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Trichomonas vaginalis is responsible for the most prevalent non-viral sexually transmitted disease worldwide, and yet the mechanisms used by this parasite to establish and maintain infection are poorly understood. We previously identified a T. vaginalis homologue (TvMIF) of a human cytokine, human macrophage migration inhibitory factor (huMIF). TvMIF mimics huMIF's role in increasing cell growth and inhibiting apoptosis in human host cells. To interrogate a role of TvMIF in parasite survival during infection, we asked whether overexpression of TvMIF (TvMIF-OE) confers an advantage to the parasite under nutrient stress conditions by comparing the survival of TvMIF-OE parasites to that of empty vector (EV) parasites. We found that under conditions of serum starvation, overexpression of TvMIF resulted in increased parasite survival. Serum-starved parasites secrete 2.5-fold more intrinsic TvMIF than unstarved parasites, stimulating autocrine and paracrine signaling. Similarly, we observed that addition of recombinant TvMIF increased the survival of the parasites in the absence of serum. Recombinant huMIF likewise increased the parasite survival in the absence of serum, indicating that the parasite may use this host survival factor to resist its own death. Moreover, TvMIF-OE parasites were found to undergo significantly less apoptosis and reactive oxygen species (ROS) generation under conditions of serum starvation, consistent with increased survival being the result of blocking ROS-induced apoptosis. These studies demonstrated that a parasitic MIF enhances survival under adverse conditions and defined TvMIF and huMIF as conserved survival factors that exhibit cross talk in host-pathogen interactions. IMPORTANCE Macrophage migration inhibitory factor (MIF) is a conserved protein found in most eukaryotes which has been well characterized in mammals but poorly studied in other eukaryotes. The limited analyses of MIF proteins found in unicellular eukaryotes have focused exclusively on the effect of parasitic MIF on the mammalian host. This was the first study to assess the function of a parasite MIF in parasite biology. We demonstrate that the Trichomonas vaginalis MIF functions to suppress cell death induced by apoptosis, thereby enhancing parasite survival under adverse conditions. Our research reveals a conserved survival mechanism, shared by a parasite and its host, and indicates a role for a conserved protein in mediating cross talk in host-pathogen interactions., (Copyright © 2018 Chen et al.)

Trichomonas vaginalis is a sexually transmitted parasite that causes vaginitis in women and itself secretes lipid mediator leukotriene B 4 (LTB 4 ). Mast cells are important effector cells of tissue inflammation during infection with parasites. Membrane-bridging SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes are critical for fusion during exocytosis. Although T. vaginalis-derived secretory products (TvSP) have been shown to induce exocytosis in mast cells, information regarding the signaling mechanisms between mast cell activation and TvSP is limited. In this study, we found that SNAP23-dependent surface trafficking of LTB 4 receptor 1 (BLT1) is required for nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-mediated exocytotic degranulation of mast cells induced by TvSP. First, stimulation with TvSP induced exocytotic degranulation and reactive oxygen species (ROS) generation in HMC-1 cells. Next, TvSP-induced ROS generation and exocytosis were strongly inhibited by transfection of BLT1 small interfering RNA (siRNA). TvSP induced trafficking of BLT1 from the cytosol to the plasma membrane. We also found that knockdown of SNAP23 abrogated TvSP-induced ROS generation, exocytosis, and surface trafficking of BLT1 in HMC-1 cells. By coimmunoprecipitation, there was a physical interaction between BLT1 and SNAP23 in TvSP-stimulated HMC-1 cells. Taken together, our results suggest that SNAP23-dependent surface trafficking of BLT1 is essential for exocytosis in human mast cells induced by T. vaginalis-secreted LTB 4 Our data collectively demonstrate a novel regulatory mechanism for SNAP23-dependent mast cell activation of T. vaginalis-secreted LTB 4 involving surface trafficking of BLT1. These results can help to explain how the cross talk mechanism between parasite and host can govern deliberately tissue inflammatory responses., (Copyright © 2016 American Society for Microbiology.)

Iron-sulfur flavoproteins (Isf) are flavin mononucleotide (FMN)- and FeS cluster-containing proteins commonly encountered in anaerobic prokaryotes. However, with the exception of Isf from Methanosarcina thermophila, which participates in oxidative stress management by removing oxygen and hydrogen peroxide, none of these proteins has been characterized in terms of function. Trichomonas vaginalis, a sexually transmitted eukaryotic parasite of humans, was found to express several iron-sulfur flavoprotein (TvIsf) homologs in its hydrogenosomes. We show here that in addition to having oxygen-reducing activity, the recombinant TvIsf also functions as a detoxifying reductase of metronidazole and chloramphenicol, both of which are antibiotics effective against a variety of anaerobic microbes. TvIsf can utilize both NADH and reduced ferredoxin as electron donors. Given the prevalence of Isf in anaerobic prokaryotes, we propose that these proteins are central to a novel defense mechanism against xenobiotics., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

In Trichomonas vaginalis, a novel nuclear localization signal spanning the folded R2R3 DNA-binding domain of a Myb2 protein was previously identified. To study whether a similar signal is used for nuclear translocation by other Myb proteins, nuclear translocation of Myb3 was examined in this report. When overexpressed, hemagglutinin-tagged Myb3 was localized to nuclei of transfected cells, with a cellular distribution similar to that of endogenous Myb3. Fusion to a bacterial tetracycline repressor, R2R3, of Myb3 that spans amino acids (aa) 48 to 156 was insufficient for nuclear translocation of the fusion protein, unless its C terminus was extended to aa 167. The conserved isoleucine in helix 2 of R2R3, which is important for Myb2's structural integrity in maintaining DNA-binding activity and nuclear translocation, was also vital for the former activity of Myb3, but less crucial for the latter. Sequential nuclear influx and efflux of Myb3, which require further extension of the nuclear localization signal to aa 180, were immediately induced after iron repletion. Sequence elements that regulate nuclear translocation with cytoplasmic retention, nuclear influx, and nuclear efflux were identified within the C-terminal tail. These results suggest that the R2R3 DNA-binding domain also serves as a common module for the nuclear translocation of both Myb2 and Myb3, but there are intrinsic differences between the two nuclear localization signals.

The morphological transformation of Trichomonas vaginalis from an ellipsoid form in batch culture to an adherent amoeboid form results from the contact of parasites with vaginal epithelial cells and with immobilized fibronectin (FN), a basement membrane component. This suggests host signaling of the parasite. We applied integrated transcriptomic and proteomic approaches to investigate the molecular responses of T. vaginalis upon binding to FN. A transcriptome analysis was performed by using large-scale expressed-sequence-tag (EST) sequencing. A total of 20,704 ESTs generated from batch culture (trophozoite-EST) versus FN-amoeboid trichomonad (FN-EST) cDNA libraries were analyzed. The FN-EST library revealed decreased amounts of transcripts that were of lower abundance in the trophozoite-EST library. There was a shift by FN-bound organisms to the expression of transcripts encoding essential proteins, possibly indicating the expression of genes for adaptation to the morphological changes needed for the FN-adhesive processes. In addition, we identified 43 differentially expressed proteins in the proteomes of FN-bound and unbound trichomonads. Among these proteins, cysteine peptidase, glyceraldehyde-3-phosphate dehydrogenase (an FN-binding protein), and stress-related proteins were upregulated in the FN-adherent cells. Stress-related genes and proteins were highly expressed in both the transcriptome and proteome of FN-bound organisms, implying that these genes and proteins may play critical roles in the response to adherence. This is the first report of a comparative proteomic and transcriptomic analysis after the binding of T. vaginalis to FN. This approach may lead to the discovery of novel virulence genes and affirm the role of genes involved in disease pathogenesis. This knowledge will permit a greater understanding of the complex host-parasite interplay.

The parabasalid protist Trichomonas vaginalis is a widespread parasite that affects humans, frequently causing vaginitis in infected women. Trichomonad mitosis is marked by the persistence of the nuclear membrane and the presence of an asymmetric extranuclear spindle with no obvious direct connection to the chromosomes. No centromeric markers have been described in T. vaginalis, which has prevented a detailed analysis of mitotic events in this organism. In other eukaryotes, nucleosomes of centromeric chromatin contain the histone H3 variant CenH3. The principal aim of this work was to identify a CenH3 homolog in T. vaginalis. We performed a screen of the T. vaginalis genome to retrieve sequences of canonical and variant H3 histones. Three variant histone H3 proteins were identified, and the subcellular localization of their epitope-tagged variants was determined. The localization of the variant TVAG_185390 could not be distinguished from that of the canonical H3 histone. The sequence of the variant TVAG_087830 closely resembled that of histone H3. The tagged protein colocalized with sites of active transcription, indicating that the variant TVAG_087830 represented H3.3 in T. vaginalis. The third H3 variant (TVAG_224460) was localized to 6 or 12 distinct spots at the periphery of the nucleus, corresponding to the number of chromosomes in G(1) phase and G(2) phase, respectively. We propose that this variant represents the centromeric marker CenH3 and thus can be employed as a tool to study mitosis in T. vaginalis. Furthermore, we suggest that the peripheral distribution of CenH3 within the nucleus results from the association of centromeres with the nuclear envelope throughout the cell cycle.

The protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, the most widespread nonviral sexually transmitted disease in humans. It possesses hydrogenosomes-anaerobic mitochondria that generate H(2), CO(2), and acetate from pyruvate while converting ADP to ATP via substrate-level phosphorylation. T. vaginalis hydrogenosomes lack a genome and translation machinery; hence, they import all their proteins from the cytosol. To date, however, only 30 imported proteins have been shown to localize to the organelle. A total of 226 nuclear-encoded proteins inferred from the genome sequence harbor a characteristic short N-terminal presequence, reminiscent of mitochondrial targeting peptides, which is thought to mediate hydrogenosomal targeting. Recent studies suggest, however, that the presequences might be less important than previously thought. We sought to identify new hydrogenosomal proteins within the 59,672 annotated open reading frames (ORFs) of T. vaginalis, independent of the N-terminal targeting signal, using a machine learning approach. Our training set included 57 gene and protein features determined for all 30 known hydrogenosomal proteins and 576 nonhydrogenosomal proteins. Several classifiers were trained on this set to yield an import score for all proteins encoded by T. vaginalis ORFs, predicting the likelihood of hydrogenosomal localization. The machine learning results were tested through immunofluorescence assay and immunodetection in isolated cell fractions of 14 protein predictions using hemagglutinin constructs expressed under the homologous SCSα promoter in transiently transformed T. vaginalis cells. Localization of 6 of the 10 top predicted hydrogenosome-localized proteins was confirmed, and two of these were found to lack an obvious N-terminal targeting signal.

Nuclear proteins usually contain specific peptide sequences, referred to as nuclear localization signals (NLSs), for nuclear import. These signals remain unexplored in the protozoan pathogen, Trichomonas vaginalis. The nuclear import of a Myb2 transcription factor was studied here using immunodetection of a hemagglutinin-tagged Myb2 overexpressed in the parasite. The tagged Myb2 was localized to the nucleus as punctate signals. With mutations of its polybasic sequences, 48KKQK51 and 61KR62, Myb2 was localized to the nucleus, but the signal was diffusive. When fused to a C-terminal non-nuclear protein, the Myb2 sequence spanning amino acid (aa) residues 48 to 143, which is embedded within the R2R3 DNA-binding domain (aa 40 to 156), was essential and sufficient for efficient nuclear import of a bacterial tetracycline repressor (TetR), and yet the transport efficiency was reduced with an additional fusion of a firefly luciferase to TetR, while classical NLSs from the simian virus 40 T-antigen had no function in this assay system. Myb2 nuclear import and DNA-binding activity were substantially perturbed with mutation of a conserved isoleucine (I74) in helix 2 to proline that altered secondary structure and ternary folding of the R2R3 domain. Disruption of DNA-binding activity alone by point mutation of a lysine residue, K51, preceding the structural domain had little effect on Myb2 nuclear localization, suggesting that nuclear translocation of Myb2, which requires an ordered structural domain, is independent of its DNA binding activity. These findings provide useful information for testing whether myriad Mybs in the parasite use a common module to regulate nuclear import.

Iron-inducible transcription of a malic enzyme gene (also reputed to be ap65-1) in Trichomonas vaginalis was previously shown to involve a Myb1 repressor and a Myb2 activator, each of which may preferentially select two closely spaced promoter sites, MRE-1/MRE-2r, which comprises overlapping promoter elements, and MRE-2f. In the present study, an iron-inducible approximately 32-kDa Myb3 nuclear protein was demonstrated to bind only the MRE-1 element. Changes in the iron supply, which produced antagonistic effects on the levels of Myb2 and Myb3 expression, also resulted in temporal and alternate entries of Myb2 and Myb3 into the ap65-1 promoter. Repression or activation of basal and iron-inducible ap65-1 transcription was detected in transfected cells when Myb3 was, respectively, substantially knocked down or overexpressed. In the latter case, increased Myb3 promoter entry was detected with concomitant decrease in Myb2 promoter entry under specific conditions, while Myb3 promoter entry was inhibited under all test conditions in cells overexpressing Myb2. In contrast, concomitant promoter entries by Myb2 and Myb3 diminished in cells overexpressing Myb1, except that Myb3 promoter entry was slightly affected under prolonged iron depletion. Together, these results suggest that Myb2 and Myb3 may coactivate basal and iron-inducible ap65-1 transcription against Myb1 through conditional and competitive promoter entries.

Trichomonas vaginalis, the protist that causes vaginal itching, has a huge genome with numerous gene duplications. Recently we found that Trichomonas has numerous genes encoding putative dolichyl-phosphate-glucose (Dol-P-Glc) synthases (encoded by ALG5 genes) despite the fact that Trichomonas lacks the glycosyltransferases (encoded by ALG6, ALG8, and ALG10 genes) that use Dol-P-Glc to glucosylate dolichyl-PP-linked glycans. In addition, Trichomonas does not have a canonical DPM1 gene, encoding a dolichyl-P-mannose (Dol-P-Man) synthase. Here we show Trichomonas membranes have roughly 300 times the Dol-P-Glc synthase activity of Saccharomyces cerevisiae membranes and about one-fifth the Dol-P-Man synthase activity of Saccharomyces membranes. Endogenous Dol-P-hexoses of Trichomonas are relatively abundant and contain 16 isoprene units. Five paralogous Trichomonas ALG5 gene products have Dol-P-Glc synthase activity when expressed as recombinant proteins, and these Trichomonas Alg5s correct a carboxypeptidase N glycosylation defect in a Saccharomyces alg5 mutant in vivo. A recombinant Trichomonas Dpm1, which is deeply divergent in its sequence, has Dol-P-Man synthase activity. When radiolabeled Dol-P-Glc is incubated with Trichomonas membranes, Glc is incorporated into reducing and nonreducing sugars of O-glycans of endogenous glycoproteins. To our knowledge, this is the first demonstration of Dol-P-Glc as a sugar donor for O-glycans on glycoproteins.

Trichomonas vaginalis is a protist that causes the most common human sexually transmitted infection. A T. vaginalis cDNA expression library was screened with pooled sera from patients with trichomoniasis. A highly reactive cDNA clone of 1,428 bp encoded a trichomonad protein of 472 amino acids with sequence identity to alpha-enolase (tv-eno1). The sequence alignment confirmed the highly conserved nature of the enzyme with 65% to 84% identity among organisms. The expression of tv-eno1 was up-regulated by contact of parasites with vaginal epithelial cells, and this is the first report demonstrating up-regulation by cytoadherence of a plasminogen-binding alpha-enolase in T. vaginalis. Immunofluorescence with monoclonal antibody of nonpermeabilized trichomonads showed tv-ENO1 on the surface. The recombinant tv-ENO1 was expressed in Escherichia coli as a glutathione S-transferase (GST)::tv-ENO1 fusion protein, which was cleaved using thrombin to obtain affinity-purified recombinant tv-ENO1 protein (tv-rENO1) detectable in immunoblots by sera of patients. Immobilized tv-rENO1 bound human plasminogen in a dose-dependent manner, and plasminogen binding by tv-rENO1 was confirmed in a ligand blot assay. The plasminogen-specific inhibitor epsilon-aminocaproic acid blocked the tv-rENO1-plasminogen association, indicating that lysines play a role in binding to tv-rENO1. Further, both parasites and tv-rENO1 activate plasminogen to plasmin that is mediated by tissue plasminogen activator. These data indicate that as with other bacterial pathogens, tv-ENO1 is an anchorless, surface-associated glycolytic enzyme of T. vaginalis.

Recent data suggest that frataxin plays a key role in eukaryote cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (FeS) cluster biosynthesis. We have now identified a frataxin homologue (T. vaginalis frataxin) from the human parasite Trichomonas vaginalis. Instead of mitochondria, this unicellular eukaryote possesses hydrogenosomes, peculiar organelles that produce hydrogen but nevertheless share common ancestry with mitochondria. T. vaginalis frataxin contains conserved residues implicated in iron binding, and in silico, it is predicted to form a typical alpha-beta sandwich motif. The short N-terminal extension of T. vaginalis frataxin resembles presequences that target proteins to hydrogenosomes, a prediction confirmed by the results of overexpression of T. vaginalis frataxin in T. vaginalis. When expressed in the mitochondria of a frataxin-deficient Saccharomyces cerevisiae strain, T. vaginalis frataxin partially restored defects in heme and FeS cluster biosynthesis. Although components of heme synthesis or heme-containing proteins have not been found in T. vaginalis to date, T. vaginalis frataxin was also shown to interact with S. cerevisiae ferrochelatase by using a Biacore assay. The discovery of conserved iron-metabolizing pathways in mitochondria and hydrogenosomes provides additional evidence not only of their common evolutionary history, but also of the fundamental importance of this pathway for eukaryotes.

Trichomonas vaginalis is one of the most common nonviral sexually transmitted human infections and, worldwide, has been linked to increased incidence of human immunodeficiency virus type 1 transmission, preterm delivery, low birth weight, cervical cancer, and vaginitis. The molecular pathways that are important in initiating host inflammatory and immune responses to T. vaginalis are poorly understood. Here we report interactions of human cervicovaginal epithelial cells with the most abundant cell surface glycoconjugate of the parasite, the T. vaginalis lipophosphoglycan (LPG). Purified LPG mediated the adhesion of parasites to human vaginal epithelial cells in a dose-dependent manner. Furthermore, T. vaginalis LPG (but not LPG from Tritrichomonas foetus, the causative agent of bovine trichomoniasis) induced a selective upregulation of chemotactic cytokines by human endocervical, ectocervical, and vaginal epithelial cells, which do not express Toll-like receptor 4/MD2. The T. vaginalis LPG triggered interleukin 8 (IL-8), which promotes the adhesion and transmigration of neutrophils across the endothelium, and macrophage inflammatory protein 3alpha, which is a chemoattractant for immune cells and is essential for dendritic cell maturation. These effects were dose dependent and sustained in the absence of cytotoxicity and IL-1beta release and utilized, at least in part, a signaling pathway independent from the Toll-like/IL-1 receptor adaptor protein MyD88.

The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.

Trichomonas vaginalis secretes putrescine that is readily detected in vaginal secretions. We wanted to examine the effect of decreased putrescine synthesis by inhibition of ornithine decarboxylase (ODC) on T. vaginalis. One reason is because inhibition of Tritrichomonas foetus ODC results in growth arrest, destruction of hydrogenosomes, and decreased amounts of hydrogenosomal enzymes. Treatment of T. vaginalis T016 with >/=20 mM 1,4-diamino-2-butanone (DAB) to inhibit ODC resulted in growth arrest, which was reversed by addition of exogenous putrescine. No similar reversal of growth arrest was achieved with the polyamines spermine or spermidine or with iron. Electron microscopic examination of control versus DAB-treated trichomonads did not reveal any adverse effects on the number and integrity of hydrogenosomes. Further, the adhesins AP65, AP51, and AP33 mediating binding to immortalized vaginal epithelial cells (VECs) share identity to enzymes of the hydrogenosome organelle, and there was no difference in amounts of adhesins between control versus DAB-treated T. vaginalis parasites. Likewise, similar patterns and extent of fluorescence were evident for the prominent AP65 adhesin. Surprisingly, DAB treatment increased by 4- to 20-fold above untreated trichomonads handled identically the level of adherence mediated by adhesins. Interestingly, the enhanced attachment to VECs was reversed by exogenous putrescine added to DAB-treated trichomonads. Equally noteworthy was that DAB-treated T. vaginalis with enhanced adherence did not possess the previously reported ability to kill host cells in a contact-dependent fashion mediated by cysteine proteinases, and total cysteine proteinase activity patterns were identical between control and DAB-treated trichomonads. Overall, these data suggest that polyamine metabolism and secreted putrescine are linked to host cell adherence and cytotoxicity.

The existence of a symbiotic relationship between Trichomonas vaginalis and Mycoplasma hominis, which is the first reported example of symbiosis between two obligate human pathogens, has been recently reported by our research group. In this work, we examined the cellular location of M. hominis in respect to T. vaginalis. By using gentamicin protection assays, double immunofluorescence, and confocal microscopy, we obtained strong evidence that M. hominis is located within protozoan cells. 5-Bromodeoxyuridine incorporation assays showed that intracellularly located mycoplasmas actively synthesize DNA. Our results demonstrate that M. hominis has the capability of entering trichomonad cells and of replicating inside the protozoon. These findings suggest that symbiosis might provide the bacteria, during human infection, with the capability to resist to environmental stresses, such as host defense mechanisms and pharmacological therapies.

The kinetics of the electron transfer reaction between reduced [2Fe-2S] ferredoxins and select nitroimidazole antimicrobial agents is reported. The ferredoxins from the protozoan Trichomonas vaginalis and the cyanobacterium Anabaena sp. strain 7120 were studied because they are the proximal electron donors to nitroimidazoles in these two organisms with significantly different nitroimidazole susceptibilities. The rates of electron transfer from Anabaena ferredoxin to all nitroimidazoles were 1 to 2 orders of magnitude lower than for T. vaginalis ferredoxin. Quantitative structure-activity analysis of the kinetic data showed that the size of the alkyl substituent on the N-1 position of the imidazole ring strongly influenced the magnitude of the electron transfer rate constant. This implies that the distance between the iron-sulfur cluster and the nitro group of the imidazole is the critical variable in determining the rate of electron transfer. A correlation between the magnitude of the one-electron transfer rate constant with the susceptibility of the host organism to the cytotoxic effects of nitroimidazoles was also discovered. These results demonstrate that reductive activation is the most crucial step in determining the toxicity of nitroimidazoles.

The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract, fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent, and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined. Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently been investigated using laboratory-induced resistant isolates. Instead of downregulation of the pyruvate:ferredoxin oxidoreductase and ferredoxin pathway as seen in G. duodenalis and T. vaginalis, E. histolytica induces oxidative stress mechanisms, including superoxide dismutase and peroxiredoxin. The review examines the value of investigating both clinical and laboratory-induced syngeneic drug-resistant isolates and dissection of the complementary data obtained. Comparison of resistance mechanisms in anaerobic bacteria and the parasitic protozoa is discussed as well as the value of studies of the epidemiology of resistance.

Trichomonas vaginalis infected with a double-stranded RNA virus undergoes phenotypic variation on the basis of surface versus cytoplasmic expression of the immunogenic protein P270. Examination of batch cultures by flow cytofluorometry with monoclonal antibody (MAb) to P270 yields both fluorescent and nonfluorescent trichomonads. Greater numbers and intensity of fluorescent organisms with surface P270 reactive with MAb were evident in parasites grown in medium depleted of iron. Placement of iron-limited organisms in medium supplemented with iron gave increased numbers of nonfluorescent trichomonads. Purified subpopulations of trichomonads with and without surface P270 obtained by fluorescence-activated cell sorting reverted to nonfluorescent and fluorescent phenotypes when placed in high- and low-iron media, respectively. No similar regulation by iron of P270 was evident among virus-negative T. vaginalis isolates or virus-negative progeny trichomonads derived from virus-infected isolates. Equal amounts of P270 were detectable by MAb on immunoblots of total proteins from identical numbers of parasites grown in low- and high-iron media. Finally, P270 was found to be highly phosphorylated in high-iron parasites. Iron, therefore, plays a role in modulating surface localization of P270 in virus-harboring parasites.

Benzimidazoles have been widely used since the 1960s as anthelmintic agents in veterinary and human medicine and as antifungal agents in agriculture. More recently, selected benzimidazole derivatives were shown to be active in vitro against two protozoan parasites, Trichomonas vaginalis and Giardia lamblia, and clinical studies with AIDS patients have suggested that microsporidia are susceptible as well. Here, we first present in vitro susceptibility data for T. vaginalis and G. lamblia using an expanded set of benzimidazole derivatives. Both parasites were highly susceptible to four derivatives, including mebendazole, flubendazole, and fenbendazole (50% inhibitory concentrations of 0.005 to 0.16 microgram/ml). These derivatives also had lethal activity that was time dependent: 90% of T. vaginalis cells failed to recover following a 20-h exposure to mebendazole at 0.17 microgram/ml. G. lamblia, but not T. vaginalis, was highly susceptible to five additional derivatives. Next, we examined in vitro activity of benzimidazoles against additional protozoan parasites: little or no activity was observed against Entamoeba histolytica, Leishmania major, and Acanthamoeba polyphaga. Since the microtubule protein beta-tubulin has been identified as the benzimidazole target in helminths and fungi, potential correlations between benzimidazole activity and beta-tubulin sequence were examined. This analysis included partial sequences (residues 108 to 259) from the organisms mentioned above, as well as the microsporidia Encephalitozoon hellem and Encephalitozoon cuniculi and the sporozoan Cryptosporidium parvum. beta-tubulin residues Glu-198 and, in particular, Phe-200 are strong predictors of benzimidazole susceptibility; both are present in Encephalitozoon spp. but absent in C. parvum.

We have investigated the effects of a novel neutrophil-activating factor released by Trichomonas vaginalis (TV-NAF) on neutrophil chemotaxis. TV-NAF was present in the supernatant from 10(7) T. vaginalis (STV) cultured in 1 ml of serum-free Hanks' balanced salt solution (HBSS) at 37 degrees C for 30 min. With a multichamber chemotactic assay, we found that there were 112 +/- 15 migrated neutrophils (mean +/- standard deviation, n = 7) for STV and 11 +/- 4 for HBSS per high-power field (x 400). STV was also able to induce neutrophil actin assembly (increased 1.5-fold), enhance expression of complement receptor type 3 (increased 5-fold), and promote intracellular calcium mobilization (increased 2.5-fold). There was no chemotactic activity in the preparation of STV from killed trichomonads. The fact that heating up to 100 degrees C or deproteinization by treatment with proteinase K at 65 degrees C for 1 h did not abolish its chemotactic activity suggests that the TV-NAF involved was not a protein. The chemotactic activity of TV-NAF was associated with the fraction containing small molecules of less than 3,000 Da. Therefore, the possibility that eicosanoid production by trichomonads is responsible for neutrophil activation was investigated. Leukotriene B4 (LTB4; 500 pg/ml) but not thromboxane B2 (< 20 pg/ml) or prostaglandin E2 (< 8 pg/ml) was found in the STV by radioimmunoassay. Production of LTB4 by trichomonads was time dependent and increased twofold when arachidonic acid (100 microM) was added but was not decreased when eicosanoid inhibitors were present. Evidence for the presence of LTB4 in STV was further provided by the fact that rabbit anti-LTB4 antiserum could abolish the chemotactic activity of STV. These studies suggest that the spontaneous release of TV-NAF, which is most likely LTB4, may activate neutrophils, presumably through a different arachidonate metabolic pathway than that in mammalian cells.

Recent work has shown that Trichomonas vaginalis produces a cell-detaching factor (CDF) that causes detachment of monolayer cells in vitro. To study the role of CDF as a pathogenic marker of disease, we studied the production of CDF in 12 clinical isolates of T. vaginalis. These isolates were also utilized in the mouse subcutaneous assay of Honigberg, which is the standard for pathogenicity of T. vaginalis. The isolates were divided into three groups based on clinical presentation (asymptomatic [n = 4], moderate [n = 4], and severe symptoms [n = 4]). CDF was assessed by harvesting the supernatant from the growth of T. vaginalis in cell culture and filtering the supernatant through a 0.45-microns-pore-size filter. The filtrate was applied in a microtiter cytotoxicity assay. The mouse subcutaneous assay did not significantly differentiate among the isolates. However, CDF was strongly associated with clinical presentation by two-way, repeated-measure analysis of variance (P = 0.025). Thus, CDF appears to correlate with clinical presentation and may be an important virulence marker in T. vaginalis pathogenesis.

Three Trichomonas vaginalis isolates with low in vivo susceptibilities to metronidazole (95% curative dose, greater than 3 X 100 mg kg-1 in subcutaneous infections in mice) were compared with strain ATCC 30001 and with four isolates exhibiting high in vivo susceptibilities (95% curative dose, less than 3 X 15 mg kg-1). Activity of pyruvate:ferredoxin oxidoreductase, anaerobic fermentation, and anaerobic intracellular accumulation of [14C]metronidazole label showed no significant isolate-dependent differences which could be correlated with drug susceptibility. The results suggest that processes providing electrons for metronidazole activation are not defective in the resistant strains. Aerobiosis, known to inhibit the antimicrobial action of metronidazole, inhibited accumulation of label more strongly in resistant isolates than in susceptible ones. No differences were detected, however, between resistant and susceptible isolates in respiration, aerobic fermentation, and the specific activity of NADH and NADPH oxidases, the main terminal oxidases of T. vaginalis. These findings suggest that the production of electrons is not diminished under aerobic conditions. The inhibitory effect of aerobic conditions on metronidazole activation, possibly due to competition for the electrons, is markedly enhanced in the resistant isolates compared to the susceptible ones. The mechanism of this effect, however, remains unknown.

Trichomonas vaginalis grown in iron-enriched medium contained increased concentrations of iron-sulfur proteins, including ferredoxin and pyruvate-ferredoxin oxidoreductase. The increases in hydrogenosomal constituents correlated with increased in vivo hydrogenosomal metabolism.

[(14)C]metronidazole used at the chemotherapeutic concentration of 10 mug/ml is taken up rapidly by the anaerobic protozoa Tritrichomonas foetus, Trichomonas vaginalis, and Entamoeba invadens kept under anaerobic conditions. It can be calculated that within 30 to 60 min the intracellular concentration of the label is 50 to 100 times higher than in the medium. The presence of air markedly suppresses the uptake in the trichomonads and abolishes it in E. invadens. The suppression disappears after anaerobic conditions are established. The rate of uptake in T. foetus is dependent on the concentration of the drug in the range studied (1 to 200 mug/ml). Analysis of double reciprocal plots suggests that the drug enters the cells predominantly or exclusively by diffusion. The major factor driving the uptake is most likely the intracellular biotransformation of the compound. If less than 3 mug of drug per mg of protein is taken up by T. foetus no decrease in viability is observed. Above this level the cytotoxic activity corresponds roughly to the amount accumulated in the cell, irrespective of whether the conditions are anaerobic or aerobic.

The interaction of alpha 1-Antitrypsin, the major serine protease inhibitor in plasma, with pathogenic Trichomonas vaginalis and the acquisition by trichomonads of this host protein from normal human plasma were investigated. alpha 1-Antitrypsin acquired by intact parasites could not be removed by repeated washings in phosphate-buffered saline. Saturation kinetics were observed after incubation of glutaraldehyde-fixed organisms with 125I-labeled alpha 1-antitrypsin. Evidence suggesting that specific parasite membrane sites were responsible for trichomonal acquisition of alpha 1-antitrypsin was obtained through competitive binding experiments using purified preparations of homologous versus heterologous plasma proteins. No evidence of degradation of bound antitrypsin by live parasites was observed. The avid binding of alpha 1-antitrypsin by pathogenic T. vaginalis after incubation in normal human plasma was demonstrated by using sensitive electrophoretic and immunodetection techniques. Radioimmunoprecipitation of intrinsically labeled, detergent-solubilized extracts of T. vaginalis incubated with monospecific antisera against alpha 1-antitrypsin and other human plasma proteins revealed the inability of parasites to biosynthesize any substance cross-reactive with host plasma proteins. Finally, T. vaginalis organisms pretreated with alpha 1-antitrypsin inhibited trypsin caseinase activity in an in vitro assay. The implications of these observations are discussed.

Acetamide forms from metronidazole in cultures of either Entamoeba histolytica or Trichomonas vaginalis as it does in cultures of bacteria which are susceptible to this drug. Under aerobic conditions, the formation of acetamide is more rapid in a strain of T. vaginalis which is more susceptible to metronidazole. Thus, it appears that the formation of acetamide may be associated with the microbiocidal action of metronidazole.

Twelve 4- and 5-nitroimidazole derivatives, including metronidazole and two of its metabolites, tinidazole, dimetridazole, and nimorazole, were tested for antitrichomonad action on Tritrichomonas foetus (KV(1)) and Trichomonas vaginalis (ATCC 30001) for mutagenicity on a nitroreductase-positive (TA 100) and a nitroreductase-deficient (TA 100-FR(1)) strain of Salmonella typhimurium, as well as for the reducibility of the nitro group by T. foetus homogenates. Compounds with activity <1% of that of metronidazole are regarded as inactive. All antitrichomonad compounds induce mutations and can be reduced. S. typhimurium TA 100 gave mutations under both aerobiosis and anaerobiosis; TA 100-FR(1), however, gave mutations only under anaerobiosis. Certain compounds that are reducible, and the nonreducible derivatives, were inactive. Metronidazole and its inactive 4-nitro analogue were reduced in a four-electron process in ferredoxin- or methyl viologen-mediated reactions with the same velocity. The results underscore the role of the reduction of the nitro group in the antitrichomonad and in the mutagenic activity of nitroimidazoles.

The primary purpose of this study was to identify the mammalian tissue cultures which were most suitable for investigations of the cytopathogenicity of Trichomonas vaginalis. A recently isolated strain of the organism was inoculated into 15 different tissue cultures which were maintained in an appropriately modified growth medium. Proliferation of the protozoon was accompanied by the progressive disintegration of cell culture monolayers. Initial focal lesions consisting of detached cells and an accumulation of trichomonads gradually enlarged until the entire monolayer was disrupted. When judged by the size of the inoculum required to obtain this effect, differences among the tissue cultures were noted. An inoculum of approximately 10(3) viable trichomonads was sufficient to completely disrupt monolayers of HeLa 229, HeLa, McCoy, HEp-2, and RK-13 cells. To obtain a comparable effect with other cells, 10- to 100-fold higher levels of inoculum were required. Polyethylene glycol concentrates from culture filtrates contained a cell-detaching factor (CDF) which caused detachment and clumping of susceptible cells. Freshly seeded cells in growth medium containing CDF failed to form a monolayer. Aggregates of cells maintained for up to 1 week in the presence of CDF remained viable and formed a monolayer after being washed and suspended in normal growth medium. The activity of the CDF was not lost during 1 week of contact with the cells. The CDF may contribute to the pathogenicity mechanisms of T. vaginalis.