The transcriptome of human sleeping sickness
Zusammenfassung der Projektergebnisse
Human trypanosomiasis is widespread in Africa. Few chemotherapies are available and modern diagnostic methods are lacking. The results of this project will provide new insights into the human trypanosome interaction, and should provide a basis for new diagnostic tools. Nearly all experiments on trypanosome metabolism and gene expression have been done with cultured parasites, and the results are used to identify targets for chemotherapy. We aimed to look for differences between trypanosomes in human blood, trypanosomes in cerebrospinal fluid, and cultured trypanosomes. Using high throughput sequencing, we planned to characterise the transcriptomes of trypanosomes taken directly from sleeping sickness patients. Some of these aims were partially achieved, and further data are pending. There were additional aims to study immunology of sleeping sickness; this was not attempted. Finally, we aimed to establish, in Uganda, bioinformatic and sample-preparation methods. This aim was achieved. Human patients often have relatively few parasites in their blood, so when RNA is prepared, the most transcripts are from human cells. In the first part of the project, current methods for isolating trypanosomes from blood cells were examined, using rat infections as the model. We showed that red blood cell lysis had severe effects on trypanosome transcriptomes. Purification of the trypanosomes through an ion exchange column also resulted in very considerable variability in transcriptomes, presumably because of mRNA degradation and stress responses that occurred during the procedure. Since purification of the parasites is not a viable option, we developed a method to amplify the trypanosome RNA specifically from a mixture with a 100-fold excess of human mRNA. This method is sufficiently reproducible to be applied to blood RNA preparations with low parasitaemias. We collected about 40 matched samples of blood and cerebrospinal fluid from sleeping sickness patients. Various methods to purify RNA were tested, using German donated blood. However when these methods were used with patient blood no RNA was obtained. Possibly the patient blood has much more RNase because of immune activation, but no RNA was obtained even when RNase inhibitor was used throughout. Eventually, we did succeed, using blood that had been collected into PAXgene tubes. Samples from these are currently at the sequencing facility. Meanwhile, we characterised genomes and transcriptomes of four trypanosome isolates from the patients, doing all the bioinformatic analysis. Transcriptomes of parasites grown to high density in rats showed high abundances of mRNAs that are enriched in the non-growing stumpy form, which is pre-adapted for growth in the Tsetse fly vector. The genomes showed interesting variations in gene copy numbers.
Projektbezogene Publikationen (Auswahl)
- (2014) Methods to determine the transcriptomes of trypanosomes in mixtures with mammalian cells: the effects of parasite purification and selective cDNA amplification. PLoS Negl Trop Dis 8: e2806
Mulindwa J, Fadda A, Mercé C, Matovu E, Enyaru J, Clayton C
(Siehe online unter https://doi.org/10.1371/journal.pntd.0002806) - (2015) Transcriptomes of newly-isolated Trypanosoma brucei rhodesiense reveal hundreds of mRNAs that are co-regulated with stumpyform markers. BMC Genomics 16: 1118
Mulindwa J, Mercé C, Matovu E, Enyaru J, Clayton C
(Siehe online unter https://doi.org/10.1186/s12864-015-2338-y)
