Expression of procyclin mRNAs during cyclical transmission of Trypanosoma brucei

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.

Figure 1. Distribution of Procyclin mRNAs Expressed by Tsetse-Derived Trypanosomes

RNA was isolated from the midguts and salivary glands at various days post infection and subjected to RT-PCR using generic primers for all procyclins. The products were cloned and individual clones assigned by hybridisation with a panel of oligonucleotides (see Materials and Methods). The total number of procyclin clones analysed at each time point is indicated within the chart. The standard deviations for the data from days 11–24 derive from two independent tsetse infections.

Figure 2. Relative Amount of Procyclin mRNA in Different Life Cycle Stages

RTQ-PCR was performed on RNA samples from bloodstream forms and midgut and salivary gland trypanosomes at various days post infection. β-tubulin was used as an internal control. The ratio of procyclin/tubulin mRNAs in a reference culture of procyclic forms was set at 1. Standard deviations derive from two to five replicas. RNA derived from independent infections (days 11–24) was quantitated separately.

Figure 3. Effect of Procyclin 3′ UTRs on Expression of a Reporter Gene during Fly Transmission

The coding region of GARP was fused to individual procyclin 3′ UTRs and stably integrated in place of the corresponding procyclin coding region. The percentage of trypanosomes expressing GARP in different compartments of the digestive tract and the salivary glands of the tsetse fly was determined by immunofluorescence analysis. For the sake of clarity the figures are set to the same scale. Blank spaces on the graphs indicate that no samples were taken from a particular tissue.