A novel EF-hand calcium-binding protein in the flagellum of the protozoan Tritrichomonas suis.

The cloning and characterization of Ts-p41, an EF-hand calcium-binding protein of the protozoan parasite Tritrichomonas suis is described. A T. suis cDNA library was screened with monospecific antibodies affinity purified on an immunoreactive 41 kDa antigen in a Triton X-114 membrane-protein fraction. The resulting cDNA fragments turned out to be derived from 2 different genes encoding closely related Ts-p41 variants. The deduced amino acid sequences contained 6 EF-hand domains perfectly matching the canonical consensus motif and a putative C-terminal prenylation site. Northern and Southern hybridizations revealed that Ts-p41 was highly expressed and encoded by a gene-family. A cDNA encoding Ts-p41 was expressed as recombinant protein in Escherichia coli. By overlay with 45Ca it was demonstrated that the native and recombinant Ts-p41 proteins bind Ca2+. In immunofluorescence, epitopes recognized by anti-Ts-p41 antibodies were distributed as well on the anterior flagella as on the recurrent flagellum of the parasite. Our findings with the parabasalid T. suis suggest that multiple EF-hand bearing calcium-binding proteins might be a common phenomenon associated with flagellar motility.

Conservation of calnexin in the early branching protozoan Tritrichomonas suis.

The cloning and characterization of Ts-p66, a calcium-binding protein representing calnexin of the protozoan parasite Tritrichomonas suis is described. A T. suis cDNA expression library was screened with monospecific antibodies affinity-purified on an immuno-reactive 66 kDa antigen in a Triton X-114 membrane-protein fraction. The deduced amino acid sequence of the resulting cDNA clones revealed that Ts-p66 belongs to the calreticulin protein family and represents calnexin of T. suis. The key structural features and sequence motifs of the calnexins were all conserved. By lectin-blotting we demonstrated that the native protein is glycosylated. Northern and Southern hybridizations showed that T. suis calnexin was highly expressed and encoded by a single or low copy number gene. A cDNA encoding Ts-p66 was expressed as recombinant protein in Escherichia coli. By overlay with 45Ca it was demonstrated that the native and recombinant proteins bind Ca(2+). Using immunofluorescence with affinity-purified antibodies, a staining pattern was observed which points towards a putative localization of Ts-p66 in the nuclear membrane and endoplasmic reticulum. Demonstration of a structurally conserved calnexin in the amitochondriate protist T. suis indicates the very early evolutionary origin of the machinery for quality control of protein folding in the endoplasmic reticulum and the molecules involved hereby.

Host-parasite interaction in bovine infection with Tritrichomonas foetus.

Tritrichomonas foetus is a parasite of particular veterinary importance causing bovine tritrichomonosis, a sexually transmitted disease leading to infertility and abortion. The present review summarizes the current knowledge on potential mechanisms of pathogenicity of T. foetus, the immunology of host-parasite interaction in bovine tritrichomonosis, and the experimental model systems of this parasitic disease.

Identification of a differentially expressed Echinococcus multilocularis protein Em6 potentially related to antigen 5 of Echinococcus granulosus.

By a strategy of differential immunological screening of an expression library constructed from adult Echinococcus multilocularis parasites, a partial cDNA sequence encoding a protein termed Em6 was isolated. This molecule displayed high sequence homology to the recombinant antigen 'Eg6' which was previously described as an immunogenic epitope of antigen 5 of E. granulosus. Further Em6 sequences and the corresponding sequences from a cattle isolate of E. granulosus were obtained by a PCR approach. By immunoblot analyses using affinity purified antibodies, expression of Em6 in fertile cysts producing protoscoleces of the E. multilocularis metacestode stage was observed. However, Em6 was absent in non-fertile metacestodes. The demonstration of a protein in E. multilocularis displaying identities to 'antigen 6' of E. granulosus could potentially contribute to the future elucidation of the relationship between antigen 5 and 'antigen 6' in the genus Echinococcus, and shed some lights on the performance of serodiagnostic assays for hydatid disease based on the respective antigens.

Stage-specific expression of the 14-3-3 gene in Echinococcus multilocularis.

A cDNA expression library representing the metacestode developmental stage of the tapeworm Echinococcus multilocularis was immunoscreened with monospecific antibodies affinity purified following differential immunoblot analysis. Using this procedure, a metacestode-specific clone was isolated representing a 14-3-3 gene of the parasite, which is present as a single copy in the parasite genome. The identity of this clone was demonstrated by cross-reactivity of the recombinant E. multilocularis 14-3-3 protein with antibodies raised against a heterologous 14-3-3 protein from Saccharomyces cerevisiae. In addition, expression of the E. multilocularis 14-3-3 gene in the mutant S. cerevisiae strain, DS9-22, resulted in complementation of the phenotypic deficiency of this strain, thus demonstrating the functionality of the respective gene product. By reverse transcription-polymerase chain reaction (RT-PCR) we showed that the E. multilocularis 14-3-3 protein is about 10-fold overexpressed in the metacestode stage compared with the expression level in the adult stage. Immunolocalization of the 14-3-3 protein in E. multilocularis metacestodes revealed its predominant presence in the germinal layer of the parasite. The results of this study, taken together with the current knowledge on the 14-3-3 protein family, suggest that this parasite molecule may contribute to the promotion of the progressive, potentially unlimited growth behaviour of the E. multilocularis metacestode within the host tissue.

Comparative genetic analysis of tritrichomonadid protozoa by the random amplified polymorphic DNA technique.

The taxonomic classification within the genus Tritrichomonas is a subject of controversy, and, potentially, separation of the tritrichomonads from cattle and swine on the species level is not valid. To tackle this hypothesis we comparatively assessed several isolates of protozoan parasites from the three Tritrichomonas species T. foetus, T. suis, and T. mobilensis by the RAPD (random amplified polymorphic DNA) technique. In this method with 20 different primers, all T. foetus and T. suis isolates resulted in identical genomic fingerprints, thus yielding additional evidence for the genetic identity of T. foetus and T. suis. In contrast, it turned out that the species T. mobilensis isolated from the squirrel monkey is genetically distinct and can clearly be discriminated from the other tritrichomonads. Consequently, the results obtained in this study support a possible future revision of the taxonomic classification of the genus Tritrichomonas.

Detection of Tritrichomonas foetus by PCR and DNA enzyme immunoassay based on rRNA gene unit sequences.

Tritrichomonas foetus is the causative agent of bovine tritrichomonosis, a sexually transmitted disease leading to infertility and abortion. Diagnosis is hampered by putative contamination of samples with intestinal or coprophilic trichomonadid protozoa which might be mistaken for T. foetus. Therefore, we developed a PCR test optimized for applicability in routine diagnosis. Amplification is based upon primers TFR3 and TFR4 directed to the rRNA gene units of T. foetus. In order to avoid potential carryover contamination by products of previous amplification reactions, conditions were adapted to the use of the uracil DNA glycosylase system. Furthermore, documentation and interpretation of results were facilitated by including a DNA enzyme immunoassay for the detection of amplification products. Specificity was confirmed with genomic material from different related trichomonadid protozoa. The high sensitivity of the test allowed the detection of a single T. foetus organism in diagnostic culture medium or about 50 parasites per ml of preputial washing fluid. The present methods are thus proposed as (i) confirmatory tests for microscopic diagnosis following diagnostic in vitro cultivation and (ii) a direct T. foetus screening test with diagnostic samples.

Comparative sequence analysis of 5.8S rRNA genes and internal transcribed spacer (ITS) regions of trichomonadid protozoa.

The taxonomic situation in the genus Tritrichomonas is the subject of controversial discussion: potentially T. foetus and T. suis, the tritrichomonads from cattle and swine, respectively, could belong to the same species. In order to shed some light on this question, a molecular biological analysis was performed. The 5.8S rRNA gene and the flanking internal transcribed spacer regions (ITS1 and ITS2) of 12 different isolates of 3 Tritrichomonas species T. foetus, T. suis and T. mobilensis were enzymatically amplified by PCR and subcloned. Also, the corresponding regions of the trichomonads Trichomonas vaginalis, T. tenax, T. gallinae and Pentatrichomonas hominis were included in this study. Sequence analysis of cloned fragments was used to compare the parasite isolates. The genus Tritrichomonas exhibited an extremely high degree of homogeneity. All T. foetus and T. suis isolates had identical sequences, and only 1 substitution was found in the ITS2 region of T. mobilensis. In contrast, the genus Trichomonas shared more diversity. The results obtained in this study support a possible future revision of the taxonomic classification of tritrichomonads.