Further studies on the surface charge of various strains ofTrichomonas vaginalis andTritrichomonas foetus

The surface charge of three strains ofTrichomonas vaginalis and five strains ofTritrichomonas foetus was determined by direct measurement of the mean cellular electrophoretic mobility (EPM) of cells suspended in solutions of different ionic strength and pH. No differences were observed in the mean EPM among the two species, although significant differences among the strains exist. Strains that are more pathogenic to mouse, as measured using the subcutaneous assay, had a surface more negative. Treatment of the parasites with trypsin or neuraminidase reduced significantly their mean EPM and increased their isoelectric point.Tritrichomonas foetus was more sensitive to the enzyme treatment thanT. vaginalis. Enzyme-treated cells recovered their normal EPM if, after enzyme treatment, they were incubated in fresh culture medium. The recovery process of trypsintreated cells was inhibited 10–20% by addition of inhibitors of either protein synthesis (puromycin) orN-glycosylation of proteins (tunicamycin) to the incubation medium, suggesting that a cytoplasmic pool of sialoglycoproteins may exist. The recovering of the EPM ofT. foetus andT. vaginalis previously treated with neuraminidase was inhibited by puromycin or tunicamycin about 40–50% and 17–30%, respectively. These observations suggest that sialoglycolipids exist on the surface of both parasite species, and that they contribute more to the surface charge ofT. vaginalis than to that ofT. foetus.     

A Freeze-Fracture Electron Microscope Study of Trichomonas vaginalis Donné and Tritrichomonas foetus (Riedmüller)1

Two strains of Trichomonas vaginalis, JH162A, with low pathogenicity, and Balt 44, with high pathogenicity, as well as one highly pathogenic strain, KV-1, of Tritrichomonas foetus were studied by freeze-fracture electron microscopy. The protoplasmic faces (PFs) of the cell membranes of all three strains of both species had similar numbers of intramembranous particles (IMPs); however, the particles in the external faces (EFs) of these membranes were least abundant in Trichomonas vaginalis strain Balt 44 and most numerous in those of strain JH162A of this species. In Tritrichomonas foetus strain KV-1 the number of IMPs in the EF was close to but somewhat lower than that in the mild strain of the human urogenital trichomonad. In both species, the anterior, but not the recurrent, flagella had rosette-like formations, consisting of ～9 to 12 IMPs on both the PFs and EFs. The numbers and distribution of the rosettes appeared to vary among different flagella and in different areas of individual flagella of a single organism belonging to either species. The freeze-fracture electron micrographs provided a more complete understanding of the fine structure of undulating membranes of Trichomonadinae, as represented by Trichomonas vaginalis, and of Tritrichomonadinae (the Tritrichomonas augusta-type), as exemplified by Tritrichomonas foetus, than was gained from previous transmission and scanning electron microscope studies. Typically three longitudinal rows of IMPs on the PF of the recurrent flagellum of Trichomonas vaginalis were noted in the area of attachment of this flagellum to the undulating membrane. The functional aspects of the various structures and differences between certain organelles revealed in the two trichomonad species by the freeze-fracture method are discussed.     

Concordance between genetic relatedness and phenotypic similarities of Trichomonas vaginalis strains

Background  

Despite the medical importance of trichomoniasis, little is known about the genetic relatedness of Trichomonas vaginalis strains with similar biological characteristics. Furthermore, the distribution of endobionts such as mycoplasmas or Trichomonas vaginalis virus (TVV) in the T. vaginalis metapopulation is poorly characterised.     

Double-stranded RNA viral infection of Trichomonas vaginalis and correlation with genetic polymorphism of isolates

Trichomonas vaginalis can be infected with double-stranded RNA (dsRNA) viruses known as T. vaginalis virus (TVV). This viral infection may have important implications for trichomonal virulence and disease pathogenesis. The objective of this study was to determine the possible correlation between the T. vaginalis genetic polymorphism and the isolate infection with TVV. The Random Amplified Polymorphic DNA (RAPD) technique was used to determine genetic differences among 37 isolates of T. vaginalis using a panel of 30 random primers and these genetic data were correlated with the infection of isolates with TVV. The trees drawn based on RAPD data showed significantly association with the presence of TVV (P = 0.028) demonstrating the existence of concordance between the genetic relatedness and the presence of TVV in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the viral enters and/or survival.     

Double-stranded RNAs associated with Fusarium proliferatum mitochondria

Many fungi harbor double-stranded (ds) RNA molecules, which can have phenotypic effects such as hypovirulence, altered colony morphology, and pigmentation. In some species of Fusarium, dsRNA molecules are found in every strain examined. We examined 100 F. proliferatum strains collected primarily from maize and sorghum in the United States, but found only four that carried dsRNAs. Each strain harbored a distinct set of dsRNAs, which ranged in size from approximately 0.7–3.1 kb. A single dsRNA band was observed from one strain, but multiple bands were observed from the other three. The strains with multiple dsRNAs transmitted these dsRNAs as sets at a high frequency (≥ 97 %) to vegetatively produced microconidia, but the single dsRNA of the fourth strain was only rarely (≤ 3 %) transmitted in this manner. None of these dsRNAs could be transmitted through sexual crosses in which the dsRNA-containing strain served as the male parent. Transmission through the female parent could not be tested as the field strains and dsRNA-free derivatives of these strains were female sterile. The dsRNAs from the strains with multiple dsRNAs were present in and protected against ribonuclease A digestion in crude mitochondrial preparations. The high transmission rate to single-conidiospore cultures, the lack of transmission through the male parent of sexual crosses, and the protection against ribonuclease A digestion are all consistent with a mitochondrial localization of the dsRNAs from the strains carrying multiple dsRNAs. dsRNAs often function as viruses in fungi, and the three F. proliferatum strains reported here join strains of Ophiostoma novo-ulmi, Rhizoctonia solani, and Cryphonectria parasitica as the only fungi known to carry dsRNAs associated with the mitochondria. Contribution number 02-495-J from the Kansas Agricultural Experiment Station (Manhattan).     

Eicosapentaenoic Acid Modulates Trichomonas vaginalis Activity

Trichomonas vaginalis is a sexually transmitted parasite and, while it is often asymptomatic in males, the parasite is associated with disease in both sexes. Metronidazole is an effective treatment for trichomoniasis, but resistant strains have evolved and, thus, it has become necessary to investigate other possible therapies. In this study, we examined the effects of native and oxidized forms of the sodium salts of eicosapentaenoic, docosahexaenoic, and arachidonic acids on T. vaginalis activity. Eicosapentaenoic acid was the most toxic with 190 and 380 μM causing approximately 90% cell death in Casu2 and ATCC 50142 strains, respectively. In contrast, oxidized eicosapentaenoic acid was the least toxic, requiring > 3 mM to inhibit activity, while low levels (10 μM) were associated with increased parasite density. Mass spectrometric analysis of oxidized eicosapentaenoic acid revealed C20 products containing one to six additional oxygen atoms and various degrees of bond saturation. These results indicate that eicosapentaenoic acid has different effects on T. vaginalis survival, depending on whether it is present in the native or oxidized form. A better understanding of lipid metabolism in T. vaginalis may facilitate the design of synthetic fatty acids that are effective for the treatment of metronidazole‐resistant T. vaginalis.     

Occurrence of virus-like particles in the Dead Sea

Electron-microscopic examination of water samples from the hypersaline Dead Sea showed the presence of high numbers of virus-like particles. Between 0.9 and 7.3 × 10⁷ virus-like particles ml⁻¹ were enumerated in October 1994 in the upper 20 m of the water column during the decline of a bloom of halophilic Archaea. Virus-like particles outnumbered bacteria by a factor of 0.9–9.5 (average 4.4). A variety of viral morphologies were detected, the most often encountered being spindle-shaped, followed by polyhedral and tailed phages. In addition, other types of particles were frequently found, such as unidentified algal scales, and virus-sized star-shaped particles. Water samples collected during 1995 contained low numbers of both bacteria and virus-like particles (1.9–2.6 × 10⁶ and 0.8–4.6 × 10⁷ ml⁻¹ in April 1995), with viral numbers sharply declining afterwards (less than 10⁴ ml⁻¹ in November 1995–January 1996). It is suggested that viruses may play a major role in the decline of halophilic archaeal communities in the Dead Sea, an environment in which protozoa and other predators are absent. Received: February 5, 1997 / Accepted: May 24, 1997     

Antigenic Heterogeneity in the 115,000 Mr Major Surface Antigen of Trichomonas vaginalis1

ABSTRACT The 115,000-molecular-weight antigen of Trichomonas vaginalis was characterized using monoclonal antibodies developed to three different strains of T. vaginalis and one strain of Tritrichomonas foetus. The antigen was found to be present on all strains or isolates of T. vaginalis examined and was demonstrated to be located on the external surface plasma membrane by agglutination assays and complement-mediated lysis assays. Characteristics of the antigen were assessed with a proteolytic enzyme and periodate oxidation. Periodate treatment of whole T. vaginalis abrogated binding for eight antibodies while use of pronase-treated antigen resulted in loss of antibody binding for two different antibodies. Screening of 19 axenized clinical isolates of T. vaginalis and one strain each of T. foetus and Giardia lamblia with type-specific antibodies delineated three major groups of T. vaginalis based on antigenic specificities (epitope distributions) within the 115,000-molecular-weight antigen. In addition, one epitope of the 115,000-molecular-weight antigen was found only on the immunizing strain. Two epitopes were present on all T. vaginalis isolates as well as T. foetus and G. lamblia. One epitope was common to all T. vaginalis except one. A minimum of six different epitopes of the 115,000-molecular-weight antigen were identified. Antigens purified with type-specific or “common” monoclonal antibodies shared the same partial peptide maps demonstrating relatedness.     

Effect of the Antimicrobial Peptide Tritrpticin on the In Vitro Viability and Growth of <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>

Antimicrobial peptides are widely distributed in nature; they play important roles in several aspects of innate immunity and may provide a basis for the design of novel therapeutic agents. In this study, C-amidated tritrpticin, a 13 amino acid tryptophan-rich antimicrobial peptide derived from a porcine cathelicidin, was tested against Trichomonas vaginalis, a protozoan that causes a serious non-viral sexually transmitted disease associated with preterm birth, low birth weight, and high risk of HIV-1 infection. Tritrpticin was selected due to its reasonably easy synthesis and because analogs with lower toxicity may be designed. Our results show that tritrpticin-NH₂ at either 100 or 200 μg/ml (52.5 or 105 μM) clearly reduces the viability and growth of Trichomonas vaginalis. Together with tritrpticin-NH₂, sodium bicarbonate further limited trichomonad growth. Additionally, a low concentration of metronidazole (5.8 μM), the most commonly used medication for Trichomonas vaginalis, was more effective against the growth of the parasite when it was combined with tritrpticin-NH₂.     

Transmissibility of viral double-stranded RNA between strains of the violet root rot fungus <Emphasis Type="Italic">Helicobasidium mompa </Emphasis>and the potential for viral dsRNA infection to this fungus using monokaryotic strains

 To examine the potential of a method of double-stranded (ds) RNA infection to Helicobasidium mompa, the transmissibility of dsRNA between strains of this fungus was investigated. Strain V70 was used as a dsRNA donor. The dsRNA recipients were six strains that were mycelially incompatible with V70, plus two monokaryotic strains. Random amplified polymorphic DNA analysis suggested that the mycelially incompatible strains were genetically different mutually; however, the analysis also suggested that V70 was genetically homogeneous with the two monokaryotic strains. When V70 was paired with either of the mycelially incompatible strains, the dsRNAs did not transmit to the recipients at all. When V70 was paired with the two monokaryotic strains, the dsRNAs were transmitted to the monokaryotic strains. The two monokaryotic strains, which had acquired dsRNAs from V70 in the previous experiment, were used as new dsRNA donors in a next experiment so that we could investigate dsRNA transmission from these monokaryotic strains to the six strains used in the previous experiment. One of the monokaryotic strains permitted dsRNA transmission to two of the six recipients. We conclude that we can infect genetically different strains of H. mompa with dsRNA using the monokaryotic strains. Received: December 12, 2002 / Accepted: January 27, 2003 Acknowledgments This research was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences. The authors are grateful to Dr. Tadanori Aimi of Tottori University for helpful discussion. Correspondence to:K. Suzaki     

Purification and some properties of strawberry mottle virus

Strawberry mottle virus (SMoV) (three isolates: HJ, 3E and N) were transmitted to Chenopodium quinoa plants by sap inoculation. All three isolates induced very similar symptoms consisting of chlorotic spots and ringspots in inoculated leaves, and vein chlorosis, mottling, and dwarfing of the upper leaves. SMoV isolate HJ was purified from infected C. quinoa by homogenisation with 10 mM phosphate buffer, pH 7.2 containing 5% Triton X-100, followed by differential, sucrose density-gradient and CsCl equilibrium density-gradient centrifugations. A fraction with a buoyant density of 1.42g^- cm^-3 after CsCl density-gradient centrifugation was highly infectious to C. quinoa and contained many isometric virus-like particles c. 37 nm in diameter. These virus-like particles were never found in fractions from uninfected preparations. Electrophoretic analysis of a fraction containing virus-like particles revealed that these particles might have a single coat protein subunit with the apparent molecular mass of 26 K daltons and one nucleic acid of 6.6 kilobases. Double-stranded RNA analysis of isolate HJ-infected or uninfected C. quinoa and Fragaria vesca var. semperflorens seedling line ‘Alpine’ plants showed that both infected plants had two infection-specific dsRNA bands of mol. wts 4.5 and 3.9 × 10⁶.     

Virus-like particle capsid proteins encoded by different L double-stranded RNAs of Saccharomyces cerevisiae: their roles in maintenance of M double-stranded killer plasmids.

The plasmid determinants of killer phenotypes in type K1 and K2 killer yeast cells are the 1.9-kilobase (kb) M1 and 1.7-kb M2 double-stranded RNAs (dsRNAs), respectively. These are dependent for their maintenance and encapsidation, in Saccharomyces cerevisiae virus ScV-M1 or ScV-M2 virus-like particles, on the capsid provided by one of a group of moderately related 4.7-kb dsRNAs called LA. The L1A and L2A dsRNAs found in naturally isolated K1 and K2 killers encode 88-kilodalton VL1A-P1 and 86-kilodalton VL2A-P1 capsids, respectively. These are competent for encapsidating homologous LA dsRNAs as well as M dsRNAs. Most strains of S. cerevisiae, including killers, contain one of a second group of closely related 4.7-kb dsRNAs called LBC. These encode their own 82-kilodalton capsid protein, VLBC-P1, which, at least in strains containing only LBC, encapsidates homologous dsRNA in ScV-LBC virus-like particles. In a K1 killer strain containing both L1A and LBC, ScV-M1 particles contain only VL1A-P1. In such strains it is probable that each virus-like particle contains a single capsid type and that each L dsRNA is encapsidated by a homologous capsid.     

Virus-like particles and double stranded RNA from killer and non-killer strains of Saccharomyces cerevisiae.

Virus-like particles and DsRNA found in extracts of killer, non-killer and suppressive non-killer strains were co-precipitated from cell extracts using an antibody prepared against purified virus-like particles isolated from a non-killer strain having only the higher molecular weight L dsRNA. The relative amount of virus-like particles correlated roughly with the amount of dsRNA: those strains with high concentrations of dsRNA had the most particles. When a preparation of particles was subjected to sucrose gradient velocity centrifugation, particles containing the S and M dsRNA could be separated from those containing the L dsRNA. These experiments taken together suggest that the L, M and S dsRNAs are separately encapsulated by the same protein coat.     

Organization of biosynthetic gene cluster for avermectin in Streptomyces avermitilis: analysis of enzymatic domains in four polyketide synthases

The analysis of the incorporation of ¹³C-labeled precursors into avermectins indicates that the avermectin aglycons are synthesized by head-to-tail condensation of various acyl groups, which is similar to the biosynthesis of other polyketides. Polyketide synthases (PKS) use the appropriate CoA ester as a primer and add acetate units from malonyl-CoA and propionate units from methylmalonyl-CoA to assemble the polyketides. Avermectin aglycons are formed by addition to the starter unit (2-methylbutyrate or isobutyrate) of 12 acyl condensations in the order P–A–A–A–A–P–P–A–P–A–P–A (P, propionyl; A, acetyl). Within the 90-kb gene cluster for avermectin biosynthesis, the central 65-kb segment was found to be required for aglycon biosynthesis by phenotypic analysis of strains containing deletion or insertion mutations in this region. A complete sequence analysis of the 65-kb segment indicated that this segment encodes avermectin PKS. The avermectin PKS genes are organized into two converging blocks of ORFs. From the results of sequencing analysis, a feature of the two regions, aveA1/aveA2 and avea3/aveA4, is that they encode four kinds of large multifunctional polypeptides containing 55 domains which possess putative fatty acid synthase-like activities. The avermectin PKS (AVES 1–4) appear to contain two, three, or four modules. AVES 1 and 2 contain two and four modules, respectively, whereas AVES 3 and AVES 4 each contains three modules. The 12 modules correspond to the 12 cycles required for synthesis of the avermectin aglycon. Journal of Industrial Microbiology & Biotechnology (2001) 27, 170–176. Received 21 September 1999/ Accepted in revised form 14 September 2000     

Association of particles that contain double-stranded RNAs with algal chloroplasts and mitochondria

Linear dsRNAs (double-stranded RNAs) belonging to several distinct size classes were found to be localized in chloroplasts and mitochondria of Bryopsis spp., raising the possibility that these dsRNAs are prokaryotic in nature. The algal cytosol and nuclei did not contain dsRNAs. The amount of the dsRNAs in the organelles appeared constant, and there were about 500 copies per chloroplast. The four major dsRNAs from Bryopsis chloroplasts were about 2 kbp (kilobase pairs) in length and originated from discrete isometric particles of about 25 nm diameter. These virus-like particles were purified by CsCl density gradient centrifugation after extraction from isolated chloroplasts with chloroformbutanol and subsequent precipitation with polyethylene glycol. They had a buoyant density of about 1.40 g · cm^–3 and contained four major and three minor proteins. Mitochondrial dsRNAs were about 4.5 kbp in length and formed less-stable particles of about 40 nm in diameter with a buoyant density of 1.47 g · cm^–3. Some observations support the hypothesis that vertical transmission of the protein-coated, non-infectious dsRNAs occurs within cell organelles. Double-stranded RNAs of various sizes were found in most green, red, and brown algae. The characteristics of the algal dsRNAs are compared with those of dsRNAs from higher plants and the biological significance of the dsRNAs in cell organelles is discussed.Abbreviations dsRNA double-stranded RNA - kbp kilobase pairs - SDS sodium dodecyl sulfate - SSC 0.15 M NaCl 0.015 M sodium citrate - PAGE polyacrylamide gel electrophoresis The authors would like to express their gratitude to Dr. T. Natsuaki, Utsunomiya University, and Dr. D. Hosokawa, Tokyo University of Agriculture and Technology, for their helpful suggestions throughout this research. They are also much indebted to Dr. B. Wang, Institute of Genetics, Academia Sinica, Beijing, PRC, for his suggestions on rice dsRNA, and to Dr. T. Kohbara, Senshu University, on Bryopsis cells. Sincere thanks are also due to Dr. T. Misonou, Yamanashi University, and Dr. K. Masuda, Akita Prefectural College of Agriculture, for supplying plant materials; to Dr. N. Sonoki, Azabu University, for nucleotide analysis of dsRNAs; and to Y. Koshino for technical assistance. This research was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.     

The killer phenomenon in Ustilago: Electron microscopy of the dsRNA encapsidated in individual virus particles

From earlier studies with the Ustilago maydis virus and other dsRNA viruses it is known that discrete dsRNA segments typical of each virus are obtained by extraction. A variation exists with respect to the encapsidation of these segments among different viruses. The encapsidation of the genome in individual particles of the Ustilago virus was examined by electron microscopy after disruption of virus particles. The study included the P6 wild-type and 2 mutants containing only part of the genome. The results indicate that most virus particles of the wild-type and the mutants contain up to 12–14×10⁶ daltons of dsRNA. Since the largest extracted molecule is 3.2×10⁶D these findings suggest that an individual particle may contain more than one segment of dsRNA. Free linear molecules that exceed in size the extracted segments were also found following the disruption of each of the 3 virus types examined. Thus, the viral genome seen segmented after extraction is organized as a concatamer in the capsid and each virus particle can contain an entire viral genome consisting of each type of the segments seen after extraction, a repeat of a single segment or a random assortment. In each case the information may be organized as a concatamer.     

The Pyrophosphate-Dependent Phosphofructokinase of the Protist, Trichomonas vaginalis, and the Evolutionary Relationships of Protist Phosphofructokinases

The pyrophosphate-dependent phosphofructokinase (PP_i-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified. The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation. The protein was fragmented and a number of peptides were sequenced. Based on this information a PCR product was obtained from T. vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones. Southern analysis indicated the presence of five genes. One open reading frame (ORF) was completely sequenced and for two others the 5′ half of the gene was determined. The sequences were highly similar. The complete ORF corresponded to a polypeptide of about 46 kDa. All the peptide sequences obtained were present in the derived sequences. The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half. The T. vaginalis enzyme was most similar to PP_i-PFK of the mitochondriate heterolobosean, Naegleria fowleri. Most of the residues shown or assumed to be involved in substrate binding in other PP_i-PFKs were conserved in the T. vaginalis enzyme. Direct comparison and phylogenetic reconstruction revealed a significant divergence among PP_i-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists. The separation of these groups is supported with a high percentage of bootstrap proportions. The short T. vaginalis PFK shares a most recent common ancestor with the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the α- and β-subunits of plant PP_i-PFKs. The third group (``X') containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorferi. The fourth group (``Y') comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences. The well-studied PP_i-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups. These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier. These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers. Received: 5 December 1997 / Accepted: 18 March 1998     

Reversible association of tetraspanin with Trichomonas vaginalis flagella upon adherence to host cells

The parasite Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection. Here, we report the cellular analyses of T. vaginalis tetraspanin 6 (TvTSP6). This family of membrane proteins has been implicated in cell adhesion, migration and proliferation in vertebrates. We observed that TvTSP6 expression is upregulated upon contact with vaginal ectocervical cells (VECs) and that parasite strains that are highly adherent to VECs express higher levels of TvTSP6 mRNA relative to poorly adherent strains. TvTSP6 is localized predominantly on the flagella of parasites cultured in the absence of host cells; however, adherence of the parasite to VECs initially results in a redistribution of the protein to intracellular vesicles and the plasma membrane of the main body of the cell. We found that a 16‐amino‐acid C‐terminal intracellular tail of TvTSP6 is necessary and sufficient for flagellar localization and protein redistribution when the parasite is in contact with VECs. Additionally, deletion of the C‐terminal tail reduced parasite migration through Matrigel, a mimic of the extracellular matrix. Together, our data support roles for TvTSP6 in parasite migration in the host and sensory reception during infection.