Analysis of a Novel Peptide That Is Capable of Inhibiting the Enzymatic Activity of the Protein Kinase A Catalytic Subunit-Like Protein from Trypanosoma equiperdum.

A 26-residue peptide possessing the αN-helix motif of the protein kinase A (PKA) regulatory subunit-like proteins from the Trypanozoom subgenera (VAP26, sequence = VAPYFEKSEDETALILKLLTYNVLFS), was shown to inhibit the enzymatic activity of the Trypanosoma equiperdum PKA catalytic subunit-like protein, in a similar manner that the mammalian heat-stable soluble PKA inhibitor known as PKI. However, VAP26 does not contain the PKI inhibitory sequence. Bioinformatics analyzes of the αN-helix motif from various Trypanozoon PKA regulatory subunit-like proteins suggested that the sequence could form favorable peptide-protein interactions of hydrophobic nature with the PKA catalytic subunit-like protein, which possibly may represent an alternative PKA inhibitory mechanism. The sequence of the αN-helix motif of the Trypanozoon proteins was shown to be highly homologous but significantly divergent from the corresponding αN-helix motifs of their Leishmania and mammalian counterparts. This sequence divergence contrasted with the proposed secondary structure of the αN-helix motif, which appeared conserved in every analyzed regulatory subunit-like protein. In silico mutation experiments at positions I234, L238 and F244 of the αN-helix motif from the Trypanozoon proteins destabilized both the specific motif and the protein. On the contrary, mutations at positions T239 and Y240 stabilized the motif and the protein. These results suggested that the αN-helix motif from the Trypanozoon proteins probably possessed a different evolutionary path than their Leishmania and mammalian counterparts. Moreover, finding stabilizing mutations indicated that new inhibitory peptides may be designed based on the αN-helix motif from the Trypanozoon PKA regulatory subunit-like proteins.

Should the enzyme name 'rhodesain' be discontinued?

Rhodesain is the generic name for the cathepsin L-like peptidase of Trypanosoma brucei rhodesiense. The term rhodesain was derived from the subspecies epithet rhodesiense which itself originated form Rhodesia, a historical region in southern Africa named after the 19th century British imperialist and white supremacist Cecil Rhodes. This tainting could be grounds for discontinuing the name, however, there are also scientific grounds. Specifically, protein sequence comparisons and frequency-based difference profiling reveal that rhodesain is essentially identical (99.87-98.44%) to the cathepsin L-like peptidases of both T. b. brucei and T. b. gambiense. Accordingly, and based on a previously proposed terminology for kinetoplastid C1 peptidases (Caffrey and Steverding, 2009), we suggest the use of the formal term, TbrCATL, to denote the cathepsin L-like peptidases of the T. brucei subspecies. The earlier and informal term, 'brucipain', could also be used.

Genome-Wide SNP Analysis Reveals Distinct Origins of Trypanosoma evansi and Trypanosoma equiperdum.

Trypanosomes cause a variety of diseases in man and domestic animals in Africa, Latin America, and Asia. In the Trypanozoon subgenus, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause human African trypanosomiasis, whereas Trypanosoma brucei brucei, Trypanosoma evansi, and Trypanosoma equiperdum are responsible for nagana, surra, and dourine in domestic animals, respectively. The genetic relationships between T. evansi and T. equiperdum and other Trypanozoon species remain unclear because the majority of phylogenetic analyses has been based on only a few genes. In this study, we have conducted a phylogenetic analysis based on genome-wide SNP analysis comprising 56 genomes from the Trypanozoon subgenus. Our data reveal that T. equiperdum has emerged at least once in Eastern Africa and T. evansi at two independent occasions in Western Africa. The genomes within the T. equiperdum and T. evansi monophyletic clusters show extremely little variation, probably due to the clonal spread linked to the independence from tsetse flies for their transmission.

First Draft Genome Sequence of the Dourine Causative Agent: Trypanosoma Equiperdum Strain OVI.

Trypanosoma equiperdum is the causative agent of dourine, a sexually-transmitted infection of horses. This parasite belongs to the subgenus Trypanozoon that also includes the agent of sleeping sickness (Trypanosoma brucei) and surra (Trypanosoma evansi). We herein report the genome sequence of a T. equiperdum strain OVI, isolated from a horse in South-Africa in 1976. This is the first genome sequence of the T. equiperdum species, and its availability will provide important insights for future studies on genetic classification of the subgenus Trypanozoon.

Phylogenetic analysis of the Trypanosoma genus based on the heat-shock protein 70 gene.

Trypanosome evolution was so far essentially studied on the basis of phylogenetic analyses of small subunit ribosomal RNA (SSU-rRNA) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. We used for the first time the 70kDa heat-shock protein gene (hsp70) to investigate the phylogenetic relationships among 11 Trypanosoma species on the basis of 1380 nucleotides from 76 sequences corresponding to 65 strains. We also constructed a phylogeny based on combined datasets of SSU-rDNA, gGAPDH and hsp70 sequences. The obtained clusters can be correlated with the sections and subgenus classifications of mammal-infecting trypanosomes except for Trypanosoma theileri and Trypanosoma rangeli. Our analysis supports the classification of Trypanosoma species into clades rather than in sections and subgenera, some of which being polyphyletic. Nine clades were recognized: Trypanosoma carassi, Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma grayi, Trypanosoma lewisi, T. rangeli, T. theileri, Trypanosoma vivax and Trypanozoon. These results are consistent with existing knowledge of the genus' phylogeny. Within the T. cruzi clade, three groups of T. cruzi discrete typing units could be clearly distinguished, corresponding to TcI, TcIII, and TcII+V+VI, while support for TcIV was lacking. Phylogenetic analyses based on hsp70 demonstrated that this molecular marker can be applied for discriminating most of the Trypanosoma species and clades.

Further evidence from SSCP and ITS DNA sequencing support Trypanosoma evansi and Trypanosoma equiperdum as subspecies or even strains of Trypanosoma brucei.

The subgenus Trypanozoon includes three species Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum, which are morphologically identical and indistinguishable even using some molecular methods. In this study, PCR-based single strand conformation polymorphism (PCR-SSCP) was used to analyze the ribosomal DNA of the Trypanozoon species. Data indicate different patterns of ITS2 fragments between T. brucei, T. evansi and T. equiperdum by SSCP. Furthermore, analysis of total ITS sequences within these three members of the subgenus Trypanozoon showed a high degree of homology using phylogenetic analysis but were polyphyletic in haplotype networks. These data provide novel nuclear evidence to further support the notion that T. evansi and T. equiperdum should be subspecies or even strains of T. brucei.

Development and evaluation of an ITS1 "Touchdown" PCR for assessment of drug efficacy against animal African trypanosomosis.

Animal African trypanosomoses (AAT) are caused by flagellated protozoa of the Trypanosoma genus and contribute to considerable losses in animal production in Africa, Latin America and South East Asia. Trypanosoma congolense is considered the economically most important species. Drug resistant T. congolense strains present a threat to the control of AAT and have triggered research into discovery of novel trypanocides. In vivo assessment of trypanocidal efficacy relies on monitoring of treated animals with microscopic parasite detection methods. Since these methods have poor sensitivity, follow-up for up to 100 days after treatment is recommended to increase the chance of detecting recurrent parasitaemia waves. Molecular techniques are more amendable to high throughput processing and are generally more sensitive than microscopic detection, thus bearing the potential of shortening the 100-day follow up period. The study presents a "Touchdown" PCR targeting the internal transcribed spacer 1 of the ribosomal DNA (ITS1 TD PCR) that enables detection and discrimination of different Trypanosoma taxa in a single run due to variations in PCR product sizes. The assay achieves analytical sensitivity of 10 parasites per ml of blood for detection of T. congolense savannah type and T. brucei, and 100 parasites per ml of blood for detection of T. vivax in infected mouse blood. The ITS1 TD PCR was evaluated on cattle experimentally infected with T. congolense during an investigational new veterinary trypanocide drug efficacy study. ITS1 TD PCR demonstrated comparable performance to microscopy in verifying trypanocide treatment success, in which parasite DNA became undetectable in cured animals within two days post-treatment. ITS1 TD PCR detected parasite recrudescence three days earlier than microscopy and had a higher positivity rate than microscopy (84.85% versus 57.58%) in 66 specimens of relapsing animals collected after treatments. Therefore, ITS1 TD PCR provides a useful tool in assessment of drug efficacy against T. congolense infection in cattle. As the assay bears the potential for detection of mixed infections, it may be applicable for drug efficacy studies and diagnostic discrimination of T. vivax and T. congolense against other pathogenic trypanosomes, including T. brucei, T. evansi and T. equiperdum.

Analysis of Molecular Profiles Among Trypanozoon Species and Subspecies By MGE-PCR Method / 中国寄生虫学与寄生虫病杂志

Objective To analyze the relationship between genetic variability and evolution among Trypanosoma brucei (including T. b. brucei, T. b. rhodesiense and T. b. gambiense), T. evansi and T. equiperdum isolates. Methods Genomic DNAs of 26 trypanosome isolates were amplified by a mobile genetic elements (MGE)-PCR technique and cluster analysis was performed based on the molecular profiles with Neighbor-Joining method. Results The genetic variability among trypanosome isolates examined was obvious with an average genetic distance of 41.2% (ranged from 0 to 100%). Similarity coefficient among T. brucei isolates was 41.15% which was lower than that between T. evansi and T. equiperdum isolates. The closest relationship was found between T. evansi and T. brucei isolates with a similarity coefficient of 62.94%. The genetic variability between T. b. rhodesiense and T. b. brucei isolates was higher than that among T. b. gambiense isolates. Conclusion Species and subspecies in Trypanozoon displayed a higher genetic variability; T. equiperdum isolates collected from China and from South America, and T. evansi isolates from China and from South America, should have a similar origin.

Human Serum Trypanosome Lytic Factor and Serum Resistance-Associated Protein of Trypanosome / 中国寄生虫学与寄生虫病杂志

Human African trypanosomiasis is caused by the infection of Trypanosoma brucei gambiense or T.b. rhodesiense, while another morphologically identical subspecies, T.b.brucei, and other closely related species, T.equiper-dum and T.evansi, are considered not infectious to human. This is highly related to the trypanosome lytic factor (TLF) found in normal human serum (NHS) and the serum resistance-associated (SRA) protein of trypanosomes infectious to human. We reviewed the research progress in TLF and its role in trypanosome lysis as well as the mechanism of SRA against the TLF.