Fucose Ameliorates Tritrichomonas sp.-Associated Illness in Antibiotic-Treated Muc2-/- Mice.

The mucus layer in the intestine plays a critical role in regulation of host-microbe interactions and maintaining homeostasis. Disruptions of the mucus layer due to genetic, environmental, or immune factors may lead to inflammatory bowel diseases (IBD). IBD frequently are accompanied with infections, and therefore are treated with antibiotics. Hence, it is important to evaluate risks of antibiotic treatment in individuals with vulnerable gut barrier and chronic inflammation. Mice with a knockout of the Muc2 gene, encoding the main glycoprotein component of the mucus, demonstrate a close contact of the microbes with the gut epithelium which leads to chronic inflammation resembling IBD. Here we demonstrate that the Muc2-/- mice harboring a gut protozoan infection Tritrichomonas sp. are susceptible to an antibiotic-induced depletion of the bacterial microbiota. Suppression of the protozoan infection with efficient metronidazole dosage or L-fucose administration resulted in amelioration of an illness observed in antibiotic-treated Muc2-/- mice. Fucose is a monosaccharide presented abundantly in gut glycoproteins, including Mucin2, and is known to be involved in host-microbe interactions, in particular in microbe adhesion. We suppose that further investigation of the role of fucose in protozoan adhesion to host cells may be of great value.

Tumor suppressor p53 regulates intestinal type 2 immunity.

The role of p53 in tumor suppression has been extensively studied and well-established. However, the role of p53 in parasitic infections and the intestinal type 2 immunity is unclear. Here, we report that p53 is crucial for intestinal type 2 immunity in response to the infection of parasites, such as Tritrichomonas muris and Nippostrongylus brasiliensis. Mechanistically, p53 plays a critical role in the activation of the tuft cell-IL-25-type 2 innate lymphoid cell circuit, partly via transcriptional regulation of Lrmp in tuft cells. Lrmp modulates Ca2+ influx and IL-25 release, which are critical triggers of type 2 innate lymphoid cell response. Our results thus reveal a previously unrecognized function of p53 in regulating intestinal type 2 immunity to protect against parasitic infections, highlighting the role of p53 as a guardian of immune integrity.

Commensal Bacteria Impact a Protozoan's Integration into the Murine Gut Microbiota in a Dietary Nutrient-Dependent Manner.

Our current understanding of the host-microbiota interaction in the gut is dominated by studies focused primarily on prokaryotic bacterial communities. However, there is an underappreciated symbiotic eukaryotic protistic community that is an integral part of mammalian microbiota. How commensal protozoan bacteria might interact to form a stable microbial community remains poorly understood. Here, we describe a murine protistic commensal, phylogenetically assigned as Tritrichomonas musculis, whose colonization in the gut resulted in a reduction of gut bacterial abundance and diversity in wild-type C57BL/6 mice. Meanwhile, dietary nutrient and commensal bacteria also influenced the protozoan's intestinal colonization and stability. While mice fed a normal chow diet had abundant T. musculis organisms, switching to a Western-type high-fat diet led to the diminishment of the protozoan from the gut. Supplementation of inulin as a dietary fiber to the high-fat diet partially restored the protozoan's colonization. In addition, a cocktail of broad-spectrum antibiotics rendered permissive engraftment of T. musculis even under a high-fat, low-fiber diet. Furthermore, oral administration of Bifidobacterium spp. together with dietary supplementation of inulin in the high-fat diet impacted the protozoan's intestinal engraftment in a bifidobacterial species-dependent manner. Overall, our study described an example of dietary-nutrient-dependent murine commensal protozoan-bacterium cross talk as an important modulator of the host intestinal microbiome.IMPORTANCE Like commensal bacteria, commensal protozoa are an integral part of the vertebrate intestinal microbiome. How protozoa integrate into a commensal bacterium-enriched ecosystem remains poorly studied. Here, using the murine commensal Tritrichomonas musculis as a proof of concept, we studied potential factors involved in shaping the intestinal protozoal-bacterial community. Understanding the rules by which microbes form a multispecies community is crucial to prevent or correct microbial community dysfunctions in order to promote the host's health or to treat diseases.

Are Tritrichomonas foetus and Tritrichomonas suis synonyms?

Tritrichomonas suis, a tritrichomonad of pigs, and the related species Tritrichomonas foetus, a tritrichomonad of cattle, are morphologically identical. The taxonomic relationship between these two tritrichomonads has been questioned ever since they were established as distinct species in 1843 and 1928, respectively. Here, we compare the similarities of morphology, ultrastructure, distribution, host specificity, characteristics of in vitro cultivation, immunology, biochemistry and analysis of molecular data from published sources between these two species. All data indicate that these two tritrichomonad species are identical. Thus, we propose that T. foetus and T. suis are synonyms.

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.

The interaction of Tritrichomonas foetus and Trichomonas vaginalis with macrophages.

The process of interaction between macrophages and Tritrichomonas foetus and Trichomonas vaginalis was analysed using light microscopy, scanning and transmission electron microscopy. The parasites attach to the macrophage surface and are ingested through a phagocytic process. Parasite-macrophage association index was higher for activated than for resident macrophages. Previous incubation of the parasites in the presence of Concanavalin A rendered their surface less negative and more hydrophobic, as evaluated by measurement of the zeta potential and contact angle, respectively. This treatment significantly increased parasite ingestion by resident, but not activated macrophages.

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.

Cytotoxic and hemolytic effects of Tritrichomonas foetus on mammalian cells.

Geographically distinct lines of Tritrichomonas foetus were assayed for their ability to cause cytotoxicity in nucleated mammalian cells and lysis of bovine erythrocytes. T. foetus was highly cytotoxic toward a human cervical cell line (HeLa) and early bovine lymphosarcoma (BL-3) but displayed low levels of cytotoxicity against African green monkey kidney (Vero) cells. In addition to variation in the extent of cytotoxicity toward different targets, differences in the levels of cytotoxicity in the same nucleated target occurred with different parasite lines. Whole T. foetus, unfractionated whole-cell extracts, and parasite-conditioned medium (RPMI 1640 without serum) all caused lysis of bovine erythrocytes. Lytic activity in the conditioned medium was substantially reduced by repeated freezing and thawing or heating to 90 degrees C for 30 min. Damage of mammalian target cells by live T. foetus could be reduced by the presence of protease inhibitors; however, such inhibitors did not diminish the lytic effects of conditioned medium. These results suggested that proteolytic enzymes were necessary for the lytic mechanism of the live parasites but were not required once lytic factors were released into the parasite-conditioned medium. They further suggested that the lytic molecules were either proteins or had proteinaceous components.

Fibronectin- and laminin-mediated endocytic activity in the parasitic protozoa Trichomonas vaginalis and Tritrichomonas foetus.

The pathogenic protozoan Tritrichomonas foetus is able to ingest polystyrene particles with a diameter up to 1.0 micron. Trichomonas vaginalis, however, ingest particles as large as 4.4 microns in diameter. The particles are found within cytoplasmic membrane-bounded vacuoles. Morphometrical analysis showed that T. vaginalis presents a higher endocytic activity than T. foetus. Coating of the polystyrene particles with cationized ferritin increases their binding to the parasite surface but does not interfere with their ingestion. In contrast, coating with laminin significantly increased the uptake of the particles by both parasites while coating with fibronectin potentiates the ingestion of the particles only by T. foetus. These observations suggest the presence of laminin- and fibronectin-binding sites on the surface of trichomonads, an observation which is in agreement with the recent description of a receptor for laminin on the surface of trichomonads.

Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells.

Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells (VECs) in vitro was investigated with fresh washed bovine VECs and log-phase cultures of T. foetus. Observation under phase-contrast microscopy showed that T. foetus usually adhered first by the posterior flagellum and later by the body. Significantly more keratinized squamous epithelial cells were detected with attached parasites than nonkeratinized round epithelial cells. The optimal pH range for attachment was 6.0 to 7.5, with peak attachment at pH 6.5 for squamous VECs. Surface-reactive bovine antiserum to T. foetus prevented adherence to bovine squamous VECs. Inhibition of adherence occurred at nonagglutinating, nonimmobilizing serum dilutions. Antiserum fractions enriched for immunoglobulin G1 inhibited adherence, but fractions enriched for immunoglobulin G2 did not. The inhibitory antiserum was specific for several medium- to high-molecular-weight membrane antigens as detected in Western blots (immunoblots). The ability of surface-reactive antibodies to prevent adherence and to agglutinate and immobilize T. foetus indicates that they may be protective.

Use of DMSO for the cryopreservation of Tritrichomonas foetus in liquid nitrogen.

Four Tritrichomonas foetus strains were successfully preserved in liquid nitrogen at -196 degrees C in the presence of 10% dimethylsulfoxide (DMSO) for 7 months (Strain 1) and 6 months (Strain 2, 3 and 4). The percentage of viable organisms after thawing ranged between 65 and 85%, depending upon the strain. The method used is described in detail.

Adherence and surface properties of Tritrichomonas mobilensis, an intestinal parasite of the squirrel monkey.

Adherence properties of the potentially enteropathogenic Tritrichomonas mobilensis were studied in vitro. Axenically cultivated trichomonads readily attached to isolated intestinal epithelial cells and mucus of the squirrel monkey. The kinetics and nature of T. mobilensis cytadherence were microscopically evaluated in cell-suspension assay using Chinese hamster ovary (CHO) cells and in microplate hemagglutination assay with human erythrocytes. Adherence of the parasites to target cells was concentration- and time-dependent; it was inhibited by sialic acid (N-acetylneuraminic or N-glycolylneuraminic acid) and sialyllactose. Neither trypsinization of the flagellates nor their exposure to low temperature (4 degrees C) affected their cytadherence capacities. The data indicate the presence of adhesin(s) with lectin properties on T. mobilensis. Agglutination of live protozoa by animal and plant lectins with various carbohydrate-binding specificities as well as the occurrence of an electron-dense cell coat on plasma membrane suggest marked glycosylation of the parasite surface.

Tritrichomonas foetus: ultrastructure and cytochemistry of endocytosis.

Tritrichomonas foetus ingests horseradish peroxidase, native ferritin, cationized ferritin, and 0.08 micron latex beads by a process which involves the formation of pinocytic vesicles. These vesicles fuse with each other and with lysosomes forming large vacuoles. Biochemical determinations on the ingestion of horseradish peroxidase and morphometric analysis on the ingestion of cationized ferritin covered latex beads indicated that T. foetus has high endocytic activity. The process of ingestion of the various tracers used was analyzed by transmission electron microscopy of thin sections and freeze fracture replicas.

The surface charge of Tritrichomonas foetus.

The surface charge of Tritrichomonas foetus was evaluated by means of the binding of colloidal iron hydroxide particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, as visualized by electron microscopy and by direct measurements of the electrophoretic mobility (EPM), of cells suspended in solutions of different ionic strength and pH. At pH 7.2, T. foetus has a negative surface charge with a mean EPM of -1.03 micrometer . s-1 . V-1 . cm. At lower pH, there is a decrease in the negative surface charge with an isoelectric point at pH 1.2. At higher pH (greater than 9.0), there is an increase in the surface charge reaching an EPM of -2.5 micrometers . s-1 . V-1 . cm. These results indicate that the surface of T. foetus contains both negatively and positively charged dissociating groups. Binding of colloidal iron hydroxide and cationized ferritin particles throughout the cell surface of the protozoon was observed. Treatment of T. foetus with neuraminidase or trypsin reduced significantly the EPM of the cells. Enzyme-treated cells recovered their normal EPM when incubated for 6 h in fresh culture medium by a process that is inhibited by puromycin.

Tritrichomonas muris in the hamster: pseudocysts and the infection of newborn.

Female golden hamsters, either in the last week of pregnancy or in the first weeks of nursing, excreted in their feces variable numbers of pseudocysts of Tritrichomonas muris. Pseudocysts examined by electron microscopy had internalization of the 3 anterior flagella and the undulating membrane with its recurrent flagellum. The undulating membrane and the associated marginal lamellae were characteristic of T. muris. Pseudocysts gradually become motile after 2 or more hours of incubation in medium. The "excysted" trophozoites were identified ultrastructurally as T. muris. Newborn hamsters were not infected with T. muris at 3 days of age, but by the 7th day essentially all were found to have infected ceca, concomitant with cecal enlargement and the appearance of adult-type feces.