A Metabolite-Triggered Tuft Cell-ILC2 Circuit Drives Small Intestinal Remodeling.

The small intestinal tuft cell-ILC2 circuit mediates epithelial responses to intestinal helminths and protists by tuft cell chemosensory-like sensing and IL-25-mediated activation of lamina propria ILC2s. Small intestine ILC2s constitutively express the IL-25 receptor, which is negatively regulated by A20 (Tnfaip3). A20 deficiency in ILC2s spontaneously triggers the circuit and, unexpectedly, promotes adaptive small-intestinal lengthening and remodeling. Circuit activation occurs upon weaning and is enabled by dietary polysaccharides that render mice permissive for Tritrichomonas colonization, resulting in luminal accumulation of acetate and succinate, metabolites of the protist hydrogenosome. Tuft cells express GPR91, the succinate receptor, and dietary succinate, but not acetate, activates ILC2s via a tuft-, TRPM5-, and IL-25-dependent pathway. Also induced by parasitic helminths, circuit activation and small intestinal remodeling impairs infestation by new helminths, consistent with the phenomenon of concomitant immunity. We describe a metabolic sensing circuit that may have evolved to facilitate mutualistic responses to luminal pathosymbionts.

The specificity of trichomonad cysteine proteinases analysed using fluorogenic substrates and specific inhibitors.

The multiple cysteine proteinases of Trichomonas vaginalis and Tritrichomonas foetus, both those retained intracellularly and those released, were separated using gelatin-SDS-PAGE, and their activity towards a range of 15 fluorogenic peptidyl aminomethylcoumarins determined together with their relative sensitivity to inhibitors. Three types of enzyme were apparent in T. vaginalis: (i) an 86-kDa enzyme active only on Z-Arg-Arg-NHMec; (ii) a 54-kDa proteinase which was most active on Z-Phe-Arg-NHMec but also able to hydrolyse N-t-Boc-Val-Leu-Lys-NHMec, Suc-Ala-Phe-Lys-NHMec, H-Pro-Phe-Arg-NHMec and Z-Arg-Arg-NHMec; and (iii) a group of six enzymes which preferentially hydrolysed substrates with bulky residues at the P2 and P3 positions. N-t-Boc-Val-Leu-Lys-NHMec and H-Leu-Val-Tyr-NHMec were the best substrates for the latter group. The 86-kDa proteinase was inactivated by E-64, but only at high concentrations, and was relatively insensitive to the peptidyl diazomethanes. The other proteinases were inhibited by low concentrations of E-64 and by Z-Phe-Ala-CHN2, and to a lesser extent by Z-Phe-Phe-CHN2. Differences between the proteinases of T. foetus were also demonstrated. All of them were active on Z-Arg-Arg-NHMec, but their activity towards other substrates varied. Three predominantly extracellular proteinases (25, 27 and 34 kDa), hydrolysed Z-Arg-Arg-NHMec specifically. Other proteinases (apparent Mr of 20,000 and 32,000) hydrolysed a number of other substrates, with the 32-kDa enzyme having greater activity towards N-t-Boc-Val-Leu-Lys-NHMec and H-Leu-Val-Tyr-NHMec than towards Z-Arg-Arg-NHMec. At a high concentration (270 microM), E-64 inhibited all of the T. foetus enzymes, but lower concentrations were less effective, with the 18-kDa proteinase being particularly insensitive. Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2 were relatively poor inhibitors. The results demonstrate that the proteinases of both species are a heterogeneous group with respect to specificity, and have highlighted significant differences between the enzymes of T. vaginalis and T. foetus. The information on the specificities will be useful for assessing the features required in proteinase inhibitors if they are to be of potential value as antitrichomonal agents.

Pseudocysts in trichomonads--new insights.

Tritrichomonas foetus and Trichomonas vaginalis, parasitic protists of the urogenital tract, display a trophozoite and a pseudocyst stage. The ultrastructure of the trophozoite was compared with the pseudocyst form. The latter appears under unfavorable environmental conditions when the flagella are internalized, and a true cell wall is not formed. Although some authors consider this form as a degenerate stage, the cell behaves as a resistant form. Pseudocysts were found in natural culture conditions and also under induction by hydroxyurea or cycles of cooling and warming cultures. They were studied by light and scanning and transmission electron microscopy, using immunofluorescence and videomicroscopy. This report presents evidence that the trichomonad pseudocysts appear under stress conditions and that they are competent to divide. Pseudocysts differ from trophozoites in that: (1) the flagella are located in endocytic vacuoles and remain beating; (2) the axostyle and the costa are not depolymerized but present a curved shape; (3) the axostyle does not exhibit staining with antitubulin antibodies when the mitotic spindle is observed; (4) the mitotic process occurs within pseudocysts but differs from that described for trophozoites; (5) a nuclear canal is formed connecting the two spindle poles; and (6) the process is reversible if the cells are transferred to fresh medium.

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.

Comparable growth of Tritrichomonas mobilensis in two commercially available culture media.

The investigation reported herein was undertaken to determine which medium is more practical for the axenic laboratory culture of trichomonads. The growth of Tritrichomonas mobilensis was monitored in 2 different types of commercially available growth media. Although Roswell Park Memorial Institute (RPMI) 1640 medium is typically used as a mammalian cell culture medium, it was found to support the growth of trichomonads as well as the American Type Culture Collection (ATCC) medium 745 under similar conditions. Environmental variables, such as temperature and pH, known to affect the success of trichomonad cultures were controlled. The mean generation times (MGTs) of T. mobilensis in the log phase of growth were 5.1 and 4.9 hr for RPMI 1640 and ATCC medium 745, respectively. A stationary phase of zero growth was reached more quickly in the ATCC medium 745 cultures, and in both media a phase of rapid attrition followed this period of static growth. In assessing the practicality of the media, total cell amplification, as well as factors such as cost, ease of preparation, and storage capacity, were considered.

A simple micropore system for experimental studies on trichomonad parasites.

A simple leak-free micropore chamber containing protozoan parasite species was implanted subcutaneously on the back of hamsters and evaluated for viability and multiplication of protozoan parasites. Trophozoites of defined strains of Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, and Tritrichomonas foetus were used; their survival and multiplication in the chambers formed the basis of evaluation. Entamoeba histolytica and G. lamblia did not survive more than 6 hr and succumbed due to cellular adhesion. Trichomonas vaginalis and T. foetus survived 3 and 6 days and multiplied a maximum of 3.6 and 26 times, respectively. This indicated that exchange of body fluids and cells needed for the survival and multiplication of trichomonads readily occurs. This preliminary observation showed that micropore chambers may be useful for chemotherapeutic and immunological studies on trichomonads in ectopic sites.

Pathogenicity of Tritrichomonas mobilensis: subcutaneous inoculation in mice.

The standard "subcutaneous mouse assay" was used to investigate the inherent pathogenicity of Tritrichomonas mobilensis, an intestinal parasite of squirrel monkeys. C57B1/6 mice given subcutaneous bilateral inocula of T. mobilensis died by day 4 postinoculation with lesions too small to be measured. Control mice similarly inoculated with pathogenic and nonpathogenic strains of Trichomonas vaginalis survived the challenge and produced lesions on day 6 with mean volumes in agreement with previous reports. CD1 mice similarly inoculated with standard and double doses of trichomonads (T. mobilensis) again produced small lesions. CD1 mice inoculated at double dosage were moribund or dead on days 5 and 6, respectively, postinoculation. Necropsies were performed on dead and sacrificed mice. Tissues were obtained from internal organs for histology and culture. Unexpectedly, trichomonads were cultured from liver and lung of C57B1/6 mice at the standard level of inoculation and liver, lung, and spleen of CD1 mice at the higher level of inoculation. Although trichomonads are normally considered surface-dwelling noninvasive organisms, the penetration of trichomonads to deep tissues is not without precedent. Tritrichomonas foetus and Trichomonas gallinae are known to invade tissues of their respective hosts. Trichomonas vaginalis has been demonstrated in subepithelial areas of both the prostate gland and cervix of humans. The ability of several species of trichomonads to invade tissues and/or migrate to other sites in their hosts suggests a need for revision of the concept of trichomonads as strictly lumen or surface-dwelling parasites.

Gastrointestinal trichomonads in horses: occurrence and identification.

A survey of horses for gastrointestinal trichomonads was conducted to determine the organism's role in equine diarrhea and to establish its proper identity and morphology. Trichomonads were found by cultural examination of feces of 101 (35%) of 289 apparently healthy horses. At necropsy, trichomonads were cultured from 11 (37%) of another 30 horses which showed no signs of diarrhea at the time of death. In 4 of the 11 horses, colonies of trichomonads numbered 30,000 to 150,000/ml of cecal fluid. Diarrhea was induced in 1 of 6 horses, with the fecal fluid containing 10,000 to 110,000 trichomonads/ml. The trichomonad was identified as Tritrichomonas equi and it appears to be a normal member of the intestinal fauna of the horse. Its role as the etiologic agent of equine diarrhea is considered doubtful. The large numbers of T equi found in diarrheic feces are considered a response to, rather than a cause of, the fluidic environment of the gastrointestinal tract.

[The presence of cysts in Tritrichomonas muris]. / Presencia de quistes en Tritrichomonas muris.

Cysts of Tritrichomonas muris are reported. The morphology of this evolution stage is described under light and electron microscopy. The biologic and epidemiologic importance of this finding is discussed.

Trichomonad gastritis in rhesus macaques (Macaca mulatta) infected with simian immunodeficiency virus.

In a retrospective study, 51 cases of gastritis (14%) were identified from among 341 necropsies performed on simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) at the New England Primate Research Center from 1993 to 2001. Protozoa were seen in the stomach of 13 monkeys (25%) with gastritis. Two histopathologic manifestations of gastritis were observed: seven cases of lymphoplasmacytic gastritis with trichomonad trophozoites within lumens of gastric glands and four cases of necrosuppurative gastritis containing intralesional periodic acid-Schiff-positive protozoa; two cases of gastritis had morphologic features of both types of gastritis. In instances of necrosuppurative and combined lymphoplasmacytic and necrosuppurative gastritis, protozoa were 4-35 microm in diameter and round to tear-shaped. Because of the unusual morphology of the protozoa in these latter cases, transmission electron microscopy and polymerase chain reaction (PCR) were used to further identify these organisms. The protozoa were definitively identified as Tritrichomonas in all cases on the basis of ultrastructural characteristics (flagella and undulating membranes) and amplification of a 347-bp product of the 5.8S ribosomal RNA gene of Tritrichomonas foetus, Tritrichomonas suis and Tritrichomonas mobilensis by PCR using DNA extracted from stomach tissue. On the basis of these observations, we conclude that Tritrichomonas can be a significant cofactor in the development of necrosuppurative gastritis in SIV-infected rhesus macaques.

Scanning electron microscopy study of Tritrichomonas augusta.

Tritrichomonas augusta is a flagellated protozoan that parasitizes amphibians and reptiles. According to scanning electron microscopy (SEM), the cell shape of T. augusta varies from slender pyriform to ovoidal. Our data show the morphological features of the trophozoites: the emergence of the anterior flagella, the structure of the undulating membrane and the position and shape of the pelta, axostyle and posterior flagellum. In addition, herein we describe spherical forms which are probably pseudocysts. The description of the external structure of T. augusta, as demonstrated by SEM, contributes to the understanding of the biology of this parasite.

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.

Experimental infections in chickens with Chilomastix gallinarum, Tetratrichomonas gallinarum, and Tritrichomonas eberthi.

Flagellates from the caeca of a diseased hen and a diseased goose were transmitted to 35 specific pathogen-free (SPF) chickens. The flagellates of chicken origin were identified as Chilomastix gallinarum, Tritrichomonas eberthi, and Tetratrichomonas gallinarum. T. eberthi was not detected in the material of goose origin. Morphologic studies did not reveal any differences between Chilomastix and Tetratrichomonas specimens from chicken or goose origin. The species from the goose were identified as C. gallinarum and T. gallinarum (Syn. T. anseris Hegner, 1929). Both trichomonad species produced pseudocysts that developed in the faeces of chickens within 3 h after excretion. Only 17% of the trichomonads excreted had reached the pseudocyst stage. All three flagellate species are infective to chickens when inoculated per rectum or per os or when consumed with chlorinated tap water. The prepatency period was always less than 24 h. SPF chickens between 2 and 30 days of age were equally susceptible. The infections persisted at a high level of intensity throughout the observation periods, i.e. up to 7 months. Of 35 inoculated SPF chickens, 2 developed disease (emaciation, ruffled feathers, diarrhoea, dilatation of the caeca). The three flagellate species were cultivated in Diamond's medium for 110 days. Cryopreserved and cultivated flagellates retained their infectivity to chickens.

Tritrichomonas foetus: partly defined cultivation medium for study of the purine and pyrimidine metabolism.

A partly defined medium was successfully designed for the cultivation of Tritrichomonas foetus, an anaerobic protozoan parasite of cattle. The medium consists of hypoxanthine, uracil, and thymidine as the sole precursors of nucleotides in T. foetus. Elimination of any one of the three precursors from the medium led to cessation of T. foetus growth. The information provided by this medium verifies our previous observations that T. foetus is incapable of de novo purine and pyrimidine synthesis, that hypoxanthine can be converted to AMP and GMP, that uracil is incorporated into all pyrimidine ribonucleotides including UDP-glucose--the precursor of glycogen synthesis, and that thymidine is the only precursor of TMP. The omission of folate from the medium, without affecting growth of T. foetus, also supports our previous finding that the parasite does not have functioning dihydrofolate reductase or thymidylate synthetase. The successful plating of T. foetus on agar plates incorporating the partly defined medium with near 100% plating efficiency makes it possible to isolate T. foetus mutants for further studies of purine and pyrimidine metabolism in this parasite.

Localization of post-translationally modified alpha-tubulin and pseudocyst formation in tritrichomonads.

The distribution of acetylated tubulin was investigated in members of the Tritrichomonadinae subfamily using a specific monoclonal antibody and indirect immunofluorescence microscopy. Species probed in this study were: Tritrichomonas foetus, T. mobilensis, T. muris, and an unnamed tritrichomonad isolated from the cecum of cotton rats (Sigmodon hispidus). Additionally, the distribution of glutamylated tubulin in T. muris was investigated using the GT 335 antibody to the glutamyl motif. Although acetylated alpha-tubulin was ubiquitously distributed throughout the axostyle and flagella of T. foetus and T. mobilensis, a distinct and unusual labeling pattern was observed in 'T. muris'-type or pseudocyst-forming trichomonads. In trophozoite stages of T. muris and the cotton rat isolate, flagella were intensely labeled, but acetylation of axostylar tubulin appeared to be limited to the anterior and posterior thirds. However, trophozoites labeled with an antibody to alpha-tubulin or glutamylated tubulin demonstrated no such discontinuity in axostylar staining. Additionally, pseudocysts labeled with anti-alpha-tubulin and anti-acetylated alpha-tubulin were all found to possess continuously fluorescent axostyles. That the mid-region axostyle remained unlabeled by anti-acetylated tubulin in trophozoites indicates possible deacetylase activity, which may have functional implications with respect to the life cycle. Glutamylated tubulin appeared to be distributed throughout the axostyle and flagella, which fluoresced brightly in T. muris.

Life cycle of Tritrichomonas muris.

Morphological and physiological studies were carried out on a flagellate, Tritrichomonas muris, in golden hamsters. The experiments confirmed that a pseudocyst and an intermediate form exist in addition to the trophozoite in the life cycle; furthermore it was shown that the pseudocyst was the infective stage, and that a true cyst did not occur.