Detection of Succinate by Intestinal Tuft Cells Triggers a Type 2 Innate Immune Circuit.

In the small intestine, type 2 responses are regulated by a signaling circuit that involves tuft cells and group 2 innate lymphoid cells (ILC2s). Here, we identified the microbial metabolite succinate as an activating ligand for small intestinal (SI) tuft cells. Sequencing analyses of tuft cells isolated from the small intestine, gall bladder, colon, thymus, and trachea revealed that expression of tuft cell chemosensory receptors is tissue specific. SI tuft cells expressed the succinate receptor (SUCNR1), and providing succinate in drinking water was sufficient to induce a multifaceted type 2 immune response via the tuft-ILC2 circuit. The helminth Nippostrongylus brasiliensis and a tritrichomonad protist both secreted succinate as a metabolite. In vivo sensing of the tritrichomonad required SUCNR1, whereas N. brasiliensis was SUCNR1 independent. These findings define a paradigm wherein tuft cells monitor microbial metabolites to initiate type 2 immunity and suggest the existence of other sensing pathways triggering the response to helminths.

Use of Ronidazole and Limited Culling To Eliminate Tritrichomonas muris from Laboratory Mice.

Tritrichomonas muris is occasionally identified during routine fecal screening of laboratory mice. Frequently, entire racks are affected, and because no effective treatment is available, culling of affected mice and rederivation by embryo transfer have been suggested. The current study evaluated whether treatment with ronidazole, a nitroimidazole efficacious against T. fetus infections in cats, combined with limited culling was effective against T. muris in laboratory mice (Mus musculus). A subset (n = 39) of mice were treated with ronidazole (400 mg/L in drinking water) for 15 d, after which 6 of the mice still shed T. muris. Consequently all mice in the affected rack received ronidazole (500 mg /L in drinking water) for 25 d. All mice were retested by using pooled samples, and those positive for T. muris (except for a valuable breeding pair) were culled. The remaining mice continued to receive ronidazole for another 17 d. At the end of the treatment period, all mice were tested (days 60 and 81) and were shown to be negative for T. muris. Over the following year, sentinel mice from the rack were tested every 3 mo and remained negative for tritrichomonads by fecal smear. Thus, a combination of limited culling and treatment with ronidazole in the drinking water successfully cleared research mice of infection with T. muris.

Metabolic differences between metronidazole resistant and susceptible strains of Tritrichomonas foetus.

Tritrichomonas foetus mutants resistant to metronidazole lack the hydrogenosomal enzymes pyruvate: ferredoxin oxidoreductase and hydrogenase. Hydrogenosomes of these organisms did not oxidize pyruvate or produce ATP in its presence. Elimination of hydrogenosomal metabolism of pyruvate was compensated by an increased rate of glycolysis. The resistant mutants excreted no organic acids and H2 as metabolic end products. Glycolysis of the resistant T. foetus KV1-1MR-100 can be summarized as 1 mol glucose----2 mol ethanol + 2 mol CO2. The parent strain KV1, excreting H2, CO2 and acidic end products, converted about 10% of glucose to ethanol. Both strains produced ethanol from pyruvate through the action of two cytoplasmic enzymes: pyruvate decarboxylase and alcohol dehydrogenase. The specific activity of the former enzyme, catalyzing nonoxidative decarboxylation of pyruvate to acetaldehyde, was nearly seven times higher in the resistant than in the parent strain. Alcohol dehydrogenase reducing acetaldehyde to ethanol was specific to NADPH; it catalyzed the reverse reaction only slowly, and displayed similar activities in both resistant and sensitive trichomonads. Development of anaerobic metronidazole resistance in T. foetus depended on the loss of pyruvate:ferredoxin oxidoreductase as well as on the ability to increase alcoholic fermentation.

Polyamine biosynthesis in trichomonads.

Trichomonas vaginalis, Tritrichomonas foetus and Trichomitus batrachorum grown in modified Diamond's medium all had high concentrations of putrescine and lower concentrations of spermidine and spermine. Ornithine decarboxylase (ODC; EC 4.1.1.17) was detectable in all three species although at significantly different levels. Trichomonas vaginalis had the highest activity (typically around 1.85 nmol min-1 (mg protein)-1), Trichomitus batrachorum the lowest (0.11 nmol min-1 (mg protein)-1). The Trichomonas vaginalis ODC had an apparent Mr of 230 000 and was severely inhibited by alpha-difluoromethylornithine (DFMO). S-Adenosyl-methionine decarboxylase (EC 4.1.1.50) could not be detected in T. batrachorum but was present in the other two species. Arginine decarboxylase was apparently absent from all three. All three trichomonad species were able to accumulate spermidine and putrescine from the medium. When T. vaginalis was grown in the presence of DFMO (4 mM), which had little effect on parasite growth, ODC activity was reduced by over 99% and the polyamine content was altered; putrescine concentrations were decreased, those of spermidine and spermine remained the same or were raised. DFMO-treated cells accumulated more exogenous putrescine than untreated control cells. The results suggest that the lack of effect of DFMO on T. vaginalis in culture was due to the parasite being able to accumulate polyamines from the growth medium. It appears, therefore, that testing DFMO and similar compounds in axenic trichomonad cultures may well not give a true indication of their effectiveness in vivo where sources of exogenous polyamines may not be available.

Purine base transport in wild-type and mycophenolic acid-resistant Tritrichomonas foetus.

The purine base transport systems of wild-type and mycophenolic acid-resistant (MPAR) Tritrichomonas foetus have been characterized. Wild-type T. foetus has two carriers, one for hypoxanthine (Km = 0.7 +/- 0.3 mM, Vm = 80 +/- 20 pmol microliters-1min-1) and guanine (Km = 0.09 +/- 0.02 mM, Vm = 17 +/- 3 pmol microliters-1min-1), and a second for xanthine (Km = 0.6 +/- 0.2 mM, Vm = 25 +/- 5 pmol microliters-1min-1). Adenine transport was not saturable (k = 0.16 +/- 0.01 min-1) and therefore appears to enter the parasite by passive diffusion through the membrane. T. foetus MPAR has lost the hypoxanthine/guanine transporter. Xanthine and adenine transport are similar in wild-type and MPAR T. foetus. No purine nucleoside transporter could be identified.

Synthesis and antitrichomonal activity of certain pyrazolo (1,5-a) pyrimidines.

Several bridgehead nitrogen heterocycles were synthesized to be screened as antimicrobial agents, modeled after nalidixic acid. The activity of these new compounds, all derivatives of 3-nitro-4,6-disubstituted pyrazolo (1,5-a)pyrimidin-7-ones (3,7,8, and 9), however, was found to be highly specific for Trichomonas foetus and completely lacking in activity against bacteria, fungi, and parasites other than Trichomonas. Of the nine componds synthesized, including the intermediate 4,6-disubstituted pyrazolo (1,5-a) pyrimidin-7-ones (2-6) and the 6-substituted or unsubstituted pyrazolo (1,5-a) pyrimidin-7-ones (1 and 4), only 6-carbethoxy-4-ethyl-3-nitropyrazolo(1,5-a)pyrimidin-7-one (7) was found to be a potent antitrichomal agent, being comparable or perhaps better than metronidazole. From a tentative structure-activity relationship study, it was apparent that the combination of the 3-mitro, 4-ethyl, and 6-carbethoxy groups imparted specific activity, wheras other substitutions imparted little or no antitrichomonal activity.

A novel mechanism of mycophenolic acid resistance in the protozoan parasite Tritrichomonas foetus.

Tritrichomonas foetus relies primarily on the salvage of hypoxanthine to supply purine nucleotides. Mycophenolic acid disrupts T. foetus growth by specifically inhibiting inosine-5'-monophosphate (IMP) dehydrogenase, thereby blocking the biosynthesis of guanine nucleotides from hypoxanthine. We have cloned a T. foetus strain (mpar) that was 50-fold more resistant to mycophenolic acid than wild type (IC50 = 1 mM for mpar vs 20 microM for wild type). None of the usual mechanisms of drug resistance could be identified. IMP dehydrogenase isolated from T. foetus mpar was indistinguishable from the wild type enzyme. No difference in mycophenolic acid uptake or metabolism was detected between the wild type and mpar strains. Mycophenolic acid (100 microM) completely blocked the conversion of adenine and hypoxanthine to guanine nucleotides in T. foetus mpar, although no inhibition of T. foetus mpar growth was observed at this concentration. These observations indicate that the major purine salvage pathways must be altered in T. foetus mpar so that guanine nucleotide biosynthesis no longer requires IMP dehydrogenase. T. foetus mpar incorporated xanthine more efficiently into the nucleotide pool relative to hypoxanthine and guanine than wild type. Xanthine incorporation via XMP provided an IMP dehydrogenase independent route to guanine nucleotides that would enable the parasite to become mycophenolic acid resistant. No difference could be detected between wild type and mpar hypoxanthine-guanine-xanthine phosphoribosyltransferases, the key enzyme in purine base incorporation into nucleotides. Two alterations were identified in the purine salvage network of mpar: it was deficient in hypoxanthine transport and had diminished adenine deaminase activity. The apparent net result of these two changes was to lower the intracellular concentration of hypoxanthine in mpar. Hypoxanthine and adenine inhibited the incorporation of xanthine into the nucleotide pool in wild type T. foetus, but not in mpar. The mpar strain, therefore, can salvage xanthine more efficiently from a mixture of purines and thus bypass the drug block at IMP dehydrogenase.

Differential effects of inhibitors of purine metabolism on two trichomonad species.

Tritrichomonas foetus and Trichomonas vaginalis are both incapable of de novo purine nucleotide synthesis. Previous studies indicated that T. foetus relies mainly on the salvage of hypoxanthine and subsequent conversion of IMP to AMP and GMP, whereas T. vaginalis depends on direct conversions of exogenous adenosine to AMP and guanosine to GMP without much interconversion between the two nucleotides. These two different types of purine salvage suggest the possibility of differential sensitivities between the two species of trichomonad flagellates toward different purine antimetabolites. Mycophenolic acid, hadacidin, 8-azaguanine, and formycin B inhibited the growth of T. foetus but had no effect on T. vaginalis. Mycophenolic acid acted by blocking conversion of IMP to GMP, hadacidin inhibited conversion of IMP to AMP, and 8-azaguanine was incorporated into the T. foetus nucleotide pool, likely via hypoxanthine phosphoribosyl transferase. Formycin B was converted to 5'-monophosphate in T. foetus and inhibited the conversion of IMP to AMP. Its precise mechanism of action on T. foetus remains, however, to be elucidated. Alanosine, whose ribonucleotide derivative is a potent inhibitor of adenylosuccinate synthetase, had no effect on the growth or hypoxanthine incorporation in T. foetus, which may be due to the lack of conversion of alanosine to the ribonucleotide because of the absence of de novo purine nucleotide synthesis in parasites. Four adenosine analogs, adenine arabinoside, tubercidin, sangivamycin, and toyocamycin, were found inhibitory to the growth of T. vaginalis but showed little effect on T. foetus growth. Further investigations suggested that these four compounds acted on T. vaginalis by blocking incorporation of adenosine into the adenine nucleotide pool.

Sensitivity of parasites to free radical damage by antiparasitic drugs.

Over the last few years a remarkable progress has been made in the understanding of parasites biochemistry, molecular biology, and immunology. This progress is especially encouraging in that emphasis on drug development is shifting from random screening towards a more rational approach. A number of peculiar aspects characteristic of parasites which are not present in other organisms and that might be exploitable for the design of specific agents have been described recently. One of these aspects is their deficiency in defense mechanisms against oxygen toxicity. Catalase is absent in many parasites. Distinct superoxide dismutases have been detected and specific inhibitors of these enzymes have been investigated. Glutathione is absent in some anaerobic protozoa. Peroxidase and reductase activities dependent on a glutathione-spermidine cofactor termed trypanothione have been detected in several trypanosomatids and apparently replace the glutathione peroxidase-glutathione reductase system of other eukaryotic cells. Free radical intermediates have been shown to be involved in the reaction of enzymes present in anaerobic protozoa. In addition, a number of antiparasitic agents have been shown to exert their actions through a free radical metabolism: nitro compounds used against trypanosomatids, anaerobic protozoa and helminths; crystal violet used in blood banks to prevent blood transmission of Chagas' disease; the antimalarial primaquine, chloroquinine, and quinhasou; and quinones active in vitro and in vivo against different parasites.

Sialic acid-specific lectin-mediated adhesion of Tritrichomonas foetus and Tritrichomonas mobilensis.

Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract producing infection associated with inflammatory changes, abortion, and infertility, Tritrichomonas mobilensis was isolated from squirrel monkey colon, and symptoms involve diarrheal complications. Both tritrichomonads produced hemagglutinins with the properties of sialic acid-specific lectins. Assays on the adherence of these protozoans to Chinese hamster ovary (CHO) cells and to bovine cervical and monkey colon mucus were performed to assess the function of the lectins in adhesion. Sialic acid at concentration as low as 2 mM inhibited the adhesion to CHO cells, less effectively to the mucus. Predigestion with Clostridium perfringens sialidase prevented the adhesion to both epithelial cells and the mucus. Inhibition of endogenous sialidases with 2,3-dehydro-2-deoxy-NeuAc increased the adhesion of T. mobilensis to CHO cells. Specific anti-T. foetus lectin (TFL) and anti-T. mobilensis lectin (TML) antibodies inhibited adhesion of the trichomonads to the epithelial cells and to the mucus. TFL histochemistry disclosed the presence of lectin ligands on keratinized vaginal epithelia, cervical mucosa, and mucin and on endometrial glands and their secretions. TML histochemistry showed reactivity with the luminal membranes of colonic glandular epithelium and less with the colonic mucin. Both lectins bound to the surface membrane of CHO cells. Anti-lectin antibodies showed granular cytoplasmic and strong membrane localization of the lectins in both tritrichomonads. Although the 2 tritrichomonads have different habitats, the results indicate that both these protozoa use lectins with sialic acid specificity for adhesion to mucosal surfaces.

Inhibition of Tritrichomonas foetus by vermiculine in vitro.

The macrolide aglycosidic antibiotic vermiculine, added to the cultivation medium to a concentration of 30 micogram/ml, has an in vitro inhibitory effect on Tritrichomonas foetus. The agent interacts rapidly with the cells, causing irreversible changes after several hours of action. The changes are not repaired on removing the agent; the cells suffer from a rapid inhibition of nucleic acid synthesis, the protein synthesis remaining intact.

In vivo effect of 2,4-dinitro-phenyl-substituted sugar hydrazones and phenylosazone against Trichomonas vaginalis and Tritrichomonas foetus.

Antitrichomonal activity of selected 2,4-dinitro-phenylhydrazones and phenylosazones of sugars was investigated in vitro and in vivo and compared with the activity of tinidazole, an effective therapeutic agent. In experimental infections with Trichomonas vaginalis and Tritrichomonas foetus in mice glycolaldehyde-2,4-dinitro-phenylhydrazone acetate and 2,3-diacetylglycerolaldehyde-2,4-dinitro-phenylhydrazone exhibited an antiparasitic effect slightly inferior to that of tinidazole. Structure-activity relationships are discussed. On the basis of the results obtained the chemotherapeutic value of the mentioned substances seems to be promising.

[The effect of nitrofuran and asymmetrically substituted urea on Trichomonas vaginalis and Tritrichomonas foetus in vitro and in vivo]. / Ucinok nitrofuránov a nesymetricky substituovaných mocovín na Trichomonas vaginalis a Tritrichomonas foetus in vitro a in vivo.

Antitrichomonal activity of selected nitrofuran derivatives and asymmetrically substituted ureas was investigated in vitro and in vivo and compared with the activity of the effective therapeutic agent tinidazole. In experimental infection with Trichomonas vaginalis and Tritrichomonas foetus in mice, some substituted urea derivatives exhibited an antiparasitic effect slightly lower than that of tinidazole. The substance N1-(3-nitrophenyl)-N3-(2-pyrimidinyl)urea proved to be the most effective of the substances tested. On the basis of preliminary toxicity tests the chemotherapeutic value of this substance appears to be promising.

Tritrichomonas foetus: stable anaerobic resistance to metronidazole in vitro.

Stable anaerobic resistance of Tritrichomonas foetus to metronidazole was induced in vitro by cultivation of trichomonads in the Diamond's TYM medium with metronidazole in concentrations sublethal to the parasites. Nine metronidazole-resistant strains were derived from four drug-susceptible clones of the T. foetus strain KV-1. Subculturing the parasites at both increasing and constant pressure of the drug resulted in development of resistance if the medium contained at least 3 micrograms ml-1 of metronidazole and the organisms were exposed to the drug for 3 to 8 months. The development of resistance was gradual and in all clones investigated proceeded through similar sequence of stages: (1) Survival without growth and subsequent reproduction at low metronidazole concentrations (1 to 5 micrograms ml-1). (2) Survival and reproduction at moderate concentrations of the drug (10 to 15 micrograms ml-1). (3) Resistance to 100 micrograms ml-1 metronidazole, unstable in absence of selective pressure of the drug. (4) Resistance to high concentrations of metronidazole, stable when the organisms were maintained under nonselective conditions. The trichomonads with fully developed resistance were able to grow in anaerobic culture at 100 micrograms ml-1 metronidazole and could be maintained indefinitely under these conditions. The minimal lethal concentrations for metronidazole obtained with these strains in an anaerobic in vitro assay were, at 48 h, 500 to 1000 micrograms ml-1. This is 100 to 400 times higher than those obtained with the parent clones. The fully developed resistance was stable in organisms maintained in the absence of the drug over 2 years. The substrains with unstable resistance regained the susceptibility to high concentrations of metronidazole after 80 to 100 transfers in drug-free media. These strains, however, retained their resistance to moderate doses of metronidazole and full resistance could be restored by subculture in the presence of 10 micrograms ml-1 metronidazole.

Assay conditions and the demonstration of nitroimidazole resistance in Tritrichomonas foetus.

Tritrichomonas foetus KV(1), a nitroimidazole-susceptible strain, and KV(1)/M100, its nitroimidazole-resistant daughter strain, differed markedly in their in vivo susceptibility to metronidazole. In vitro susceptibility testing in multiwell plates and tubes with different trichomonad media containing no, or low concentrations of, ascorbate demonstrated that the resistant strain behaves like the susceptible one, if tested under anaerobic conditions (deep cultures in tubes or multiwell plates in anaerobic jars), but shows resistance if tested in the presence of air (multiwell plates exposed to air). In media containing high concentrations of ascorbate, no resistance was observed even in air. The results suggest that the two strains differ in the regulation of internal redox systems and underscore the role testing methods may play in the in vitro detection of nitroimidazole-resistant protozoan parasites.

Tritrichomonas foetus: freeze-fracture cytochemistry using polymyxin B.

The general structure of Tritrichomonas foetus incubated in the presence of the peptide antibiotic polymyxin B, which interacts specifically with anionic phospholipids, was analyzed using transmission electron microscopy of thin sections, and freeze-fracture replicas. Polymyxin B induced morphological changes in the plasma membrane of the parasites with the formation of membrane blebs with a diameter varying from 65 nm to 1.5 micron. Freeze-fracture images of the membrane lining the blebs showed that their inner membrane half is smooth. However, membrane particles, with a density similar to that observed on the E face of the plasma membrane, were seen on the outer half of this membrane.

[Effect of glycolysis inhibitors on the uptake of metronidazole by the protozoa Tritrichomonas foetus and Entamoeba invadens]. / L'action des inhibiteurs de la glycolyse sur l'absorption du métronidazole par les protozoaires Tritrichomonas foetus et Entamoeba invadens.

Uptake of metronidazole by the anaerobic protozoa, Tritichomonas foetus and Entamoeba invadens is dependent on the energy metabolism of these organisms. The inhibitors of glycolysis, iodoacetamide and sodium fluoride, inhibit the uptake. An atmosphere of hydrogen eliminates this inhibition in the hydrogenase-containing T. foetus but not in E. invadens which lacks the enzyme.