Abstract
BackgroundTrypanosoma brucei brucei, the parasite causing Nagana in domestic animals, is closely related to the parasites causing sleeping sickness, but does not infect humans. In addition to its importance as a pathogen, the relative ease of genetic manipulation and an innate capacity for RNAi extend its use as a model organism in cell and infection biology. During its development in its mammalian and insect (tsetse fly) hosts, T. b. brucei passes through several different life-cycle stages. There are currently four life-cycle stages that can be cultured: slender forms and stumpy forms, which are equivalent to forms found in the mammal, and early and late procyclic forms, which are equivalent to forms in the tsetse midgut. Early procyclic forms show coordinated group movement (social motility) on semi-solid surfaces, whereas late procyclic forms do not.ResultsRNA-Seq was performed on biological replicates of each life-cycle stage. These constitute the first datasets for culture-derived slender and stumpy bloodstream forms and early and late procyclic forms. Expression profiles confirmed that genes known to be stage-regulated in the animal and insect hosts were also regulated in culture. Sequence reads of 100–125 bases provided sufficient precision to uncover differential expression of closely related genes. More than 100 transcripts showed peak expression in stumpy forms, including adenylate cyclases and several components of inositol metabolism. Early and late procyclic forms showed differential expression of 73 transcripts, a number of which encoded proteins that were previously shown to be stage-regulated. Moreover, two adenylate cyclases previously shown to reduce social motility are up-regulated in late procyclic forms.ConclusionsThis study validates the use of cultured bloodstream forms as alternatives to animal-derived parasites and yields new markers for all four stages. In addition to underpinning recent findings that early and late procyclic forms are distinct life-cycle stages, it could provide insights into the reasons for their different biological properties.
Highlights
Trypanosoma brucei brucei, the parasite causing Nagana in domestic animals, is closely related to the parasites causing sleeping sickness, but does not infect humans
Faithful expression of stage-specific markers in culturederived trypanosomes To place our analysis of the transcriptomes of early and late procyclic forms in a wider context, we compared them to culture-derived slender and stumpy bloodstream forms of a tsetse-transmissible pleomorphic strain of T. b
As well as validating the use of cultured slender and stumpy bloodstream forms as alternatives to animal-derived parasites, in compliance with 3R principles, it provides the first comparison of the transcriptomes of early and procyclic forms and identifies new stage-regulated transcripts
Summary
Trypanosoma brucei brucei, the parasite causing Nagana in domestic animals, is closely related to the parasites causing sleeping sickness, but does not infect humans. There are currently four life-cycle stages that can be cultured: slender forms and stumpy forms, which are equivalent to forms found in the mammal, and early and late procyclic forms, which are equivalent to forms in the tsetse midgut. There are two populations of procyclic forms: early procyclic forms are found for up to a week after transmission, and are positive for the surface protein GPEET procyclin, while late procyclic forms, which are responsible for persistent infection of the midgut, are GPEET-negative. These two forms cannot be distinguished by their morphology. To complete the life cycle trypanosomes must undergo several more rounds of differentiation, culminating in the delivery of infectious metacyclic forms to a new mammalian host when the tsetse takes a blood meal
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