P-cresol Production Research Articles

Catalytic Mechanism of the Glycyl Radical Enzyme 4-Hydroxyphenylacetate Decarboxylase from Continuum Electrostatic and QC/MM Calculations

Using continuum electrostatics and QC/MM calculations, we investigate the catalytic cycle of the glycyl radical enzyme 4-hydroxyphenylacetate decarboxylase, an enzyme involved in the fermentative production of p-cresol from tyrosine in clostridia. On the basis of our calculations, we propose a five-step mechanism for the reaction. In the first step, the substrate 4-hydroxyphenylacetate is activated by an unusual concerted abstraction of an electron and a proton. Namely, Cys503 radical abstracts an electron from the substrate and Glu637 abstracts a proton. Thus in total, a hydrogen atom is abstracted from the substrate. In the second step, the carboxylic group readily splits off from the phenoxy-acetate radical anion to give carbon dioxide. This decarboxylation step is coupled to a proton transfer from Glu637 back to the phenolic hydroxyl group which leads to a p-hydroxybenzyl radical. The remaining steps of the reaction involve a rotation of the Cys503 side chain followed by a proton transfer from Glu505 to Cys503 and a hydrogen atom transfer from Cys503 to the p-hydroxybenzyl radical to form p-cresol. The calculated mechanism agrees with experimental data suggesting that both Cys503 and Glu637 are essential for the catalytic function of 4-hydroxyphenylacetate decarboxylase and that the substrate requires a hydroxyl group in para-position to the acetate moiety.

P-Cresyl Sulfate Promotes Insulin Resistance Associated with CKD

The mechanisms underlying the insulin resistance that frequently accompanies CKD are poorly understood, but the retention of renally excreted compounds may play a role. One such compound is p-cresyl sulfate (PCS), a protein-bound uremic toxin that originates from tyrosine metabolism by intestinal microbes. Here, we sought to determine whether PCS contributes to CKD-associated insulin resistance. Administering PCS to mice with normal kidney function for 4 weeks triggered insulin resistance, loss of fat mass, and ectopic redistribution of lipid in muscle and liver, mimicking features associated with CKD. Mice treated with PCS exhibited altered insulin signaling in skeletal muscle through ERK1/2 activation. In addition, exposing C2C12 myotubes to concentrations of PCS observed in CKD caused insulin resistance through direct activation of ERK1/2. Subtotal nephrectomy led to insulin resistance and dyslipidemia in mice, and treatment with the prebiotic arabino-xylo-oligosaccharide, which reduced serum PCS by decreasing intestinal production of p-cresol, prevented these metabolic derangements. Taken together, these data suggest that PCS contributes to insulin resistance and that targeting PCS may be a therapeutic strategy in CKD.

The analysis of para-cresol production and tolerance in Clostridium difficile 027 and 012 strains

BackgroundClostridium difficile is the major cause of antibiotic associated diarrhoea and in recent years its increased prevalence has been linked to the emergence of hypervirulent clones such as the PCR-ribotype 027. Characteristically, C. difficile infection (CDI) occurs after treatment with broad-spectrum antibiotics, which disrupt the normal gut microflora and allow C. difficile to flourish. One of the relatively unique features of C. difficile is its ability to ferment tyrosine to para-cresol via the intermediate para-hydroxyphenylacetate (p-HPA). P-cresol is a phenolic compound with bacteriostatic properties which C. difficile can tolerate and may provide the organism with a competitive advantage over other gut microflora, enabling it to proliferate and cause CDI. It has been proposed that the hpdBCA operon, rarely found in other gut microflora, encodes the enzymes responsible for the conversion of p-HPA to p-cresol.ResultsWe show that the PCR-ribotype 027 strain R20291 quantitatively produced more p-cresol in-vitro and was significantly more tolerant to p-cresol than the sequenced strain 630 (PCR-ribotype 012). Tyrosine conversion to p-HPA was only observed under certain conditions. We constructed gene inactivation mutants in the hpdBCA operon in strains R20291 and 630Δerm which curtails their ability to produce p-cresol, confirming the role of these genes in p-cresol production. The mutants were equally able to tolerate p-cresol compared to the respective parent strains, suggesting that tolerance to p-cresol is not linked to its production.ConclusionsC. difficile converts tyrosine to p-cresol, utilising the hpdBCA operon in C. difficile strains 630 and R20291. The hypervirulent strain R20291 exhibits increased production of and tolerance to p-cresol, which may be a contributory factor to the virulence of this strain and other hypervirulent PCR-ribotype 027 strains.

Pediocin A modulates intestinal microflora metabolism in swine in vitro intestinal fermentations

The aim of this study was to investigate in vitro the effects of pediocin A [a bacteriocin produced by lactic acid bacteria (LAB) Pediococcus pentosaceus FBB61] on microbial metabolism in the small and large intestine of pigs. Pediocin A was partially purified by ion exchange chromatography and added to an in vitro fermentation system. The intestinal inoculum was collected from pigs immediately after slaughter, diluted with a buffer, and dispensed into fermentation syringes and vessels of the 2 experimental groups: 1) Bac+ = cecal liquor + predigested diet + pediocin A (final concentration 160 activity units/mL); 2) Bac- = cecal liquor + predigested diet + partially purified supernatant of P. pentosaceus FBB61-2. Intestinal microbial growth was monitored using the cumulative gas production technique; the kinetics of fermentation, bacterial counts, VFA, ammonia, polyamines, and p-cresol production were analyzed. Pediocin A had almost no effects on small intestine fermentation parameters, whereas in the cecum pediocin A decreased gas production (-16%; P < 0.05), ammonia, and VFA production (-52 and -21%, respectively, after 24 h; P < 0.001) compared with the control group. Significant inhibition of clostridia and LAB occurred in cecal fermentations: the Bac+ group yielded a decreased number of clostridia and LAB in cecal fermentations (8.19 and 7.80 cfu/mL, respectively) compared with Bac- (9.32 and 8.95 cfu/mL, respectively; P < 0.001). The low clostridia counts in the pediocin-treated group may also explain the reduced concentration of the carcinogenic compound p-cresol (-88%; P < 0.01). Our results suggest that pediocin A could be an alternative to replace antibiotic growth promoters for the prophylaxis of enteric diseases and to improve production of farm animals.

Fasting Enhances p-Cresol Production in the Rat Intestinal Tract

p-Cresol is a metabolite of aromatic amino acid metabolism produced by intestinal microflora, and its formation is influenced by intestinal conditions. Fasting drastically changes intestinal conditions. However, the effect of fasting on p-cresol production is unclear. In this study, serum and cecal p-cresol levels were determined in non-fasted rats and in rats fasting for either 12 or 18 h. Serum p-cresol increased significantly with 12-h fasting (3.44 +/- 2.15 nmol/ml; P<0.05) and 18-h fasting (5.40 +/- 2.20; P<0.001) as compared to the level in the non-fasted rats (1.02 +/- 0.50). Cecal p-cresol levels of the 12-h fasted (272.6 +/- 313.2 nmol/cecum) and 18-h fasted rats (436.6 +/- 190.8; P<0.01) were higher than those in non-fasted rats (27.1 +/- 21.9). The total cecal protein in content did not change with 18-h fasting. However, the cecal protein concentration increased significantly with fasting (P<0.001), and correlated closely with total cecal p-cresol contents (P<0.001). These results indicate that fasting enhances p-cresol production in the rat cecum, resulting in accumulation of serum p-cresol. We presume that the increase in p-cresol produced by fasting is related to the enhancement of bacterial nitrogen metabolism via an increased concentration of endogenous protein in the cecum.

New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria

During the last decade, an increasing number of new enzymes containing glycyl radicals in their active sites have been identified and biochemically characterised. These include benzylsuccinate synthase (Bss), 4-hydroxyphenylacetate decarboxylase (Hpd) and the coenzyme B12-independent glycerol dehydratase (Gdh). These are involved in metabolic pathways as different as anaerobic toluene metabolism, fermentative production of p-cresol and glycerol fermentation. Some features of these newly discovered enzymes are described and compared with those of the previously known glycyl radical enzymes pyruvate formate-lyase (Pfl) and anaerobic ribonucleotide reductase (Nrd). Among the new enzymes, Bss and Hpd share the presence of small subunits, the function of which in the catalytic mechanisms is still enigmatic, and both enzymes contain metal centres in addition to the glycyl radical prosthetic group. The activating enzymes of the novel systems also deviate from the standard type, containing at least one additional Fe-S cluster. Finally, the available whole-genome sequences of an increasing number of strictly or facultative anaerobic bacteria revealed the presence of many more hitherto unknown glycyl radical enzyme (GRE) systems. Recent studies suggest that the particular types of these enzymes represent the ends of different evolutionary lines, which emerged early in evolution and diversified to yield remarkably versatile biocatalysts for chemical reactions that are otherwise difficult to perform in anoxic environments.

Degradation of tryptophan and related indolic compounds by ruminal bacteria, protozoa and their mixture in vitro.

In vitro experiments were conducted to examine the degradation of d- and l-isomers of tryptophan (Trp) and 10 related indolic compounds by mixed rumen bacteria (B), protozoa (P) and a combination of the two (BP). The analyses were carried out by HPLC. d-Trp (1.0 mM) was not degraded by rumen microorganisms during the 24-h incubation period. The net degradation of 1 mM l-Trp was 46.5%, 8.7% and 80.0% by B, P and BP suspensions, respectively. Trp was degraded into indoleacetic acid, indolelactic acid and indole by rumen bacteria and protozoa, and into skatole, p-cresol and indolepropionic acid by rumen bacteria only. Of them, indoleacetic acid was the major product of Trp found in B (15.4%) and P (3.1%), and skatole in BP (43.2%). This is the first report of the production of indolelactic acid and p-cresol from Trp by rumen microbes. Starch, d-glucose, salinomycin and monensin inhibited the production of skatole and indole from Trp, and skatole from indoleacetic acid by rumen bacteria.

Time Course for the Modulation of Hepatic Cytochrome P450 after Administration of Ethylbenzene and Its Correlation with Toluene Metabolism

The goal of the present study was to examine the time course for changes in P450 expression and hydrocarbon metabolism after acute treatment with the simple aromatic hydrocarbon ethylbenzene (EB) and to correlate these alterations with the changes observed in alkylbenzene metabolism. Male Holtzman rats were treated with a single intraperitoneal injection of EB, and the effects on specific P450-dependent activities, immunoreactive P450 isozyme levels, and RNA levels were measured at various times after injection. Toluene was used as the test alkylbenzene for examination of the EB-mediated changes on in vitrohydrocarbon metabolism. In untreated rats, toluene was metabolized almost entirely by aliphatic hydroxylation (to benzyl alcohol); however, in EB-treated rats, significant quantities of benzyl alcohol, o-cresol, and p-cresol were produced. Interestingly, 5–10 h after EB treatment, there was a 40% decrease in benzyl alcohol production. By 24 h, rates of benzyl alcohol formation returned to control levels, whereas there was a 7-fold increase in o-cresol and a greater that 50-fold increase in p-cresol production. The changes in the disposition of toluene were then correlated with changes in particular P450 isozymes. Several P450 isozymes were induced after EB administration. P450 2B1/2-dependent testosterone 16β-hydroxylation and P450 2B1/2-immunoreactive protein were elevated 30-fold after EB administration, reaching maxima by 24 h and remaining elevated 48 h after exposure. Changes in P450 2B1 and 2B2 RNA preceded those of the proteins. Similar results were observed with P450 1A1. P450 2E1 RNA levels were elevated after a single EB injection. However, the elevation in P450 2E1-dependent activities and immunoreactive protein levels preceded the changes in RNA, suggesting that multiple steps are affected by EB exposure. In contrast to the increases in some isozymes, P450 2C11 protein was rapidly suppressed (within the first 2–10 h) after hydrocarbon exposure, suggestive of a destabilization of the protein. When comparing the changes in P450 isozymes to alterations in toluene metabolism, the immediate suppression in aliphatic hydroxylation of toluene (in the first 5–10 h) was consistent with the decrease in P450 2C11. Subsequent to this effect, P450 2B1/2 and 2E1 were induced, which elevated production of this metabolite to control levels. The increase in the aromatic hydroxylation of toluene to both o, and p-cresol was consistent with the induction of P450s 2B1/2, 2E1, and 1A1.

Anaerobic biogenesis of phenol and p-cresol from l-tyrosine

Biological production of p-cresol occurred in an anaerobic medium containing protein after the medium was inoculated with material from one of several anaerobic systems, including swine waste, petroleum-contaminated sediment, municipal sewage sludge and pristine pond sediment. Addition of the amino acid l-tyrosine was accompanied by an increase in p-cresol concentration and the appearance of phenol. When a defined medium containing [U- 14C] l-tyrosine was inoculated with petroleum-contaminated sediment, radioactivity recovered as phenol and p-cresol accounted for 4.6% and 22% of the original, respectively, showing that l-tyrosine was a precursor for both compounds. The results indicate that microorganisms that degrade l-tyrosine with the production of phenol or p-cresol occur in widely diverse anaerobic systems. The question is raised of whether phenol and p-cresol from this source could mimic low-level petrochemical pollution.

Presumptive identification of Clostridium difficile by detection of p-cresol in prepared peptone yeast glucose broth supplemented with p-hydroxyphenylacetic acid

Prereduced, anaerobically sterilized peptone yeast glucose broth was supplemented with p-hydroxyphenylacetic acid and used for the presumptive identification of Clostridium difficile. Two hundred eighty-two organisms were grown in this medium for 18 h and tested for p-cresol production by gas-liquid chromatography. All 49 stock and reference strains of C. difficile and 19 organisms confirmed as C. difficile produced p-cresol. p-Cresol was not produced by 53 negative control or 161 test organisms. The system was convenient and effective.

Production of p-cresol by Clostridium difficile on different basal media

The production of p-cresol by Clostridium difficile on a variety of agar basal media was investigated using gas-liquid chromatography. None of the basal media studied supported the production of large amounts of p-cresol. The addition of 0–1%p-hydroxyphenylacetic acid to a basal medium stimulated the production of p-cresol. If detection of p-cresol is to be used for the presumptive identification of C1. difficile then the basal medium should be supplemented with p-hydroxyphenylacetic acid.

Norleucine-tyrosine broth for rapid identification of Clostridium difficile by gas-liquid chromatography.

A new medium, norleucine-tyrosine (NT) broth, was developed for rapid identification of Clostridium difficile on the basis of caproic acid and p-cresol production. The NT broth consists of 0.5% Trypticase (BBL Microbiology Systems, Cockeysville, Md.), 0.5% yeast extract (Difco Laboratories, Detroit), 0.2% L-norleucine and 0.2% L-tyrosine (wt/vol; final concentrations), and a mixture of salts. The procedure for demonstrating caproic acid and p-cresol production involves extracting NT broth cultures with ether or chloroform and analyzing the extracts with a gas-liquid chromatograph equipped with a thermal conductivity detector, as is customary for analysis of volatile fatty acids. A total of 120 strains of C. difficile from diverse geographic locations were tested by this procedure, and they all produced caproic acid and p-cresol in NT broth. No other Clostridium species or other microorganisms tested have been found to produce both products in NT broth.

The effects of antibiotics in the weanling pig diet on growth and the excretion of volatile phenolic and aromatic bacterial metabolites

The purpose of this study was to investigate the effects of dietary antibiotic supplementation on the fecal urinary excretion of volatile phenolic and aromatic bacterial metabolites by the weanling pig, and to determine if a relationship exists between an exposure to these metabolites and growth performance. Wealing pigs were fed a basal diet, supplemented with either 110 ppm chlortetracycline, 110 ppm sulfamethazine and 55 ppm penicillin, 40 ppm lincomycin sulfate, or no antibiotics, for 30 days. Pigs on the chlortetracycline-sulfamethazine-penicillin diet on the average tended to grow at a faster rate, attained a higher percentage weight gain, and weighed slightly more than pigs on either the lincomycin sulfate or no antibiotic diets. Under all treatments, p-cresol was the predominant metabolite of the volatile phenolic and aromatic metabolites detected in feces and urine, with the urine accounting for 88% of its total daily excretion. Pigs on the chlortetracycline-sulfamethazine-penicillin diet excreted less urinary p-cresol than pigs on either the lincomycin sulfate or no antibiotic diets. Total p-cresol excretion expressed on the metabolic body size, resulted in significant treatment differences. Regression analysis of percentage body weight gain on urinary p-cresol excretion gave a negative correlation coefficient (r = -0.73). The results suggest that intestinal p-cresol production may be responsible for depressing the growth of the weanling pig.

Rapid detection and presumptive identification of Clostridium difficile by p-cresol production on a selective medium.

A modification of a selective medium for Clostridium difficile is described. The ability of Cl difficile to produce p-cresol from p-hydroxy phenyl acetic acid provides a means for the rapid, sensitive detection and presumptive identification of this species in faecal cultures.