Abstract
Patterns of gene expression in cultured Trypanosoma brucei bloodstream and procyclic forms have been extensively characterized, and some comparisons have been made with trypanosomes grown to high parasitaemias in laboratory rodents. We do not know, however, to what extent these transcriptomes resemble those in infected Tsetse flies - or in humans or cattle, where parasitaemias are substantially lower. For clinical and field samples it is difficult to characterize parasite gene expression because of the large excess of host cell RNA. We have here examined two potential solutions to this problem for bloodstream form trypanosomes, assaying transcriptomes by high throughput cDNA sequencing (RNASeq). We first purified the parasites from blood of infected rats. We found that a red blood cell lysis procedure affected the transcriptome substantially more than purification using a DEAE cellulose column, but that too introduced significant distortions and variability. As an alternative, we specifically amplified parasite sequences from a mixture containing a 1000-fold excess of human RNA. We first purified polyadenylated RNA, then made trypanosome-specific cDNA by priming with a spliced leader primer. Finally, the cDNA was amplified using nested primers. The amplification procedure was able to produce samples in which 20% of sequence reads mapped to the trypanosome genome. Synthesis of the second cDNA strand with a spliced leader primer, followed by amplification, is sufficiently reproducible to allow comparison of different samples so long as they are all treated in the same way. However, SL priming distorted the abundances of the cDNA products and definitely cannot be used, by itself, to measure absolute mRNA levels. The amplification method might be suitable for clinical samples with low parasitaemias, and could also be adapted for other Kinetoplastids and to samples from infected vectors.
Highlights
African trypanosomes live in several niches - in the blood, tissue fluids and brain of patients with second stage sleeping sickness, and in various parts of the Tsetse fly digestive tract
Most experiments on African trypanosomes, - including those designed to look for new drugs - have studied parasites either from culture, or from laboratory rodents with high parasitaemias
Most experiments on African trypanosomes - including those designed to look for new drugs - have studied parasites either from culture, or from laboratory rodents
Summary
African trypanosomes live in several niches - in the blood, tissue fluids and brain of patients with second stage sleeping sickness, and in various parts of the Tsetse fly digestive tract. Most experiments on African trypanosomes, - including those designed to look for new drugs - have studied parasites either from culture, or from laboratory rodents with high parasitaemias. Drugs have to kill parasites that are living in humans or ruminants. These parasites are very difficult to study because parasitaemias are low, so we have no idea whether their metabolism is really the same as that of parasites in culture. The ideal way to assess differences between these parasites and the standard lab models would be to characterise their proteomes or metabolomes, but the numbers of parasites are insufficient. The amounts of RNA are sufficient, for transcriptome analysis, especially since amplification techniques are available
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