Schematic diagram of the multi-compartmental metabolism of amino acids in the gut and extraintestinal tissues of mono-gastric animals. AA, amino acids; AHLs, acyl homoserine lactones; AI-2, autoinducer-2; Ala, alanine; BCAA, branched chain amino acid; BCFA, branched-chain fatty acids; BCKA, branched-chain ketoacid; CO 2 , carbon dioxide; GSH, glutathione; GSSG, glutathione disulfide; Glu, glutamate; Gln, glutamine; Glu / Gln Cycle, Glutamate / Glutamine Cycle; Gly, glycine; H 2 S, hydrogen sulfide; alpha-KG, alpha-ketoglutarate; LPS, lipopolysacharrides; Lys, lysine; Met, methionine; NDC, non-digestible 

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... the large intestine, one bacterial species might also affect the nutrient metabolism of its “neighbors” indirectly. When germ-free mice were colonized with Eubacterium rectale and Bacteriodes thetaiotaomicron (members belonging to the two dominant bacterial phyla present in the human distal gut microbiota), E . rectale adapted to the presence of B . thetaiotaomicron by reducing the production of its glycan-degrading enzymes, increasing expression of select AA and sugar transporters, and synthesizing glutamine/glutamate and D- alanine (56). However, the importance of the microbe- microbe interactions (e.g., feedback loops or molecular cross-talk) in intestinal AA metabolism is unknown. Microbial metabolites may modulate gene expression in bacteria and their production of enzymes related to AA metabolism in the digestive tract. Compared to the laboratory medium, when the probiotic bacteria Lactobacillus plantarum pass through the GI tract of mice, expression of the genes involved in the acquisition and synthesis of AA increased dramatically (57). For instance, glutamate-5-semialdehyde dehydrogenase participates in the conversion of glutamate to proline and argininosuccinate synthase converts citrulline to arginine (57). Further studies showed that several genes (e.g. argininosuccinate synthase) displayed intestinal compartment-specific activity (small intestine > colon) (58). However, proteomic studies on the interactions between Lactobacillus fermentum and intestinal epithelium cells indicated that, compared to laboratory medium, L . fermentum reduced the production of proteins related to arginyl-tRNA synthetase (protein syhthesis) and aspartate- semialdehyde dehydrogenase (glycine, serine and threonine metabolism and lysine biosynthesis) when exposed to the lumen of the rabbit jejunum (59). The contradictory results may be due to the physiological characteristics of different Lactobacillus species, different animal models or diets used. Recent work on the functionality of the human ileostomy effluent microbiota using the metatranscriptomics approach revealed that about 25% of the sequences retrieved from the mRNA enriched cDNA- amplified fragment length polymorphism profiles were related to metabolism and about 20% of these metabolism- related genes encoded proteins participating in transport and metabolism of AA (20). Examples include alanine dehydrogenase produced from bacteria belonging to the order of Bacteriodales, aspartate kinase from the order of Clostridiales, glutamate synthase and AA transporters from the order of Lactobacillales, and succinylglutamate desuccinylase from the order of Enterobacteriales (20). These results indicate that bacteria in the small intestine are active in the metabolism of nitrogenous compounds and have developed adaptive strategies to survive and propagate during the co-evolution with its host. Increasing evidence indicates that the gut microbiota plays an important role in the physiology and metabolism of enterocytes, thereby regulating the nitrogen recycling within the gut and the whole body nitrogen metabolism. For example, during in vitro incubation with L . fermentum, Caco-2 cells increased the production of proteins that were beneficial for gut integrity, including voltage-dependent anion channel 1, glutathione transferase, and heat shock protein gp96 (59). Glutathione transferase family enzymes detoxify xenobiotics and, therefore, reduce the oxidative stress of cells (60). These enzymes can also remove toxic metabolites and may interact with ATP-binding cassette transporters (60, 61). These findings suggest that enterocytes could respond and adapt to the gut microbiota either by regulating the transport of nutrients or by transforming diet- or gut bacteria-derived substances to sustain mucosal integrity and function. Therefore, enterocytes likely develop strategies to maintain their protein balance by controlling nitrogen cycling within the gut, while benefiting its bacterial ecosystem. The anabolism and catabolism of AA by both enterocytes and perhaps gut bacteria limit the availability of dietary AA to extraintestinal tissues (44, 47). Meanwhile, nitrogenous substances (e.g., digestive enzymes, bile, mucins, cell debris and urea) secreted into the gut lumen are subjected to digestion or fermentation in the small intestine and the large intestine (5, 7). It is likely that gut bacteria utilize the ammonia generated from the catabolism of extracellular and intracellular proteins, AA, or urea for the synthesis of bacterial protein (5, 6, 7, 37, 43, 44). This could be regarded as a form of nitrogen recycling, which is of nutritional importance especially for animals fed a low- protein diet. Although the absorption of AA is limited in the large intestine (44), AA synthesized in this segment of the gut may provide AA to luminal bacteria (7, 62). At the whole-body level, the modulation of host metabolism by gut microbiota may occur in multiple tissues and cell types (Figure 1). Studies aimed at comparing the multi-compartmental metabolic profiles of conventional mice and its germ-free counterparts showed that, in the presence of gut microbiota, concentrations of creatinine in urine, of hypotaurine in the liver, of alanine and creatine in the small intestine were substantially reduced (62). However, the presence of the gut microbiota resulted in increased levels of oxidized glutathione in the liver, of tyrosine, glutamate, alanine and aspartate in the small intestine, and of creatine, glutamine, and aspartate in the colon (62). It is well known that creatinine and creatine are closely related to muscle mass and arginine metabolism (63). Also, glutamine/glutamate, aspartate, and glutathione play important role in the regulation of nitrogen and energy balance in tissues and at the whole body, including the fluxes of the citric acid cycle and the urea cycle, as well as protein synthesis and degradation (2, 3, 48, 62, 64, 65). Therefore, it could be concluded that nitrogen and AA metabolism in the gut microbiota and the underlying metabolic interactions with the host may be important in the regulation of host protein and energy balance. Based on marked differences in intestinal microbial AA metabolism among subjects, the concept of personalized requirements of dietary proteins should not be neglected (8). However, the linkage between the multi-compartmental metabolic profiles and host health/disease is still not clear. The development of certain biomarkers and databases will help to better understand, predict, prevent and treat life- threatening metabolic diseases. Apart from the nutritional importance of nitrogenous substances to the growth of gut bacteria, AA metabolism in gut bacteria may serve as an important “survival strategy” for the bacteria to adapt and survive in the gut and also “cross-talk” to their neighboring species and eukaryotic host. Therefore, it is important to define the role of AA metabolism in the survival of gut bacteria as well as the impact to their surrounding environments and the consequences on host health and disease. Gut bacteria have many mechanisms to cope with harsh, variable, rapidly changing environment of the digestive tract. Stresses, such as low pH, nutrient limitation, and starvation, are now taken into consideration (66). It was found that bacteria in the dental plaque ecosystems maintained the neutral pH of their environment (acid- neutralizing activity) by the catabolism of different AA thus showed a beneficial effect to the prevention of caries. For instance, in Fusobacterium nuleatum, rates of glutamate and aspartate fermentation as well as ammonia production were higher at pH 5.0 than at pH 5.5 (67). In Streptococcus gordonii , the expression of arginine deiminase was induced by low pH, which resulted in elevated production of ammonia (68). These findings can aid in development of new strategies to prevent caries and improve oral health (67, 68). In the GI tract, a low pH can be induced by either hydrochloric acid secreted from the stomach epithelium or short chain fatty acids (formerly known as volatile fatty acids, VFA) from the bacterial fermentation of carbohydrates. This may result in acid stress in gut bacteria, therefore affecting the survival and gut transition of potential pathogenic bacteria. Early studies demonstrated that the decarboxylation of gamma-aminobutyric acid (GABA) and putrescine by Bacteroides fragilis was optimal under acidic conditions (pH 6.0) (69). When the pH of the medium was changed from 7.0 to 6.0, the preferred AA for decarboxylation in Clostridium perfringens shifted from arginine and GABA to lysine and putrescine (69). In Escherichia coli , two acid resistance systems have been discovered namely the acid resistance system 2 and 3 (AR2 and AR3), depending on the catabolism of AA (70). The AR2 and AR3 require extracellular glutamate and arginine, respectively. However, both AA can be decarboxylated to alter the membrane potential from a net inside negative to a net inside positive charge, leading to an increase in internal pH ( ∆ pH) (70). The intestinal metabolism of AA may also be important for the survival of gut bacteria under conditions of nutrient limitation and starvation during gut transition. When oxygen is limiting, the production of aspartase increased in Campylobacter jejuni (71). As a consequence, the conversion of aspartate to fumarate and ammonia was augmented with subsequent increases in fumarate utilization and bacterial growth (71). In C . jejuni , aspartate is one of the key substances in the metabolic pathway that controls the utilization (including catabolism) of AA and ATP production. Therefore, aspartase is critical for the survival of C . jejuni in the lumen of the small intestine under anaerobic conditions (71). In the large intestine, low levels of luminal nutrients can trigger the starvation and death of bacteria especially in the presence of high concentrations of ...

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... macromolecules (e.g., glycan ligands) mediate signaling pathways in gut epithelial cells and regulate the host immune function (73- 75). Additionally, it has been reported that the Rgg proteins produced by the pathogenic Streptococcus pyogenes strain regulate the synthesis and metabolism of virulence factors in the bacteria and the effects are associated with the catabolism of arginine and serine (76). In Streptococcus bovis HC5, the production of a broad-spectrum antibiotic bovicin HC5 inhibited the growth of pure cultures of hyper ammonia-producing bacteria as well as AA degradation and ammonia production by mixed ruminal bacteria (77). Recent findings have indicated that some small molecules produced by gut bacteria play crucial role in quorum sensing and biological processes, such as biofilm formation and expression of virulence factors (78). Among these, the acyl homoserine lactones (AHLs) and autoinducer-2 (AI-2) are derived from S-adenosyl methionine, an important metabolite of methionine (79). It is now known that AI-2 is essential for the adherence to HeLa cells, mobility, and expression of virulence genes in enteropathogenic E . coli (80). In addition, formation of AI- 2 affects the attachment of L . acidophilus to Caco-2 cells in vitro (81). The production of AI-2 in Streptococcus suis is stimulated in the addition of 0.5% sodium chloride or glucose (82). Furthermore, E . coli interact with neighboring species by adjusting the production and consumption of AI- 2 (83). Because AI-2 is produced and recognized both by gram-positive and gram-negative bacteria, pro- and anti- AI-2 interactions among gut bacteria and their association with the eukaryotic host within the digestive tract can affect the microbial community composition and host health (78). Current data suggest that AHL molecules mediate the interactions between gut bacteria and the host. Also, N-3-oxododecanoyl homoserine lactone produced by Pseudomonas aeruginosa accelerates apoptosis in macrophages and neutrophils, while possessing an immunomodulatory effect on human peripheral blood mononuclear cells (84, 85). Conversely, as one of defense mechanisms, host cells can inactivate (via degradation) the AHL molecules produced by P . aeruginosa in vitro (86). Although it is not clear whether similar AHLs-mediated interactions occur within the digestive tract, the above findings raise important questions about the physiological significance of the microbial metabolites of methionine and related AA. Namely, can these metabolites serve as potential biomarkers for monitoring the microbe-microbe and microbe-host interactions at different niches along the digestive tract, as well as the health status and disease of the host. Identification of the multi-compartmental metabolic profiles of quorum-sensing signals and related molecules will aid in the development of diagnostic and therapeutic tools for the well-beings of both humans and animals (87). Polycationic nitrogenous substances (e.g., polyamines) are important products of AA metabolism in luminal microbes of the small intestine. Ornithine, which is derived from arginine, proline, glutamate, and glutamine, is decarboxylated by ornithine decarboxylase to produce putrescine. Putrescine is also formed from arginine sequentially via arginine decarboxylase (to yield agmatine) and decarboxylation of agmatine by agmatinase (to generate putrescine). Putrescine is converted into spermidine and spermine via spermidine synthase and spermine synthase, respectively. Polyamines have long been recognized to important roles in cell physiology. For example, polyamines act as antioxidants to protect cells from oxidative damage. As positively charged compounds, polyamines participate in many cellular processes through binding with RNA, DNA, nucleotide triphosphate, proteins, and other negatively charged molecules. Through these interactions, polyamines regulate gene expression, signal transduction, ion channel function, DNA and protein synthesis, and apoptosis. Thus, polyamines are essential for proliferation, differentiation, and function of bacteria and enterocytes. When cells are stimulated with growth factors, one of the first crucial events is the induction of polyamine synthesis, which precedes increases in DNA replication and protein synthesis. Thus, an increase in polyamine levels is associated with enhanced growth of intestinal mucosal cells and microorganisms. Conversely, depletion of cellular polyamines arrests cell growth. Bacteria thriving in the digestive tract mainly rely on the utilization of substances of food origin. Therefore, diets have profound influence on bacterial metabolism, and specific dietary components may have selective effects on the population and metabolism of the microbiota. Over the last decades, there has been interest in effects of some dietary factors, such as protein, non- digestible carbohydrates, probiotics, synbiotics or phytochemicals on gut bacteria and host health (Figure 1). These results are highlighted in the following sections. In the small intestine of mono-gastric animals, the daily requirement of AA for the first-pass metabolism and absorption of AA are constant under normal conditions. When AA are supplemented to the diet, the entry of AA into the portal vein increases (88). However, adaptive metabolism of the small intestine may occur in response to dietary protein intake. When high protein intake exceeds the capacity of digestive enzymes and AA transporters, some of protein and AA in the diet would enter the large intestine. To date, data on the regulation of AA metabolism in the small intestine and its luminal bacteria is limited. Also, little is known about high protein diet on the hindgut ecology (19). Research showed that approximately 3-12 grams of dietary protein and peptides entered the human large intestine every day and served as nitrogen sources for the gut microbiota (14, 89). High levels of proteins and peptides in the large intestine could lead to an increased production of ammonia due to the actions of proteases, peptidases, deaminases, and deiminase produced by gut bacteria (23, 90, 91). When subjects are fed a high-protein diet, levels of sulfide and branched-chain fatty acids are elevated due to the bacterial fermentation of sulfur- containing AA and branched-chain AA (92, 93), but butyrate concentrations and numbers of butyrate-producing bacteria are decreased in the large intestine as well in the feces (94). It is widely regarded that butyrate is the main energy source for colonic epithelial cell, thus, a decrease in butyrate concentration and an increase in concentrations of ammonia and sulfide may explain the detrimental effect of high protein diet on the large intestine (e.g., increased incidence of colon cancer). The structure and AA composition of proteins can affect their hydrolysis in the GI tract and nitrogen metabolism in gut bacteria. Results of in vitro study indicate that compared with casein, sulfate production from the bacterial fermentation of bovine serum albumin is higher and that dietary protein from the meat is an important substrate for sulfide generation (93). In addition, AA, such as tryptophan, proline, tyrosine, and isoleucine, strongly inhibit the protease activity of Clostridium sporogenes (90). In Selenomonas ruminantium , the urease activity is higher when serine or threonine is used as a substrate. However, urease activity decreases markedly when histidine is added to medium (95). These findings suggest that proteins, peptides or AA in the large intestine not only affect the production of toxic substances, but also regulate the bacterial fermentation, AA degradation, and nitrogen recycling. Furthermore, the active metabolism of nitrogenous substances by bacteria in the digestive tract especially in the large intestine is also influenced by the availability and structure of carbohydrates (4, 89). Non-digestible carbohydrates (NDC) or dietary fibers are normal constituents of most foods derived from plants. They escape digestion in the small intestine as the host lacks the necessary degrading enzymes and pass into the large intestine. NDC include polysaccharides such as resistant starch, pectin, inulin, guar gum, wheat bran, cellulose, lignin, and oligosaccharides (89). According to the definition, prebiotics belong to the category of NDC and selectively stimulate the growth of potentially beneficial bacteria such as Bifidobacterium and Lactobacillus (89, 96). NDC affect the AA metabolism in gut bacteria either by regulating the bacterial composition and abundance or by providing the carbon source for the growth of microorganisms. In the three-stage continuous culture, the addition of inulin and galacto-oligosaccharides increased the numbers of lactobacilli in the proximal colon, together with a small increase in bifidobacteria, peptostreptococci, enterococci and Clostridium perfringens (97). The two carbohydrates stimulated the synthesis of nitroreductase and azoreductase (97). At neutral pH, the addition of starch to the media significantly decreased the bacterial production of amines, phenolic and indolic compounds, and branched-chain fatty acids both by pure and mixed bacteria cultures (14, 53, 69, 98). Similarly, the reduced production of phenolic and indolic compounds was reported for oligofructose added to the pure cultures of Bifidobacterium and Bacteroides (55). Recent studies on the effect of prebioctics on AA metabolism at the whole- body level revealed that galacto-oligosaccharides increased the urinary excretion of carnitine and taurine as well as fecal excretion of glycine in human infants, while the addition of prebiotics decreased the urinary excretion of lysine and alpha-keto-isocaproate (99). The findings indicate that NDC affect AA metabolism within the digestive tract and at the whole-body level. However, the underlying mechanism and effects on are not clear. Nonetheless, many gut bacteria ...