Invariant surface glycoprotein 65 of Trypanosoma brucei is a complement C3 receptor.

African trypanosomes are extracellular pathogens of mammals and are exposed to the adaptive and innate immune systems. Trypanosomes evade the adaptive immune response through antigenic variation, but little is known about how they interact with components of the innate immune response, including complement. Here we demonstrate that an invariant surface glycoprotein, ISG65, is a receptor for complement component 3 (C3). We show how ISG65 binds to the thioester domain of C3b. We also show that C3 contributes to control of trypanosomes during early infection in a mouse model and provide evidence that ISG65 is involved in reducing trypanosome susceptibility to C3-mediated clearance. Deposition of C3b on pathogen surfaces, such as trypanosomes, is a central point in activation of the complement system. In ISG65, trypanosomes have evolved a C3 receptor which diminishes the downstream effects of C3 deposition on the control of infection.

A novel strategy to identify the location of necessary and sufficient cis-acting regulatory mRNA elements in trypanosomes.

Expression of nearly all protein coding genes in trypanosomes is regulated post-transcriptionally, predominantly at the level of mRNA half-life. The identification of cis-acting elements involved in mRNA stability has been hindered by a lack of ability to screen for loss-of-regulation mutants. The method described in this article allows the region containing the necessary and sufficient elements within a mRNA to be identified and uses antibiotic resistance genes as both selectable markers and reporters. In the case of unstable mRNAs, the strategy can be extended by performing a screen for spontaneous loss-of-function mutants in regulatory parts of a mRNA. The method was validated by using the GPI-PLC mRNA, which is unstable in procyclic form trypanosomes and showed that the 3'UTR of the GPI-PLC mRNA contains all elements required for developmentally regulated instability. Loss-of-instability mutants all contained deletions within the 2300-nucleotide-long 3'UTR, and their analysis showed that a deletion including the last 800 nt of the gene stabilized the mRNA. The method is nonpresumptive, allows far more rapid screening for cis-elements than existing procedures, and has the advantage of identifying functional mutants. It is applicable to all eukaryotes using polycistronic transcription.

Developmentally regulated instability of the GPI-PLC mRNA is dependent on a short-lived protein factor.

The expression of the vast majority of protein coding genes in trypanosomes is regulated exclusively at the post-transcriptional level. Developmentally regulated mRNAs that vary in levels of expression have provided an insight into one mechanism of regulation; a decrease in abundance is due to a shortened mRNA half-life. The decrease in half-life involves cis-acting elements in the 3' untranslated region of the mRNA. The trans-acting factors necessary for the increased rate of degradation remain uncharacterized. The GPI-PLC gene in Trypanosoma brucei encodes a phospholipase C expressed in mammalian bloodstream form, but not in the insect procyclic form. Here, it is reported that the differential expression of the GPI-PLC mRNA also results from a 10-fold difference in half-life. Second, the instability of the GPI-PLC mRNA in procyclic forms can be reversed by the inhibition of protein synthesis. Third, specifically blocking the translation of the GPI-PLC mRNA in procyclic forms by the inclusion of a hairpin in the 5' untranslated region does not result in stabilization of the mRNA. Thus, the effect of protein synthesis inhibitors in stabilizing the GPI-PLC mRNA operates in trans through a short-lived factor dependent on protein synthesis.