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.     

Chelex-100法及酚氯仿法提取阴道毛滴虫DNA的比较

目的-比较Chelex-100法和酚氯仿法提取阴道毛滴虫基因组DNA。方法-分别用Chelex-100法和酚氯仿法提取阴道毛滴虫基因组DNA,用PCR法检测DNA提取的有效性。结果两种方法提取的DNA经PCR扩增均有特定的条带。结论-两种方法均能提取阴道毛滴虫DNA。Chelex-100方法简便、省时,较适用于分子生物学研究及临床PCR扩增使用。     

Inhibition of the β-carbonic anhydrase from the protozoan pathogen Trichomonas vaginalis with sulphonamides

Sulphonamides and their isosteres are classical inhibitors of the carbonic anhydrase (CAs, EC 4.2.1.1) metalloenzymes. The protozoan pathogen Trichomonas vaginalis encodes two such enzymes belonging to the β-class, TvaCA1 and TvaCA2. Here we report the first sulphonamide inhibition study of TvaCA1, with a series of simple aromatic/heterocyclic primary sulphonamides as well as with clinically approved/investigational drugs for a range of pathologies (diuretics, antiglaucoma, antiepileptic, antiobesity, and antitumor drugs). TvaCA1 was effectively inhibited by acetazolamide and ethoxzolamide, with K_Is of 391 and 283 nM, respectively, whereas many other simple or clinically used sulphonamides were micromolar inhibitors or did not efficiently inhibit the enzyme. Finding more effective TvaCA1 inhibitors may constitute an innovative approach for fighting trichomoniasis, a sexually transmitted infection, caused by T. vaginalis.     

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。     

Activation of the β-carbonic anhydrase from the protozoan pathogen Trichomonas vaginalis with amines and amino acids

We report the first activation study of the β-class carbonic anhydrase (CA, EC 4.2.1.1) encoded in the genome of the protozoan pathogen Trichomonas vaginalis, TvaCA1. Among 24 amino acid and amine activators investigated, derivatives incorporating a second carboxylic moiety, such as L-Asp, L- and D-Glu, were devoid of activating effects up to concentrations of 50 µM within the assay system, whereas the corresponding compounds with a CONH₂ moiety, i.e. L-Gln and L-Asn showed modest activating effects, with activation constants in the range of 26.9 − 32.5 µM. Moderate activation was observed with L- and D-DOPA, histamine, dopamine, serotonin, (2-Aminoethyl)pyridine/piperazine and morpholine (K_A‘s ranging between 8.3 and 14.5 µM), while the best activators were L-and D-Trp, L-and D-Tyr and 4-amino-Phe, which showed K_A‘s ranging between 3.0 and 5.1 µM. Understanding in detail the activation mechanism of β-CAs may be relevant for the design of enzyme activity modulators with potential clinical significance.     

Molecular cloning of actin genes in Trichomonas vaginalis and phylogeny inferred from actin sequences

The parasitic protozoan Trichomonas vaginalis is known to contain the ubiquitous and highly conserved protein actin. A genomic library and a cDNA library have been screened to identify and clone the actin gene(s) of T. vaginalis. The nucleotide sequence of one gene and its flanking regions have been determined. The open reading frame encodes a protein of 376 amino acids. The sequence is not interrupted by any introns and the promoter could be represented by a 10 bp motif close to a consensus motif also found upstream of most sequenced T. vaginalis genes. The five different clones isolated from the cDNA library have similar sequences and encode three actin proteins differing only by one or two amino acids. A phylogenetic analysis of 31 actin sequences by distance matrix and parsimony methods, using centractin as outgroup, gives congruent trees with Parabasala branching above Diplomonadida.     

荆芥体外抗阴道毛滴虫作用机制的实验研究

目的探讨荆芥体外杀灭阴道毛滴虫的作用机制。方法将浓度为1∶4的荆芥水提液作用于体外培养的阴道毛滴虫,并采用透射电镜观察经药物作用2 h和4 h后阴道毛滴虫的超微结构变化。结果荆芥作用后阴道毛滴虫多聚核糖体解聚,粗面内质网脱颗粒,胞质内可见大量空泡;随药物作用时间延长,核膜不完整,核质变疏松;最终,胞膜破损,内容物外溢,虫体死亡。结论荆芥可破坏阴道毛滴虫的内膜系统,具有较强的抗滴虫作用。     

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.     

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 analy...     

阴道毛滴虫多克隆抗体的制备及Fd基因的原核表达

目的 将阴道毛滴虫铁氧还蛋白（ferredoxin,Fd）基因在大肠埃希菌中诱导表达。方法制备阴道毛滴虫可溶性抗原,多点注射免疫家兔,获得的血清用ELISA测定其抗体效价。将原核表达重组质粒pET3C-Fd转化入大肠埃希菌BL21（DE3）感受态细胞中,异丙基-β-D-硫代半乳糖苷（IPTG）诱导蛋白质表达。结果制备出抗阴道毛滴虫多克隆抗体,抗体效价在1：8000以上,用于免疫印迹实验。经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹（Western blot）分析,重组质粒在大肠埃希菌中表达出Fd。结论在大肠埃希菌中表达出了Fd。     

阴道毛滴虫铁氧还蛋白与甲硝唑抵抗的研究进展

氢化酶体是阴道毛滴虫重要的代谢器官,该器官内存在的铁氧还蛋白不仅是虫体代谢过程中主要的电子传递介体,而且也在甲硝唑的激活中起关键作用。近年来阴道毛滴虫的甲硝唑抵抗株在临床和实验室都有报道,实验研究发现活化药物的铁氧还蛋白减少或缺失,因此对铁氧还蛋白与甲硝唑抵抗的相关性研究越来越受到医学及药学界的重视。本文总结近年来该领域的研究成果及发展动态,以期对滴虫药物抵抗的发生机制以及滴虫病防治的研究提供有价值的资料。     

探讨荆芥体外抗阴道毛滴虫的效果

目的探讨荆芥体外抗阴道毛滴虫的效果。方法将不同浓度的荆芥水提液作用于体外培养的阴道毛滴虫,于药物作用后不同时问记录毛滴虫的死亡率,并在光镜下观察药物作用前后毛滴虫的形态变化,同时与白头翁和青蒿的体外杀虫效果相比较。结果荆芥的杀虫效果与药物浓度和作用时间呈正相关。荆芥水提液触杀阴道毛滴虫的最低有效浓度为1：4。3种中药中,白头翁的杀虫效果最好,与另2种中药相比较差异有非常显著性（P〈0．01）;荆芥与青蒿的作用效果接近（P〉0．05）。荆芥作用后毛滴虫体内充满大量颗粒和空泡,部分虫体裂解、内容物外溢。结论荆芥具有较强的抗阴道毛滴虫作用。     

Involvement of Macrophages in Proliferation of Prostate Cancer Cells Infected with Trichomonas vaginalis

Macrophages play a key role in chronic inflammation, and are the most abundant immune cells in the tumor microenvironment. We investigated whether an interaction between inflamed prostate cancer cells stimulated with Trichomonas vaginalis and macrophages stimulates the proliferation of the cancer cells. Conditioned medium was prepared from T. vaginalis-infected (TCM) and uninfected (CM) mouse prostate cancer (PCa) cell line (TRAMP-C2 cells). Thereafter conditioned medium was prepared from macrophages (J774A.1 cell line) after incubation with CM (MCM) or TCM (MTCM). When TRAMP-C2 cells were stimulated with T. vaginalis, protein and mRNA levels of CXCL1 and CCL2 increased, and migration of macrophages toward TCM was more extensive than towards CM. Macrophages stimulated with TCM produced higher levels of CCL2, IL-6, TNF-α, their mRNAs than macrophages stimulated with CM. MTCM stimulated the proliferation and invasiveness of TRAMP-C2 cells as well as the expression of cytokine receptors (CCR2, GP130, CXCR2). Importantly, blocking of each cytokine receptors with anti-cytokine receptor antibody significantly reduced the proliferation and invasiveness of TRAMP-C2 cells. We conclude that inflammatory mediators released by TRAMP-C2 cells in response to infection by T. vaginalis stimulate the migration and activation of macrophages and the activated macrophages stimulate the proliferation and invasiveness of the TRAMP-C2 cells via cytokine-cytokine receptor binding. Our results therefore suggested that macrophages contribute to the exacerbation of PCa due to inflammation of prostate cancer cells reacted with T. vaginalis.     

Interaction between facilitated diffusion of glucose across the plasma membrane and its metabolism in Trichomonas vaginalis

Abstract The parasitic protist Trichomonas vaginalis transport glucose across the plasma membrane by facilitated diffusion. The K _m of the transporter for glucose was 1.6 mM. The uptake of labelled glucose in a minimal medium not allowing growth reached saturation only after 2.5 h, indicating the turnover of storage carbohydrate. Organisms grown on glucose showed higher activities both of the transporter and of the subsequent metabolic pathway than organisms grown on maltose. At low external glucose concentrations the transport step was rate limiting, at higher levels a subsequent enzymatic step. The uptake mechanism for glucose of T. vaginalis resembled that of parasitic kinetoplastid protist and Entamoeba histolytica .     

Effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis

Here we report the effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) hydrolysis was 5- to 7-fold higher for the fresh clinical strain, when compared with the ATCC (American Type Culture Collection) strain. ATP hydrolysis was activated in the presence of metronidazole in the ATCC strain, whilst it was inhibited 33% by 50 microM tinidazole in a fresh clinical isolate. The treatment of cells in the presence of metronidazole for 2 h inhibited ATP and ADP hydrolysis, whilst treatment with tinidazole inhibited ATP and ADP hydrolysis only in the fresh clinical isolate. The drugs did not change the ecto-5'-nucleotidase activity for both strains. Our results suggest that the modulation of extracellular ATP and ADP levels during treatment with these drugs could be a parasitic defence strategy as a survival mechanism in an adverse environment.     

阴道毛滴虫黏附蛋白33基因原核表达载体的构建及表达

目的构建阴道毛滴虫黏附蛋白33基因原核表达载体并诱导其体外表达。方法pMD-18T-ap33重组质粒和pUC18空质粒经BamH Ⅰ和XbaⅠ限制性内切酶双酶切,将ap33基因亚克隆入pUC18载体并进行筛选和鉴定。重组质粒经IPTG诱导,SDS-PAGE电泳及Western-blot杂交鉴定重组蛋白。结果经双酶切及PCR鉴定,构建的重组质粒为阳性重组子,诱导出的重组蛋白大小约为Mr36000,与理论值基本相符。结论成功构建重组质粒并获得体外表达。     

阴道毛滴虫氢化酶体腺苷酸激酶基因的克隆及序列分析

目的-克隆阴道毛滴虫氢化酶体腺苷酸激酶(AK)基因,并测定其序列,进行序列分析。方法-根据AK基因已知序列设计合成一对引物,应用PCR技术从阴道毛滴虫基因组DNA中扩增出AK基因,并将其克隆入pMD18-T simple载体。阳性克隆的重组质粒经酶切及PCR鉴定后,用双脱氧链末端终止法进行基因序列测定。应用BLAST软件辅助分析所测基因与Genbank中阴道毛滴虫氢化酶体AK序列的同源性。结果-PCR扩增得到特异的阴道毛滴虫氢化酶体腺苷酸激酶基因序列。酶切及PCR鉴定获得了正确的PT-AK重组质粒。测序表明,所克隆的AK基因大小为690bp,编码229个氨基酸。序列分析表明,所测基因与Genbank中阴道毛滴虫氢化酶体AK序列具有高度同源性(99．9％)。结论-克隆了阴道毛滴虫氢化酶体腺苷酸激酶基因,序列测定及同源性分析表明,所测基因与Genbank中阴道毛滴虫氢化酶体AK序列具有高度同源性。     

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.     

Trichomonas vaginalis: Dominant G2 Period and G2 Phase Arrest in a Representative of an Early Branching Eukaryotic Lineage

ABSTRACT. Eukaryotic mitotic cell cycles have been extensively studied in yeasts and vertebrate cells but little is known about cell cycle mechanisms in early branches of the eukaryotic lineage. Trichomonas vaginalis represents one of the earliest branching eukaryotic lineages available for study. In contrast with most yeasts and vertebrate cells, the T. vaginalis G2 period was prolonged, comprising 50 to 58% of the cell population. Hydroxyurea, aphidicolin, and excess thymidine, all of which arrest yeasts and vertebrate cells at the G1/S phase boundary, had no effect on the T. vaginalis cell cycle, probably due to the known absence of synthetic pathways. The antimicrotubule mitotic inhibitors, colchicine and nocodazole, induced G2 phase synchrony. Metronidazole, a therapeutic reagent, also caused G2 phase arrest. These observations suggest that T. vaginalis is similar to yeasts and vertebrate cells in G2 and M phases, but the parasite's G1/S phase transition is distinctive. The results also suggest potentially therapeutic, anti-trichomonad activity of microtubule inhibitors such as nocodazole. The cultured parasite may prove useful as a model for the mitotic cell cycle in the absence of G1/S phase transitional activities universal in yeasts and vertebrate cells.