Expression of Procyclin mRNAs during Cyclical Transmission of Trypanosoma brucei

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Abstract
Trypanosoma brucei, the parasite causing human sleeping sickness, relies on the tsetse fly for its transmission. In the insect, EP and GPEET procyclins are the major surface glycoproteins of procyclic (midgut) forms of the parasite, with GPEET predominating in the early procyclic form and two isoforms of EP in the late procyclic form. EP procyclins were previously detected on salivary gland trypanosomes, presumably epimastigotes, by immunoelectron microscopy. However, no procyclins could be detected by mass spectrometry when parasites were isolated from infected glands. We have used qualitative and quantitative RT-PCR to analyse the procyclin mRNAs expressed by trypanosomes in the tsetse midgut and salivary glands at different time points after infection. The coding regions of the three EP isoforms (EP1, EP2 and EP3) are extremely similar, but their 3′ untranslated regions contain unique sequences that make it possible to assign the cDNAs amplified by this technique. With the exception of EP2, we found that the spectrum of procyclin mRNAs expressed in the midgut mirrors the protein repertoire of early and established procyclic forms. Surprisingly, procyclin mRNAs, including that of GPEET, are present at relatively high levels in salivary gland trypanosomes, although the proteins are rarely detected by immunofluorescence. Additional experiments using transgenic trypanosomes expressing reporter genes or mutant forms of procyclin point to a mechanism of translational or post-translational control, involving the procyclin coding regions, in salivary gland trypanosomes. It is widely accepted that T. brucei always has a coat of either variant surface glycoprotein or procyclin. It has been known for many years that the epimastigote form does not have a variant surface glycoprotein coat. The finding that this life cycle stage is usually negative for procyclin as well is new, and means that the paradigm will need to be revised. The tropical parasite Trypanosoma brucei relies on the tsetse fly for its transmission between mammals. The parasite first establishes an infection in the fly midgut then migrates and colonizes the salivary glands. A paradigm in trypanosome biology is that parasites in contact with the mammalian host are covered by variant surface glycoproteins, enabling them to evade the immune system, while all other life cycle stages are covered by repetitive glycoproteins known as procyclins. In this publication the authors investigated the expression pattern of procyclins in all life cycle stages in infected tsetse flies, including epimastigote and metacyclic forms in the salivary glands. These stages are usually neglected as, in contrast to trypanosomes from the midgut, they cannot be cultured. The researchers showed that the vast majority of trypanosomes in the salivary glands have no procyclin coat although the levels of procyclin messenger RNAs are comparatively high. Experiments with reporter genes indicated that procyclin expression in the glands is regulated by the coding region. The lack of procyclins on salivary gland trypanosomes leads to the prediction that the epimastigote form possesses a novel surface coat that may be responsible for the tight binding of the parasite to gland tissue.