed that the production of deoxycholate from cholate by 7α-dehydroxylating gut bacteria occurs (Savidge and Sorg, Reference Savidge and Sorg2019). Broad-spectrum antibiotics block the production of secondary BAs and kill the 7α-dehydroxylating bacteria, thereby enabling C. difficile to colonise the gut (Savidge and Sorg, Reference Savidge and Sorg2019). BSH enzymes expressed by the GM and bile salt metabolism affect the immune and metabolic processes via engaging host receptors. Therefore, inhibiting BSH enzymes would enable the dissection of the role of bile salts in host–microbe interactions. Screening a library of compounds, Adhikari et al. zeroed in on a covalent suicide inhibitor containing an α-fluoromethyl ketone moiety (13 in Figure 3), which reacts with the active site cysteine of BSH enzymes, as way to globally modulate BSH and understand their physiological roles (Adhikari et al., Reference Adhikari, Seegar, Ficarro, McCurry, Ramachandran, Yao, Chaudhari, Ndousse-Fetter, Banks and Marto2020).

Glucuronidase inhibitors

β-Glucuronidase (GUS) enzymes harboured by gut microbes can cause severe toxicity reactions to certain pharmaceuticals including cancer drugs; therefore, GUS inhibitors have been developed (14, 15 in Figure 3) to ameliorate these toxic side effects. Pellock et al. reported the discovery of piperazine-based GUS inhibitors by combining chemical biology, protein structural data and mass spectrometry with cell-based assays (Pellock et al., Reference Pellock, Creekmore, Walton, Mehta, Biernat, Cesmat, Ariyarathna, Dunn, Li and Jin2018). Their GUS inhibitors interrupt the catalytic cycle of the enzyme and are substrate-dependent, binding to the catalytic intermediate by means of a piperazine-linked glucuronide. The inhibitor-glucuronide conjugates were detected by LC-MS (Santilli et al., Reference Santilli, Dawson, Whitehead and Whitehead2018).

Carbohydrate metabolism

The prospects for chemical precision editing of the GM are improving due to an expansion in the knowledge of its metabolism. GM diversity is promoted by the metabolism of complex plant polysaccharides. Selective manipulation of polysaccharide metabolism without microbicidal effects has been achieved using a small molecule inhibitor, acarbose (16 in Figure 3), which abolished the ability of Bacteroides thetaiotaomicron and B. fragilis to utilise potato starch and pullulan by interfering with the starch utilisation system (). Shifting the GM metabolic activity selectively in this non-lethal fashion alleviated colitis. Until recently, it was not known if single bacterial species or a small community is needed to drive the degradation of any highly complex polysaccharide. The most complex polysaccharide characterised in the gut environment is rhamnogalacturonan-II, which is depolymerised by B. thetaiotaomicron with the cleavage of 20 out of its 21 distinct glycosidic bonds (Ndeh et al., Reference Ndeh, Rogowski, Cartmell, Luis, Baslé, Gray, Venditto, Briggs, Zhang and Labourel2017). Further analysis revealed that several previously unknown bacterial enzymes were responsible for the degradation of rhamnogalacturonan-II.

Miscellaneous inhibitors

Zhu et al. showed that dysbiosis-linked gut inflammation caused by the expansion of facultative anaerobic Proteobacteria could be blocked via tungstate administration, which inhibits molybdenum-cofactor respiratory chain enzymes (Zhu et al., Reference Zhu, Winter, Byndloss, Spiga, Duerkop, Hughes, Büttner, de Lima Romão, Behrendt and Lopez2018). The GM composition was undisturbed when tungstate was administered under homeostatic conditions. Recurrent infections of the urinary tract caused by UPEC occur in 30–50% of patients even after antibiotic treatment. This persistence is linked to the type 1 pilus adhesin, FimH, which binds mannose and aids the colonisation of the bladder surface. Type 1 pili were also shown to aid UPEC colonisation in the gut, and the administration of the high affinity FimH inhibitor mannoside M4284 (17 in Figure 3) reduced gut colonisation and urinary tract infection caused by genetically distinct UPEC isolates, without disrupting the GM composition (Spaulding et al., Reference Spaulding, Klein, Ruer, Kau, Schreiber, Cusumano, Dodson, Pinkner, Fremont and Janetka2017).

Enzyme-based sulphated metabolome analysis

Sulphated compounds are derived from gut microbial transformation of dietary material and are related to disease states. Using an arylsulphatase enzyme to hydrolyse sulphated compounds and mass spectrometry-based metabolite analysis, Correia et al. have characterised and validated 235 sulphated metabolites in a single study, which were the products of gut microbiota and subsequent host transformations and discovered 11 previously unknown sulphated metabolites (Correia et al., Reference Correia, Jain, Alotaibi, Young Tie Yang, Rodriguez-Mateos and Globisch2020). The metabolites reported in this study could form the basis of classification of human subjects as harbouring high or low sulphate metabolising microbiota for future cohort studies. Further, the arylsulphatase-based method may be useful for discovering novel sulphated metabolites.

BSH and BA-based chemoproteomics

As mentioned, BAs are secreted by the liver and are further converted into secondary BAs by the action of the GM. The latter participate in several processes, including the metabolism of glucose and lipids, and immune homeostasis. The key reaction of secondary BA biosynthesis is catalysed by BSH. BSH are bacterial cysteine hydrolases whose activity precedes other kinds of BA transformations (Devlin and Fischbach, Reference Devlin and Fischbach2015). Parasar et al. developed a strategy based on the covalent labelling of the active site cysteine using a substrate analogue (Parasar et al., Reference Parasar, Zhou, Xiao, Shi, Brito and Chang2019). When the substrate analogue is covalently bound, biorthogonal click chemistry could be applied to attach either a fluorescent contrast agent or a biotin affinity tag to the enzyme-bound analogue. In the first case, in situ imaging could be performed following gel electrophoresis, and in the second case, affinity purification was performed using streptavidin (the samples were subsequently analysed using proteomics).

While the expression of metagenomic fragments in well-studied model microbes showed that at least three distinct phyla possess BSH activities in the GM (Jones et al., Reference Jones, Begley, Hill, Gahan and Marchesi2008), genome-based strategies suffer from the issues of potential toxicity, incomplete coverage, incomplete BGC expression, unintended changes in enzyme levels and tissue localisation, all of which led to deviations from the physiologically relevant states of the BSH enzymes. By comparison, the covalent modification of the active sites of BSH enzymes coupled with proteomics has avoided many of the pitfalls associated with genome-based methods and enabled the direct identification of these enzymes.

While BAs promote CR, little was known about the target proteins affected in the gut pathogens inhibited by BA action. Photoaffinity probes based on chenodeoxycholic acid (CDCA) were able to crosslink many host and pathogen proteins in Salmonella enterica serovar Typhimurium infection models, of which direct protein inhibition by CDCA probes was reported for HilD, a key regulator of Salmonella pathogenesis and virulence (Yang et al., Reference Yang, Stein and Hang2023). Chemical proteomics and photoaffinity labelling based on lithocholic acid were also used to identify a previously unknown BA-binding transcription factor called BapR in C. difficile (Forster et al., Reference Forster, Yang, Tai, Hang and Shen2022).

Direct lysine-acylation chemoproteomics

In a 2022 report, abundant post-translational lysine-acylation by reactive acyl-CoA species was discovered, whereby the acyl motifs found on several differentially expressed proteins corresponded to the metabolism of specific carboxylic acids in syntrophic bacteria (Muroski et al., Reference Muroski, Fu, Nguyen, Wofford, Mouttaki, James, McInerney, Gunsalus, Loo and Ogorzalek Loo2022). The importance of cross-feeding in the gut environment, the abundance of SCFA and the ability to analyse the proteome for post-translational modifications without highly biased pre-enrichment, direct analysis of lysine acylation in the GM has good potential to shed light on metabolomic aspects.

Vitamin affinity probe chemoproteomics

Bacteroidetes are one of the four major GM phylas; their genomes usually encode several B₁₂-dependent enzymes, although they lack the ability of de novo cobamide synthesis (Shelton et al., Reference Shelton, Seth, Mok, Han, Jackson, Haft and Taga2019). It is therefore likely that they could harbour B₁₂ transport proteins different at the sequence level from canonical E. coli counterparts. The use of B₁₂-based affinity probes and subsequent application of chemoproteomics in B. thetaiotaomicron samples revealed the presence of proteins without previously unknown functions; one of these proteins, BtuH2, was shown to capture and transport B₁₂ directly in vitro and responsible for gut fitness of these bacteria in gnotobiotic mice (Putnam et al., Reference Putnam, Abellon-Ruiz, Killinger, Rosnow, Wexler, Folta-Stogniew, Wright, van den Berg and Goodman2022).

Cell-surface receptors

G protein-coupled receptors (GPCRs) are the largest membrane protein family in humans and sense their ligands through a mechanism outlined in Figure 4. GPCR complexes contain a transmembrane subunit (green in Figure 4) that binds a small molecule (ligand) at the cell surface, whereas a linked trimeric G-protein bound to GDP is located inside the cell. Once the ligand has been captured by the receptor subunit, then a conformational change occurs in the complex, allowing GTP to bind the trimeric G-protein, which usually dissociates, triggering an intracellular response via further downstream events. There are a variety of GPCRs in the gut for various microbial metabolites, such as SCFA (Husted et al., Reference Husted, Trauelsen, Rudenko, Hjorth and Schwartz2017), BAs (Lefebvre et al., Reference Lefebvre, Cariou, Lien, Kuipers and Staels2009), and several other types of effectors (Cohen et al., Reference Cohen, Kang, Chu, Huang, Gordon, Reddy, Ternei, Craig and Brady2015). Gut bacteria synthesise molecules such as commendamide, which mimic the human (endogenous) ligands of GPCRs (Kroeze et al., Reference Kroeze, Sassano, Huang, Lansu, McCorvy, Giguère, Sciaky and Roth2015).

Figure 4.

Molecular mechanism of G protein-coupled receptors on the cell surface. The ligand binds to the receptor protein, causing the G-protein subunits to disassemble and exchange bound GDP with GTP. The G-protein α-subunit is bound to the receptor, whereas the other subunits signal to other proteins involved in intracellular responses. GTP hydrolysis drives the dissociation of the α-subunit from the receptor and a return to the GDP-bound multi-subunit G-protein complex.

A forward genetic screen (i.e., trying to identify genes leading to a phenotype) based on the Tango β-arrestin recruitment assay (PRESTO-Tango) was able to measure the activation processes of almost all the non-olfactory human GPCRs (Chen et al., Reference Chen, Nwe, Yang, Rosen, Bielecka, Kuchroo, Cline, Kruse, Ring and Crawford2019) and revealed several novel GPCR ligands such as l-phenylalanine secreted in the GM (Colosimo et al., Reference Colosimo, Kohn, Luo, Piscotta, Han, Pickard, Rao, Cross, Cohen and Brady2019). Several other ligands that bind GPCRs (including in immune and nerve cells) such as phenylpropanoic acid, cadaverine, 9-10-methylenehexadecanoic acid, and 12-methyltetradecanoic acid were identified in a high-throughput screening of 241 GPCRs (Kovatcheva-Datchary et al., Reference Kovatcheva-Datchary, Shoaie, Lee, Wahlström, Nookaew, Hallen, Perkins, Nielsen and Bäckhed2019), using seven gut microbes to represent a simplified human microbiome consortium (Subramanian et al., Reference Subramanian, Huq, Yatsunenko, Haque, Mahfuz, Alam, Benezra, DeStefano, Meier and Muegge2014; Kovatcheva-Datchary et al., Reference Kovatcheva-Datchary, Shoaie, Lee, Wahlström, Nookaew, Hallen, Perkins, Nielsen and Bäckhed2019).

Nuclear receptors

The major nuclear receptors in the gut are the AHR, the farnesoid X receptor (FXR), and the pregnane X receptor (PXR).

Peroxisome proliferator-activated receptors

Peroxisome proliferator-activated receptors (PPARs) are found throughout the gut tissue and have roles in fatty acid sensing, metabolism, and modulation of immunity; PPARα is crucial for fatty acid and branched chain amino acid catabolism in the mitochondria and peroxisomes (Grabacka et al., Reference Grabacka, Pierzchalska, Płonka and Pierzchalski2021), while PPARγ is important in innate immunity (Croasdell et al., Reference Croasdell, Duffney, Kim, Lacy, Sime and Phipps2015). Double agonists of both these receptors have been successful in animal models of Citrobacter rodentium and DSS-induced colitis of reducing tissue damage and bacterial loads leading to infection clearance and resolved inflammation compared with agonists of each receptor separately (Katkar et al., Reference Katkar, Sayed, Anandachar, Castillo, Vidales, Toobian, Usmani, Sawires, Leriche and Yang2022). PPARα and γ activation has been reported for keto- and hydroxy-octadecanoic acid species, which were produced by Lactiplantibacillus plantarum (Goto et al., Reference Goto, Kim, Furuzono, Takahashi, Yamakuni, Yang, Li, Ohue, Nomura and Sugawara2015). Oleoylethanolamide, an endogenous PPAR ligand, can be administered exogenously in mice to shift the microbiota in the colon to a higher Bacteroidetes/Firmicutes ratio, with corresponding increases in Bacteroides, Prevotella, and Parabacteroides and decreases in Bacillus and Lactobacillus strains (Di Paola et al., Reference Di Paola, Bonechi, Provensi, Costa, Clarke, Ballerini, De Filippo and Passani2018). The GM has also been modulated by synthetic agonists, such as fenofibrate, which led to increased SCFA levels in serum and tissues in mice fed HFDs (Wang et al., Reference Wang, Yu, Liu, Yang, Feng, Wu, Xu, Zhu and Li2022). Dysbiosis induced by either high-fructose diets or HFD in mice could be remediated by the PPAR agonist Wy-16434, whereby the Bacteroidetes/Firmicutes ratio increased (reduced Proteobacteria and increased Actinobacteria) (Silva-Veiga et al., Reference Silva-Veiga, Miranda, Martins, Daleprane, Mandarim-de-Lacerda and Souza-Mello2020; Silva-Veiga et al., Reference Silva-Veiga, Miranda, Vasques-Monteiro, Souza-Tavares, Martins, Daleprane and Souza-Mello2022).