tetracycline and Trypanosomiasis--African

Dual submicromolar trypanocidal-antiplasmodial compounds have been identified by screening and chemical synthesis of 4-aminoquinoline-based heterodimeric compounds of three different structural classes. In Trypanosoma brucei, inhibition of the enzyme trypanothione reductase seems to be involved in the potent trypanocidal activity of these heterodimers, although it is probably not the main biological target. Regarding antiplasmodial activity, the heterodimers seem to share the mode of action of the antimalarial drug chloroquine, which involves inhibition of the haem detoxification process. Interestingly, all of these heterodimers display good brain permeabilities, thereby being potentially useful for late stage human African trypanosomiasis. Future optimization of these compounds should focus mainly on decreasing cytotoxicity and acetylcholinesterase inhibitory activity.

Topics: Aminoquinolines; Animals; Antimalarials; Brain; Cell Line; Dimerization; Hemeproteins; Humans; Malaria, Falciparum; NADH, NADPH Oxidoreductases; Plasmodium falciparum; Rats; Trypanocidal Agents; Trypanosoma brucei brucei; Trypanosomiasis, African

Here, we present a simple modular extendable vector system for introducing the T7 RNA polymerase and tetracycline repressor genes into Trypanosoma brucei. This novel system exploits developments in our understanding of gene expression and genome organization to produce a streamlined plasmid optimized for high levels of expression of the introduced transgenes. We demonstrate the utility of this novel system in bloodstream and procyclic forms of Trypanosoma brucei, including the genome strain TREU927/4. We validate these cell lines using a variety of inducible experiments that recapture previously published lethal and non-lethal phenotypes. We further demonstrate the utility of the single marker (SmOx) TREU927/4 cell line for in vivo experiments in the tsetse fly and provide a set of plasmids that enable both whole-fly and salivary gland-specific inducible expression of transgenes.

Topics: Animals; Cells, Cultured; DNA-Directed RNA Polymerases; Genes, Reporter; Genetic Vectors; Plasmids; Promoter Regions, Genetic; Protein Synthesis Inhibitors; Repressor Proteins; RNA Interference; Tetracycline; Transgenes; Trypanosoma brucei brucei; Trypanosomiasis, African; Tsetse Flies; Viral Proteins

We investigated the roles played by the cysteine proteases cathepsin B and cathepsin L (brucipain) in the pathogenesis of Trypansoma brucei brucei in both an in vivo mouse model and an in vitro model of the blood-brain barrier. Doxycycline induction of RNAi targeting cathepsin B led to parasite clearance from the bloodstream and prevent a lethal infection in the mice. In contrast, all mice infected with T. brucei containing the uninduced Trypanosoma brucei cathepsin B (TbCatB) RNA construct died by day 13. Induction of RNAi against brucipain did not cure mice from infection; however, 50% of these mice survived 60 days longer than uninduced controls. The ability of T. b. brucei to cross an in vitro model of the human blood-brain barrier was also reduced by brucipain RNAi induction. Taken together, the data suggest that while TbCatB is the more likely target for the development of new chemotherapy, a possible role for brucipain is in facilitating parasite entry into the brain.

Topics: Animals; Cathepsin B; Cathepsin L; Cysteine Endopeptidases; Genes, Reporter; Humans; Life Cycle Stages; Mice; Models, Animal; Plasmids; Protozoan Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Tetracycline; Transfection; Trypanosoma brucei brucei; Trypanosomiasis, African

In Kinetoplastida, trypanothione and trypanothione reductase (TRYR) provide an intracellular reducing environment, substituting for the glutathione-glutathione reductase system found in most other organisms. To investigate the physiological role of TRYR in Trypanosoma brucei, we generated cells containing just one trypanothione reductase gene, TRYR, which was under the control of a tetracycline-inducible promoter. This enabled us to regulate TRYR activity in the cells from less than 1% to 400% of wild-type levels by adjusting the concentration of added tetracycline. In normal growth medium (which contains reducing agents), trypanosomes containing less than 10% of wild-type enzyme activity were unable to grow, although the levels of reduced trypanothione and total thiols remained constant. In media lacking reducing agents, hypersensitivity towards hydrogen peroxide (EC50 = 3.5 microM) was observed compared with the wild type (EC50 = 223 microM). The depletion of TRYR had no effect on susceptibility to melarsen oxide. The infectivity and virulence of the parasites in mice was dependent upon tetracycline-regulated TRYR activity: if the trypanosomes were injected into mice in the absence of tetracycline, no infection was detectable; and when tetracycline was withdrawn from previously infected animals, the parasitaemia was suppressed.

Topics: Animals; Anti-Bacterial Agents; Antiprotozoal Agents; Arsenicals; Cell Cycle; Cell Division; Doxycycline; Enzyme Inhibitors; Mice; Mice, Inbred BALB C; Mutation; NADH, NADPH Oxidoreductases; Oxidative Stress; Promoter Regions, Genetic; Sulfhydryl Compounds; Tetracycline; Trypanosoma brucei brucei; Trypanosomiasis, African; Virulence

Inability of T7 RNA polymerase to processively transcribe higher eukaryotic chromatin is interpreted as a correlate of its reported inhibition by nucleosomes on reconstituted templates in vitro . We used chromosomally integrated reporter cassettes to examine features of T7 transcription in a lower eukaryotic system. Luciferase reporters were targeted to rDNA in transgenic Trypanosoma brucei stably expressing the phage polymerase. Because trypanosome mRNAs are capped by RNA splicing in trans , T7 transcription could be gauged by luciferase activity. In contrast to findings from higher eukaryotes, T7 transcription is vigorous and processive on chromatin templates in T.brucei , surpassing levels achieved with endogenous promoters, including those recruiting RNA polymerase I. This may be a reflection of intrinsic differences in chromatin structure between differently evolved eukaryotes or of an integration site that is exceptionally permissive for T7 transcription due to a local accessible chromatin conformation. T7 transcription could be manipulated to achieve different levels of constitutive expression, through the use of promoter mutations. Moreover, T7 initiation could be regulated by the prokaryotic Tet repressor and elongation halted by T7 terminator sequences. We have exploited these features to construct a robust inducible expression system, whose utility potentially extends to other trans -splicing organisms.

Topics: Animals; Bacteriophage T7; Chromatin; DNA-Directed RNA Polymerases; DNA, Protozoan; DNA, Ribosomal; Doxycycline; Gene Expression Regulation; Genes, Reporter; Mice; Operator Regions, Genetic; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Promoter Regions, Genetic; Proteins; RNA, Antisense; Terminator Regions, Genetic; Tetracycline; Transcription, Genetic; Transfection; Trypanosoma brucei brucei; Trypanosomiasis, African; Viral Proteins

Topics: Africa; Asia; Brucellosis; Chloramphenicol; Chloroquine; Fever; Humans; Leishmaniasis, Visceral; Liver Abscess, Amebic; Malaria; Metronidazole; Plasmodium falciparum; Tetracycline; Travel; Trypanosomiasis, African; Tuberculosis; Typhoid Fever; United Kingdom

Topics: Animals; Anti-Bacterial Agents; Puromycin; Tetracycline; Trypanosomiasis; Trypanosomiasis, African

Topics: Animals; Anti-Bacterial Agents; Antibiotics, Antitubercular; Mice; Puromycin; Tetracycline; Trypanosoma brucei gambiense; Trypanosomiasis; Trypanosomiasis, African