ABSTRACT
The eukaryotic protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis. Polyamine biosynthesis is essential in T. brucei, and the polyamine spermidine is required for synthesis of a novel cofactor called trypanothione and for deoxyhypusine modification of eukaryotic translation initiation factor 5A (eIF5A). eIF5A promotes translation of proteins containing polyprolyl tracts in mammals and yeast. To evaluate the function of eIF5A in T. brucei, we used RNA interference (RNAi) to knock down eIF5A levels and found that it is essential for T. brucei growth. The RNAi-induced growth defect was complemented by expression of wild-type human eIF5A but not by a Lys-50 mutant that blocks modification by deoxyhypusine. Bioinformatics analysis showed that 15% of the T. brucei proteome contains 3 or more consecutive prolines and that actin-related proteins and cysteine proteases were highly enriched in the group. Steady-state protein levels of representative proteins containing 9 consecutive prolines that are involved in actin assembly (formin and CAP/Srv2p) were significantly reduced by knockdown of eIF5A. Several T. brucei polyprolyl proteins are involved in flagellar assembly. Knockdown of TbeIF5A led to abnormal cell morphologies and detached flagella, suggesting that eIF5A is important for translation of proteins needed for these processes. Potential specialized functions for eIF5A in T. brucei in translation of variable surface glycoproteins were also uncovered. Inhibitors of deoxyhypusination would be expected to cause a pleomorphic effect on multiple cell processes, suggesting that deoxyhypusine/hypusine biosynthesis could be a promising drug target in not just T. brucei but in other eukaryotic pathogens.
Subject(s)
Lysine/analogs & derivatives , Peptide Initiation Factors/metabolism , Protein Processing, Post-Translational , Protozoan Proteins/metabolism , RNA, Messenger/metabolism , RNA, Protozoan/metabolism , RNA-Binding Proteins/metabolism , Trypanosoma brucei brucei/metabolism , Amino Acid Sequence , Animals , Escherichia coli/genetics , Escherichia coli/metabolism , Flagella/genetics , Flagella/metabolism , Flagella/ultrastructure , Gene Knockdown Techniques , Humans , Lysine/metabolism , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Molecular Sequence Data , Peptide Initiation Factors/antagonists & inhibitors , Peptide Initiation Factors/genetics , Peptides/metabolism , Proteome/genetics , Proteome/metabolism , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/genetics , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics , RNA, Protozoan/antagonists & inhibitors , RNA, Protozoan/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/ultrastructure , Eukaryotic Translation Initiation Factor 5AABSTRACT
The var-gene encoding Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is known to play a major role in the pathogenicity of the P. falciparum parasite. The protein enables the parasite to adhere to the endothelial linings of small blood vessels (cytoadherence) as well as to non-infected erythrocytes (rosetting), thus preventing clearance from the bloodstream. The development and spread of resistance towards most anti-malarial drugs used for treatment and prevention of the most severe form of malaria truly emphasise the importance of a continuous research and development of new drugs. In this study we use Systematic Evolution of Ligands by EXponential enrichment (SELEX) methodology to isolate high-affinity ligands (aptamers). To validate the results from the SELEX in vitro selection, different aptamers have been selected against PfEMP1 in a live cell assay of P. falciparum strain FCR3S1.2, a highly rosetting strain. We have been able to show the rosette disrupting capacity of these SELEX-aptamers at concentrations of 33 nM and with 100% disruption at 387 nM. The described results show that RNA aptamers are promising candidates for adjunct therapy in severe malaria.
Subject(s)
Antimalarials/isolation & purification , Aptamers, Nucleotide/genetics , Aptamers, Nucleotide/isolation & purification , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/genetics , Animals , Models, Molecular , Nucleic Acid Conformation , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics , RNA, Protozoan/antagonists & inhibitors , RNA, Protozoan/genetics , Rosette Formation , SELEX Aptamer TechniqueABSTRACT
RNA interference is a powerful method for inhibition of gene expression in Trypanosoma brucei (Ngo, H., Tschudi, C., Gull, K., and Ullu, E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14687-14692). Here we describe a vector (pZJM) for in vivo tetracycline-inducible synthesis of double-stranded RNA (dsRNA) in stably transformed cells. The dsRNA is synthesized from opposing T7 promoters. We tested the vector with genes involved in processes such as kinetoplast DNA replication, mitochondrial mRNA synthesis, glycosyl phosphatidylinositol biosynthesis, glycosome biogenesis, and polyamine biosynthesis. In most cases the induction of dsRNA caused specific and dramatic loss of the appropriate mRNA, and in many cases there was growth inhibition or cell death. One striking phenotype was the loss of kinetoplast DNA after interference with expression of a topoisomerase II. The gene being analyzed by this procedure need not even be fully sequenced. In fact, many of the genes we tested were derived from partial sequences in the T. brucei genome data base that were identified by homology with known proteins. It takes as little as 3 weeks from identification of a gene sequence in the data base to the appearance of a phenotype.