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Metabolism of ornithine from diet confers competitive advantage to Cd and leads to reduced host inflammation a, The ornithine oxidative degradation pathway is significantly enriched in mice monocolonized with WT Cd on standard mouse chow compared to mice monocolonized with WT Cd provided a fully defined diet lacking ornithine (row-normalized z score for microarray data from ref. ³²; n = 4 mice per group). b, WT Cd has a competitive advantage in conventional mice over ∆oraSE strain in standard diet background, but not in a fully defined diet devoid of ornithine (unpaired two-tailed Student’s t-test, n = 3 mice per group; mean ± s.e.m.). c, 1% ornithine supplementation (w/v) to conventional mice on a fully defined ornithine-free diet provides a competitive advantage to WT Cd (pairwise Student’s t-tests; mean ± s.e.m., n = 5 mice per group). d, WT Cd achieves a higher absolute abundance than ∆oraSE in cecal contents of gnotobiotic mice fed an ornithine-free diet supplemented with ornithine in drinking water. c.f.u., colony-forming unit. e,f, ∆oraSE infection leads to higher levels of lipocalin-2 in serum in gnotobiotic mice harboring a defined consortium of bacteria (e) or conventional mice fed standard diet (f). For d–f data were analyzed by unpaired two-tailed Student’s t-tests; mean ± s.e.m., n = 5 mice per group. Source data
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The enteric pathogen Clostridioides difficile ( Cd ) is responsible for a toxin-mediated infection that causes more than 200,000 recorded hospitalizations and 13,000 deaths in the United States every year ¹ . However, Cd can colonize the gut in the absence of disease symptoms. Prevalence of asymptomatic colonization by toxigenic Cd in healthy popul...
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... The reason is related to the fact that ornithine is important nutrient utilized by C. difficile for its colonization. Ornithine oxidative metabolism in C. difficile supports its non-inflammatory asymptomatic colonization in mice 40 . In addition, the modulation of the availability of arginine and ornithine levels by T.mu are likely to be sensed by both the host and the gut microbial community, which can, in return, influence C. difficile functions. ...
... This is correlated with a relatively reduced ornithine level in gut lumen of T.mu-colonized mice after CDI (Fig. 5F and Supplementary Fig. 6A). In accordance with this, it has been reported that mice resistant to CDI and inflammation exhibit enhanced ornithine oxidative metabolism and have relatively lower ornithine level in the gut lumen 40 . Clearly, there is still much to be learned about the complex interplay between T.mu and C. difficile. ...
... These lines of evidence suggest that modulating intestinal arginine metabolism can influence the host immunity at least during CDI. In addition, previous reports indicate an effect of ornithine and arginine on C. difficile virulence 34,40,45,46 . Consistently, our data show that dietary manipulation of arginine/ornithine impacts C. difficile toxin levels. ...
Antibiotic-induced dysbiosis is a major risk factor for Clostridioides difficile infection (CDI), and fecal microbiota transplantation (FMT) is recommended for treating CDI. However, the underlying mechanisms remain unclear. Here, we show that Tritrichomonas musculis (T.mu), an integral member of the mouse gut commensal microbiota, reduces CDI-induced intestinal damage by inhibiting neutrophil recruitment and IL-1β secretion, while promoting Th1 cell differentiation and IFN-γ secretion, which in turn enhances goblet cell production and mucin secretion to protect the intestinal mucosa. T.mu can actively metabolize arginine, not only influencing the host’s arginine-ornithine metabolic pathway, but also shaping the metabolic environment for the microbial community in the host’s intestinal lumen. This leads to a relatively low ornithine state in the intestinal lumen in C. difficile-infected mice. These changes modulate C. difficile’s virulence and the host intestinal immune response, and thus collectively alleviating CDI. These findings strongly suggest interactions between an intestinal commensal eukaryote, a pathogenic bacterium, and the host immune system via inter-related arginine-ornithine metabolism in the regulation of pathogenesis and provide further insights for treating CDI.
... Consistent with these findings, another study found that arginineornithine metabolism was a top pathway in individuals colonized by C. difficile (Pruss et al., 2022). E. faecalis also significantly increased uropathogenic E. coli biofilm growth and survival in vitro and in vivo in a mouse wound infection model by exporting L-ornithine (Keogh et al., 2016). ...
Arginine‐ornithine metabolism plays a crucial role in bacterial homeostasis, as evidenced by numerous studies. However, the utilization of arginine and the downstream products of its metabolism remain undefined in various gut bacteria. To bridge this knowledge gap, we employed genomic screening to pinpoint relevant metabolic targets. We also devised a targeted liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) metabolomics method to measure the levels of arginine, its upstream precursors, and downstream products in cell‐free conditioned media from enteric pathobionts, including Escherichia coli, Klebsiella aerogenes, K. pneumoniae, Pseudomonas fluorescens, Acinetobacter baumannii, Streptococcus agalactiae, Staphylococcus epidermidis, S. aureus, and Enterococcus faecalis. Our findings revealed that all selected bacterial strains consumed glutamine, glutamate, and arginine, and produced citrulline, ornithine, and GABA in our chemically defined medium. Additionally, E. coli, K. pneumoniae, K. aerogenes, and P. fluorescens were found to convert arginine to agmatine and produce putrescine. Interestingly, arginine supplementation promoted biofilm formation in K. pneumoniae, while ornithine supplementation enhanced biofilm formation in S. epidermidis. These findings offer a comprehensive insight into arginine‐ornithine metabolism in enteric pathobionts.
... The gene for ornithine carbamoyltransferase, which modulates ornithine levels, is one such gene. Microbes capable of breaking down ornithine demonstrate a competitive advantage [66]. This raises the question: could this enzyme also confer a survival benefit to Sutterella wadsworthensis in a healthy gut, and might SwASST4 play a role in this dynamic? ...
Sulfonation, primarily facilitated by sulfotransferases, plays a crucial role in the detoxification pathways of endogenous substances and xenobiotics, promoting metabolism and elimination. Traditionally, this bioconversion has been attributed to a family of human cytosolic sulfotransferases (hSULTs) known for their high sequence similarity and dependence on 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as a sulfo donor. However, recent studies have revealed the presence of PAPS-dependent sulfotransferases within gut commensals, indicating that the gut microbiome may harbor a diverse array of sulfotransferase enzymes and contribute to detoxification processes via sulfation. In this study, we investigated the prevalence of sulfotransferases in members of the human gut microbiome. Interestingly, we stumbled upon PAPS-independent sulfotransferases, known as aryl-sulfate sulfotransferases (ASSTs). Our bioinformatics analyses revealed that members of the gut microbial genus Sutterella harbor multiple asst genes, possibly encoding multiple ASST enzymes within its members. Fluctuations in the microbes of the genus Sutterella have been associated with various health conditions. For this reason, we characterized 17 different ASSTs from Sutterella wadsworthensis 3_1_45B. Our findings reveal that SwASSTs share similarities with E. coli ASST but also exhibit significant structural variations and sequence diversity. These differences might drive potential functional diversification and likely reflect an evolutionary divergence from their PAPS-dependent counterparts.
... Many of these impact C. difficile fitness and pathogenesis. For example, bile acids, metals, amino acids, sugars, organic acids, and short-chain fatty acids (SCFAs) affect C. difficile in vitro and in animal models (4)(5)(6)(9)(10)(11)(12)(13)(14)(15). SCFAs (in particular, acetate, propionate, and butyrate) are major metabolic end-products of microbiome metabolism and are primarily generated by microbial degradation of dietary fiber. ...
The gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile, but the molecular basis of this colonization resistance is incompletely understood. A prominent class of gut microbiome-produced metabolites important for colonization resistance against C. difficile is short-chain fatty acids (SCFAs). In particular, one SCFA (butyrate) decreases the fitness of C. difficile in vitro and is correlated with C. difficile -inhospitable gut environments, both in mice and in humans. Here, we demonstrate that butyrate-dependent growth inhibition in C. difficile occurs under conditions where C. difficile also produces butyrate as a metabolic end product. Furthermore, we show that exogenous butyrate is internalized into C. difficile cells and is incorporated into intracellular CoA pools where it is metabolized in a reverse (energetically unfavorable) direction to crotonyl-CoA and ( S )−3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA. This internalization of butyrate and reverse metabolic flow of a butyrogenic pathway(s) in C. difficile coincides with alterations in toxin release and sporulation. Together, this work highlights butyrate as a marker of a C. difficile -inhospitable environment to which C. difficile responds by releasing its diarrheagenic toxins and producing environmentally resistant spores necessary for transmission between hosts. These findings provide foundational data for understanding the molecular and genetic basis of how C. difficile growth is inhibited by butyrate and how butyrate alters C. difficile virulence in the face of a highly competitive and dynamic gut environment.
IMPORTANCE
The gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile, but the molecular basis of this colonization resistance is incompletely understood, which hinders the development of novel therapeutic interventions for C. difficile infection (CDI). We investigated how C. difficile responds to butyrate, an end-product of gut microbiome community metabolism which inhibits C. difficile growth. We show that exogenously produced butyrate is internalized into C. difficile , which inhibits C. difficile growth by interfering with its own butyrate production. This growth inhibition coincides with increased toxin release from C. difficile cells and the production of environmentally resistant spores necessary for transmission between hosts. Future work to disentangle the molecular mechanisms underlying these growth and virulence phenotypes will likely lead to new strategies to restrict C. difficile growth in the gut and minimize its pathogenesis during CDI.
... For instance, ornithine enters the urea cycle in the body and is a precursor of arginine, citrulline, and proline, as well as a direct precursor of polyamines (spermidine, spermine, and putrescine), which can promote growth hormone secretion and young animal growth. Polyamines are momentary sources of cellular energy that reduce intestinal damage and lower inflammation, thereby alleviating weaning stress (Pruss et al., 2022;Wang et al., 2021). In addition, ornithine significantly increases the gene expression of CD36 in the liver, which alleviates chronic inflammation induced by obesity (Park et al., 2020). ...
Fat is one of the three macronutrients and a significant energy source for piglets. It plays a positive role in maintaining intestinal health and improving production performance. During the weaning period, physiological, stress and diet-related factors influence the absorption of fat in piglets, leading to damage to the intestinal barrier, diarrhea and even death. Signaling pathways, such as fatty acid translocase (CD36), pregnane X receptor (PXR), and AMP-dependent protein kinase (AMPK), are responsible for regulating intestinal fat uptake and maintaining intestinal barrier function. Therefore, this review mainly elaborates on the reasons for diarrhea induced by insufficient fat absorption and related signaling pathways in weaned-piglets, with an emphasis on the intestinal fat absorption disorder. Moreover, we focus on introducing nutritional strategies that can promote intestinal fat absorption in piglets with insufficient fat absorption-related diarrhea, such as lipase, amino acids, and probiotics.
... We did observe a decrease in arginine/ornithine metabolism in nutrient-limited conditions due to the presence of bile acid-altering enzymes. Ornithine metabolism is important for gut pathogen colonization including C. difficile physiology and pathogenesis (56,57). However, a limitation of our study is that we did not test for changes in the global metabolic transcriptome in the knockout strain due to bile acids and therefore cannot determine if the absence of bile acid-altering enzymes impacts amino acid metabolism of amino acids freed by bile salt hydrolases. ...
Bacteroides thetaiotaomicron ( B. theta ) is a Gram-negative gut bacterium that encodes enzymes that alter the bile acid pool in the gut. Primary bile acids are synthesized by the host liver and are modified by gut bacteria. B. theta encodes two bile salt hydrolases, as well as a hydroxysteroid dehydrogenase. We hypothesize that B. theta modifies the bile acid pool in the gut to provide a fitness advantage for itself. To investigate each gene’s role, different combinations of genes encoding bile acid-altering enzymes ( bshA , bshB , and hsdhA ) were knocked out by allelic exchange, including a triple KO. Bacterial growth and membrane integrity assays were done in the presence and absence of bile acids. To explore if B. theta’s response to nutrient limitation changes due to the presence of bile acid-altering enzymes, RNA sequencing (RNASeq) analysis of wild type (WT) and triple knockout (KO) strains in the presence and absence of bile acids was done. WT B. theta is more sensitive to deconjugated bile acids such as cholate, chenodeoxycholate (CDCA), and deoxycholate (DCA) compared with the triple KO. These deconjugated bile acids also decreased membrane integrity of both WT and triple KO. The presence of bshB is detrimental to growth in conjugated forms of CDCA and DCA. RNASeq analysis also showed that bile acid exposure impacts multiple metabolic pathways in B. theta , but DCA significantly increases expression of many genes in carbohydrate metabolism, specifically those in polysaccharide utilization loci or PULs, in nutrient-limited conditions. This study suggests that bile acids B. theta encounters in the gut may signal the bacterium to increase or decrease its utilization of carbohydrates. Further study looking at the interactions between bacteria, bile acids, and the host may inform rationally designed probiotics and diets to ameliorate inflammation and disease.
IMPORTANCE
Recent work on bile salt hydrolases (BSHs) in Gram-negative bacteria, such as Bacteroides, has primarily focused on how they can impact host physiology. However, the benefits bile acid metabolism confers to the bacterium that performs it are not well understood. In this study, we set out to define if and how Bacteroides thetaiotaomicron ( B. theta ) uses its BSHs and hydroxysteroid dehydrogenase to modify bile acids to provide a fitness advantage for itself in vitro and in vivo . Genes encoding bile acid-altering enzymes were able to impact how B. theta responds to nutrient limitation in the presence of bile acids, specifically carbohydrate metabolism, affecting many polysaccharide utilization loci. This suggests that B. theta may be able to shift its metabolism, specifically its ability to target different complex glycans including host mucin, when it comes into contact with specific bile acids in the gut.
... Polyamines are important to both prokaryotic and eukary otic biology (43,44). C. difficile was recently shown to use host-derived ornithine, the precursor of polyamines, to support non-inflammatory colonization (45). A separate study found high levels of ornithine in the GI tracts of CDI model mice and CDI patients, and supplementation of drinking water with arginine, the precursor for ornithine, decreased C. difficile pathogenesis in mice (46). ...
Clostridioides difficile is the most common cause of nosocomial gastrointestinal tract bacterial infections. We lack fully effective reliable treatments for this pathogen, and there is a critical need to better understand how C. difficile interacts with our immune system. Group 3 innate lymphocytes (ILC3s) are rare immune cells localized within mucosal tissues that protect against bacterial infections. Upon activation, ILC3s secrete high levels of the cytokine interleukin-22 (IL-22), which is a critical regulator of tissue responses during infection. C. difficile toxin B (TcdB), the major virulence factor, directly activates ILC3s, resulting in high IL-22 levels. We previously reported that polyamines are important in the activation of ILC3s by the innate cytokine interleukin-23 (IL-23) but did not identify a specific mechanism. In this study, we examine how a pathogen impacts a metabolic pathway important for immune cell function and hypothesized that polyamines are important in TcdB-mediated ILC3 activation. We show that TcdB upregulates the polyamine biosynthesis pathway, and the inhibition of the pathway decreases TcdB-mediated ILC3 activation. Two polyamines, putrescine and spermidine, are involved. Spermidine is the key polyamine in the hypusination of eukaryotic initiation factor 5A (eIF5A), and the inhibition of eIF5A reduced ILC3 activation. Thus, there is potential to leverage polyamines in ILC3s to promote activation of ILC3s during C. difficile infection and other bacterial infections where ILC3s serve a protective role.
... The morphological colony characteristics of pure isolates, including shape, margin, size, and color, were assessed on nutrient agar medium. Manual work was used to perform the biochemical analysis, which included the assays of catalase [20], urease [21], citrate [22], and motility in an ornithine medium [23]. By the manufacturer's instructions, standard biochemical tests like lysine iron agar, motility, flagella, methyl red (MR), Voges Proskauer (VP), H 2 S, gas from glucose, nitrate reduction, gas, oxidase, and pigment were also conducted [24]. ...
The rate of infectious diseases started to be one of the major mortality agents in the healthcare sector. Exposed to increased bacterial infection by antibiotic-resistant bacteria became one of the complications that occurred for bone marrow transplant patients. Nanotechnology may provide clinicians and patients with the key to overcoming multidrug-resistant bacteria. Therefore, this study was conducted to clarify the prevalence of MDR bacteria in bone marrow transplant recipients and the use of Ag 2 O/ZnO nanocomposites to treat participants of diarrhea brought on by MDR bacteria following bone marrow transplantation (BMT). Present results show that pathogenic bacteria were present in 100 of 195 stool samples from individuals who had diarrhea. Phenotypic, biochemical, and molecular analysis clarify that Proteus mirabilis and Salmonella typhi were detected in 21 and 25 samples, respectively. Successful synthesis of Ag 2 O/ZnO nanocomposites with a particle enables to inhibition of both pathogens. The maximum inhibitory impact was seen on Salmonella typhi . At low doses (10 ⁻⁵ g/l), it prevented the growth by 53.4%, while at higher concentrations (10 ⁻¹ g/l), Salmonella typhi was inhibited by 95.5%. Regarding Proteus mirabilis , at (10 ⁻⁵ g/l) Ag 2 O/ZnO, it was inhabited by 78.7%, but at higher concentrations (10 ⁻¹ g/l), it was inhibited the growth by 94.6%. Ag 2 O/ZnO nanocomposite was therefore found to be the most effective therapy for MDR-isolated bacteria and offered promise for the treatment of MDR bacterial infections that cause diarrhea.
... Our stable isotope analysis indicated that ornithine is primarily an end product of growth and is relatively inaccessible as a carbon source for E. lenta. However, ornithine is a favorable carbon and/or energy source for numerous other gut microbes [23], including as a substrate for Stickland metabolism by gut bacteria including Clostridioides difficile [6,66,67]. Therefore, production of ornithine by E. lenta may promote the growth of other proteolytic bacteria in the surrounding gut ecosystem. ...
Human gut bacteria perform diverse metabolic functions with consequences for host health. The prevalent and disease-linked Actinobacterium Eggerthella lenta performs several unusual chemical transformations, but it does not metabolize sugars and its core growth strategy remains unclear. To obtain a comprehensive view of the metabolic network of E. lenta, we generated several complementary resources: defined culture media, metabolomics profiles of strain isolates, and a curated genome-scale metabolic reconstruction. Stable isotope-resolved metabolomics revealed that E. lenta uses acetate as a key carbon source while catabolizing arginine to generate ATP, traits which could be recapitulated in silico by our updated metabolic model. We compared these in vitro findings with metabolite shifts observed in E. lenta-colonized gnotobiotic mice, identifying shared signatures across environments and highlighting catabolism of the host signaling metabolite agmatine as an alternative energy pathway. Together, our results elucidate a distinctive metabolic niche filled by E. lenta in the gut ecosystem. Our culture media formulations, atlas of metabolomics data, and genome-scale metabolic reconstructions form a freely available collection of resources to support further study of the biology of this prevalent gut bacterium.
... Enterococcus species, such as Enterococcus faecalis, convert arginine to ornithine using the arginine deiminase pathway. The resulting gut lumen featuring high ornithine and low arginine favors C. difficile pathogenesis: C. difficile ferments ornithine for energy; meanwhile, arginine limitation may provide an environment cue for C. difficile to increase toxin production (Pruss et al., 2022;Smith et al., 2022). Other than Enterococci, commensal gut bacteria such as Clostridium sardiniense and Paraclostridium bifermentans can modulate C. difficile infection via the same pathway (Girinathan et al., 2021). ...
Clostridioides difficile is a gram-positive, spore-forming, obligate anaerobe that infects the colon. C. difficile is estimated to cause nearly half a million cases in the United States annually, with about 29,000 associated deaths. Unfortunately, the current antibiotic treatment is not ideal. While antibiotics can treat the infections, they also disrupt the gut microbiota that mediates colonization resistance against enteric pathogens, including C. difficile; disrupted gut microbiota provides a window of opportunity for recurrent infections. Therefore, therapeutics that restore the gut microbiota and suppress C. difficile are being evaluated for safety and efficacy. This review will start with mechanisms by which gut bacteria affect C. difficile pathogenesis, followed by a discussion on biotherapeutics for recurrent C. difficile infections.
