The mitotic spindle and associated membranes in the closed mitosis of trichomonads

In the present work, we followed the several phases of Tritrichomonas foetus and Trichomonas vaginalis cell cycles using immunofluorescence, serial thin sections, three-dimensional (3D) reconstruction, and transmission electron microscopy. In motile trichomonad cells or in pseudocyst forms, the nuclear envelope persists throughout mitosis, and the spindle is extranuclear. We found three types of spindle microtubules: pole-to-nucleus microtubules which are attached to the nuclear envelope, pole-to-pole microtubules forming a cylindrical, cytoplasmic groove on the nuclear compartment in pseudocysts of T. foetus cells, and pole-to-cytosol microtubules which extend freely into the cytoplasm. We demonstrated that: (1) in T. foetus, the spindle is assembled from an MTOC located at the base of the costa, underneath one of the basal bodies; (2) the spindle presents an unusual arc shape during the initial phases of mitosis in motile trophozoites; (3) the spindle microtubules are glutamylated, but not acetylated; (4) several membranes similar to those of the endoplasmic reticulum follow the spindle microtubules; (5) finger-like projections extend from the nucleus towards the cell poles in pseudocysts and multinucleated cells; and (6) vesicles formed in between the two nuclear membranes are seen in the course of mitosis in both trophozoite and pseudocyst forms.     

Polypeptides of hydrogenosome-enriched fractions from rumen ciliate protozoa and trichomonads: Immunological studies

Abstract The evolution of hydrogenosomes, energy-generating organelles of rumen ciliate protozoa and the flagellate trichomonads has been the subject of much speculation. Polypeptides of the hydrogenosome-enriched fractions from the rumen ciliates, Dasytricha ruminantium, Isostricha spp., Polyplastron multivesiculatum and Eudiplodinium maggii were separated by SDS-PAGE and compared to analogous polypeptide preparations from Tritrichomonas foetus . Immunoblotting with antisera specific to the hydrogenosomes of T. foetus identified common immunoreactive polypeptides present at estimated molecular masses of 28, 35, 38, 44, 48, 58, 100 and 120 kDa. That at 120 kDa corresponds to a single subunit of the purified pyruvate: ferredoxin oxidoreductase from the hydrogenosome of Trichomonas vaginalis .     

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 anti-tubulin 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.     

Purification of cysteine proteinases from trichomonads using bacitracin-sepharose

Abstract Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus . It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteines from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 μg of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10⁹ cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusuall features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.     

An Improved Method for the Isolation of Highly Polymerized Native Deoxyribonucleic Acid from Certain Protozoa*

A relatively simple phenol extraction method, with EDTA as the nuclease inhibitor, is described for the isolation of purified, highly polymerized native DNA from Trichomonas vaginalis, Trichomonas gallinae, and Tritrichomonas foetus; it is applicable also to Tetrahymena pyriformis. RNase Tl, RNase A (Worthington's R), pronase, and α-amylase digestions constitute important steps in obtaining satisfactory yields of DNA. High degree of polymerization of the isolation product was estimated by hyperchromicity at O.D.₂₆₀ after DNase treatment and by CsCl gradient analysis. The double-stranded condition of the DNA samples was estimated by the latter method and by denaturation with NaOH, and the molecular weight by sucrose gradient analysis. Purity of the samples was determined spectrophotometrically and by chemical analyses for protein and glycogen. DNA percent recovery was estimated by the diphenylamine reaction.     

Structure of Trichomonads as Revealed by Scanning Electron Microscopy*†

SYNOPSIS.
A scanning electron microscope (SEM) study of Hypotrichomonas acosta (Moskowitz), Trichomonas vaginalis Donné. Pentatrichomonas hominis (Davaine), and Tritrichomonas foetus (Riedmüller) provided new information about the structure of the periflagellar canal: emergence of the flagella from the cell body; structure of the undulating membrane; and position, shape, and size of the pelta. Of special interest were the spatial relationships of the attached part of the recurrent flagellum and the accessory filament in Hypotrichomonas and in the members of Trichomonadinae, i.e. Trichomonas and Pentatrichomonas.     

乳酸杆菌造成的微环境对阴道毛滴虫的影响

目的:研究乳酸杆菌产生的微环境对阴道毛滴虫的影响,为新型微生态制剂的开发提供可靠依据.方法:于滴虫生长高峰期分别将0.25 ml,0.5 ml,1.0 ml,2.5 ml已培养、鉴定好的浓度为3.0×108/ml乳酸杆菌加入到最适pH值肝浸汤培养基中,观察不同乳酸杆菌浓度的培养基中滴虫死亡情况.结果:乳酸杆菌浓度为0.5×108/ml与0.14×108/ml、0.27×108/ml、1.0×108/ml比较,在其浓度为0.5×108/ml时滴虫的死亡率明显要高.结论:体外试验中对阴道毛滴虫生长的抑制作用最强的乳酸杆菌浓度为0.5×108/ml.     

A further proteomic study on the effect of iron in the human pathogen Trichomonas vaginalis

Iron is an essential element to support the growth and survival of Trichomonas vaginalis. It plays a critical role in the host-parasite interaction, and modulates the expression of virulence factors in this protozoan. In this work, parasites grown in iron-rich and iron-depleted media were analyzed by (i) light and scanning electron microscopy and (ii) 2-DE and MS. Withdrawal of iron from the culture medium resulted in dramatic changes in both the morphology and in the proteome pattern of T. vaginalis. Trophozoites underwent transformation from ellipsoid or amoeboid forms to rounded cells, whose flagella and axostyle were internalized. Forty-five proteins differentially expressed in parasites cultivated in the absence of iron were identified. In iron-depleted parasites, enzymes involved in energetic metabolism, proteolysis and hydrogenosomal iron-sulfur (Fe-S) proteins were down-regulated or even suppressed. Among up-regulated proteins, six isoforms of actin were detected. In addition, phosphoenolpyruvate carboxykinase, putative lactate dehydrogenase, and putative adenosine triphosphatase were also up-regulated or were exclusively observed in gels related to iron-depleted parasites. Our data demonstrate that iron has a pivotal role in the regulation of the morphological transformation of T. vaginalis and modulates the expression of both Fe-S and non-Fe-S proteins in the parasite.     

Molecular prevalence and zoonotic potential of trichomonads from oral cavities in household dogs

This study investigated the molecular prevalence of oral trichomonads in household dogs. Of the 144 dogs, 21 (14.6%, 21/144) tested positive for oral trichomonads. The prevalence was significantly higher in dogs with severe gingivitis (gingival index 3: 30.0%, 8/26) than that in normal dogs (gingival index 0: 2.7%, 1/37). Therefore, an interaction between oral trichomonads and the development of periodontal disease is suggested. Of the 21 positive samples, 16 isolates were T. brixi, four isolates were T. tenax, and one was Tetratrichomonas sp. Considering T. tenax is recognized as a zoonotic agent, transmission between dogs and humans cannot be neglected.     

Trichomonas vaginalis: observation of coexistence of multiple viruses in the same isolate

Trichomonas vaginalis is a flagellated, parasitic protozoan that inhabits the urogenital tract of humans. Some isolates of T. vaginalis are infected with a double-stranded RNA (dsRNA) virus, which was described in the literature as homogeneous icosahedral viral particles with an isometric symmetry and 33 nm in diameter. This study examined in detail the viral particles in T. vaginalis isolate 347 and describes a heterogeneous population of viral particles. The different dsRNA viruses were only observed after a change in the technique. The sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The detected viruses ranged in size from 33 to 200 nm. Among the shapes observed were filamentous, cylindrical, and spherical particles. These results show that T. vaginalis may be a reservoir for several different dsRNA viruses simultaneously.     

Cell death in trichomonads: new insights

Tritrichomonas foetus is an amitochondriate parasite that possesses hydrogenosomes, unusual anerobic energy-producing organelles. In these organisms the “mitochondrial cell death machinery” is supposed to be absent, and the mechanisms that lead to cell demise remain to be elucidated. The presence of a cell death program in trichomonads has already been reported, suggesting the existence of a caspase-like execution pathway in such organisms. Here we demonstrate the alterations provoked by the fungicide griseofulvin and raise the possibility that other cell death pathways may exist in T. foetus. Dramatic changes in trichomonads morphology are presented after griseofulvin treatment, such as intense plasma membrane and nuclear envelope blebbing, nucleus fragmentation, and an abnormal number of oversized vacuoles. One important finding was the exposition of phosphatidylserine (PS) in the outer leaflet of the plasma membrane in cells after drug treatment, and also the presence of a high amount of misshapen flagella and tubulin precipitates as vacuolar contents, suggesting an autophagic process of abnormal cellular elements. Interestingly, immunoreactivity for activated caspase-3 was not detected during griseofulvin treatment, a finding distinct from the observed when this cell was treated with H₂O₂. The possibility of the existence of different pathways to cell death in trichomonads is discussed.     

Detection of Trichomonas Vaginalis Infection in Chronic Prostatitis/Chronic Pelvic Pain Syndrome Patients by Rapid Immunochromatographic Test

This study aims to evaluate associations between the immunochromatographic rapid test technique and Trichomonas vaginalis (TV) infection in patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) in Taiwan. All patients received post-prostate massage urine (VB3) Trichomonas rapid tests. The demographic characteristics and urogenital symptoms of CP/CPPS were recorded. Routine urinalysis of VB3 was also performed, and laboratory examination results of semen were recorded if available. A total of 29 patients with TV infection and 109 without TV infection were enrolled, which reflected that the prevalence in patients with TV infection was approximately 21%. Patients with TV infection displayed a significantly higher frequency of suprapubic/lower abdominal pain (p = 0.034), semen leukocyte > 5/high-power field (HPF) (p = 0.020), and an inflammatory type (category IIIA) (p = 0.005) than patients without TV infection. A higher prevalence of TV infection was found in patients with category IIIA (47.37%). No significant difference was found in the symptom duration and other clinical symptoms. In conclusion, the high prevalence of TV infection was revealed in CP/CPPS patients using the VB3 rapid Trichomonas test, especially in CP/CPPS patients with category IIIA. Thus, rapid TV testing might be vital for CP/CPPS patients in the hospital.     

A systematic identification of Kolobok superfamily transposons in Trichomonas vaginalis and sequence analysis on related transposases

Transposons are sequence elements widely distributed among genomes of all three kingdoms of life, providing genomic changes and playing significant roles in genome evolution. Trichomonas vaginalis is an excellent model system for transposon study since its genome ( ～ 160 Mb) has been sequenced and is composed of ～65% transposons and other repetitive elements. In this study, we primarily report the identification of Kolobok-type transposons (termed tvBac) in T. vaginalis and the results of transposase sequence analysis. We categorized 24 novel subfamilies of the Kolobok element, including one autonomous subfamily and 23 non-autonomous subfamilies. We also identified a novel H2CH motif in tvBac transposases based on multiple sequence alignment. In addition, we supposed that tvBac and Mutator transposons may have evolved independently from a common ancestor according to our phylogenetic analysis. Our results provide basic information for the understanding of the function and evolution of tvBac transposons in particular and other related transposon families in general.     

Unexpected Presence of Blastocystis Subtype 1–3 DNA in Human Vaginal and Sperm Samples Coinfected with Trichomonas vaginalis

There have been few reports on extra-enteric infections by Blastocystis STs and none have been molecularly identified in samples from human reproductive organs. We report for the first time the identification of 3 different subtypes of Blastocystis (ST1–3) in vaginal and sperm samples, from patients infected with Trichomonas vaginalis. Blastocystis STs were identified by PCR-sequencing and by phylogenetic inferences using 28 vaginal swab samples and 7 sperm samples from patients trichomoniasis. Blastocystis STs were identified in 6 of 28 vaginal swabs (21.4%) and in 3 of 7 sperm samples (42.8%). In both biological samples, STs 1–3 were found; one vaginal sample showed subtype co-infection with ST1 and ST3. High genetic variation was observed in the sequences obtained and no specific clustering in the phylogenetic trees was detected. Most of the haplotypes identified were placed far from the main dispersal centers. Our finding suggested that incorrect cleaning of the genital area or a contamination by combination of anal and vaginal intercourse.     

乳酸杆菌的代谢产物对体外培养的阴道毛滴虫的影响

目的研究乳酸杆菌代谢产物对体外培养的阴道毛滴虫的杀伤作用。方法检测不同浓度的乳酸杆菌代谢产物对体外培养的阴道毛滴虫在不同作用时间下的杀伤率。结果乳酸杆菌代谢产物浓度10%,作用时间2 h、4 h和6 h体外培养毛滴虫的杀伤率分别为26.43%、37.47%和46.35%;浓度25%时杀伤率分别为43.56%、74.65%和90.15%;浓度50%杀伤率分别为92.36%、95.23%和99.01%。结论乳酸杆菌代谢产物的浓度越高,对体外培养的毛滴虫的杀伤力越大,作用时间越长,效果越好。     

Iron uptake mechanisms of pathogenic bacteria

Abstract: Most of the iron in a mammalian body is complexed with various proteins. Moreover, in response to infection, iron availability is reduced in both extracellular and intracellular compartments. Bacteria need iron for growth and successful bacterial pathogens have therefore evolved to compete successfully for iron in the highly iron-stressed environment of the host's tissues and body fluids. Several strategies have been identified among pathogenic bacteria, including reduction of ferric to ferrous iron, occupation of intracellular niches, utilisation of host iron compounds, and production of siderophores. While direct evidence that high affinity mechanisms for iron acquisition function as bacterial virulence determinants has been provided in only a small number of cases, it is likely that many if not all such systems play a central role in the pathogenesis of infection.     

Excretory-Secretory Products of Trichomonas vaginalis Cause Apoptosis in Mouse Sperm in Vitro

Excretory-secretory products (ESP) of T. vaginalis have been shown to inhibit sperm motility, viability, and functional integrity, leading to a decreased fertilization rate in vitro. This study investigated whether T. vaginalis induce apoptosis and ultrastructural changes of sperm using flow cytometry and electron microscopy. Incubation of sperm with T. vaginalis ESP increased phosphatidylserine externalization and DNA fragmentation, and decreased mitochondrial membrane potential. Transmission electron microscopy of sperm incubated with ESP revealed abnormal features such as distorted heads, broken necks, and acrosomes exocytosis. This is the first report that demonstrates a direct impact of T. vaginalis ESP on sperm apoptosis and architecture in vitro.