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Genetic exchange in Trypanosoma brucei is now well characterized. It is a key tool that has enabled an understanding of important parasite genetic traits and underpinned the Trypanosoma brucei genome project. However, a key aspect that has eluded us is the point in the trypanosome life cycle where genetic exchange occurs. Research using green and red fluorescent trypanosomes for visualizing genetic crosses has now identified this stage. [Copyright &y& Elsevier]

Background: Vacuolar H+-ATPase (V-ATPase) is a highly conserved protein complex which hydrolyzes ATP and pumps protons to acidify vacuolar vesicles. Beyond its role in pH maintenance, the involvement of V-ATPase in endocytosis is well documented in mammals and plants but is less clear in Trypanosoma brucei. Methods: In this study, the subcellular localization of V-ATPase subunit B (TbVAB) of T. brucei was assessed via in situ N-terminal YFP-tagging and immunofluorescence assays. Transgenic bloodstream forms (BSF) of T. brucei were generated which comprised either a V-ATPase subunit B (TbVAB) conditional knockout or a V-ATPase subunit A (TbVAA) knockdown. Acridine orange and BCECF-AM were employed to assess the roles of V-ATPase in the pH regulation of BSF T. brucei. The endocytic activities of three markers were also characterized by flow cytometry analyses. Furthermore, trypanosomes were counted from trypanolysis treatment groups (either containing 1% or 5% NHS) and endocytosed trypanosome lytic factor (TLF) was also analyzed by an immunoblotting assay. Results: TbVAB was found to localize to acidocalcisomes, lysosomes and probably also to endosomes of BSF of T. brucei and was demonstrated to be essential for cell growth. TbVAB depletion neutralized acidic organelles at 24 hours post-tetracycline depletion (hpd), meanwhile the steady state intracellular pH increased from 7.016 ± 0.013 to 7.422 ± 0.058. Trypanosomes with TbVAB depletion at 24 hpd were found to take up more transferrin (2.068 ± 0.277 fold) but less tomato lectin (49.31 ± 22.57%) by endocytosis, while no significant change was detected in dextran uptake. Similar endocytic dysregulated phenotypes were also observed in TbVAA knockdown cells. In addition, TbVAB depleted trypanosomes showed a low uptake of TLF and exhibited less sensitive to lysis in both 1% and 5% NHS treatments. Conclusions: TbVAB is a key component of V-ATPase and was found to play a key function in endocytosis as well as exhibiting different effects in a receptor/cargo dependent manner in BSF of T. brucei. Besides vacuolar alkalinization, the dysregulation of endocytosis in TbVAB depleted T. brucei is considered to contribute to the reduced sensitivity to lysis by normal human serum. [ABSTRACT FROM AUTHOR]

Trehalose, a non-permeating cryoprotective agent (CPA), has been documented as less toxic and highly efficient at cryopreserving different kinds of cells or organisms. In the present study, trehalose was evaluated for its application in cryopreservation of both Trypanosoma brucei procyclic and bloodstream form cells. The cryopreservation efficiency was determined by the motility of trypanosomes after thawing, as well as a subsequent recovery and infectivity assessment. The viability of trypanosomes from cultivation that were frozen in a serial concentrations of trehalose showed similar results to classical CPAs of glycerol and DMSO. Nevertheless, trypanosomes cryopreserved in 0.2 M trehalose showed the best growth characteristic during subsequent cultivation. In addition, CPA cocktails with trehalose and permeating CPA glycerol or DMSO were developed and evaluated. Interestingly, trypanosomes in host (mouse) blood cryopreserved in 0.4 M trehalose plus 5% glycerol showed higher infectivity than those preserved in trehalose/DMSO cocktails as well as individually. Further investigations showed that, in comparison with slow freezing at −80 °C, flash freezing in liquid nitrogen provided better cryopreservation for bloodstream form cells than slow freezing. In conclusion, trehalose is an easy, safe and efficient CPA for cryopreservation of T. brucei and potentially for other protozoan species and cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cancer is a general name for more than 100 malignant diseases. It is postulated that all cancers start from a single abnormal cell that grows out of control. Untreated cancers can cause serious conse- quences and deaths. Great progress has been made in cancer research that has significantly improved our knowledge and understanding of the nature and mechanisms of the disease, but the origins of cancer are far from being well understood due to the limitations of suitable model systems and to the complexities of the disease. In view of the fact that cancers are found in various species of vertebrates and other metazoa, here, we suggest that cancer also occurs in parasitic protoz- oans such as Trypanosoma brucei, a blood parasite, and Toxoplasma gondii, an obligate intracellular pathogen. Without treatment, these protozoan cancers may cause severe disease and death in mammals, including humans. The simpler genomes of these single-cell organ- isms, in combination with their complex life cycles and fascinating life cycle differentiation processes, may help us to better understand the origins of cancers and, in particular, leukemias. [ABSTRACT FROM AUTHOR]

Animal trypanosomiasis, caused by the members of subgenus Trypanozoon (Trypanosoma brucei brucei, T. evansi and T. equiperdum), has reduced animal productivity leading to significant negative economic impacts in endemic regions. Due to limited drug discovery and the emergence of drug-resistance over many recent decades, novel and effective compounds against animal trypanosomiasis are urgently required. This study was conducted to evaluate the antitrypanosomal potential of a batch of carbazole aminoalcohol derivatives. Among them, we found that the most effective compound was H1402, which exhibited potent trypanocidal efficacy against the bloodstream-form of T. b. brucei (EC 50 = 0.73 ± 0.05 µM) and presented low cytotoxicity against two mammalian cell lines with CC 50 > 30 µM. Using a murine model of acute infection, oral administration with H1402 demonstrated a complete clearance of T. b. brucei and all the infected mice were cured when they were treated twice daily for 5 days at a dose of 100 mg/kg. Furthermore, parasites were not detected in mice infected with T. evansi and T. equiperdum (the causative agents of surra and dourine, respectively, in animals) within 30 days following the same regimen with H1402. In addition, H1402 caused severe morphological and ultrastructural destruction to trypanosomes, as well as causing phosphatidylserine externalization, which are suggested to be the most likely cause of cell death. Overall, the present data demonstrated that H1402 could be promising as a rapid, safe and orally active lead compound for the development of new chemotherapeutics for animal trypanosomiasis. [ABSTRACT FROM AUTHOR]