Project Summary
Trypanosoma brucei causes human African trypanosomiasis, which is frequently fatal without treatment.
Few drugs are available for treating this disease, most of which have severe side-effects and are difficult to
administer, while drug-resistant T. brucei infection has been on the rise. In addition, T. brucei also causes
animal African trypanosomiasis, which has been a significant economic burden in sub-Sahara Africa. It is
therefore important to further study T. brucei pathogenesis and identify better targets for future development of
anti-parasite agents. T. brucei regularly switches its major surface antigen, VSG, to evade its mammalian host
immune response. VSG switching is a major pathogenesis mechanism that enables T. brucei to
establish and maintain a long-term infection. However, how VSG switching is initiated naturally in T.
brucei is still not clear. T. brucei has >2,500 VSG genes and pseudogenes, all of which are located at
subtelomeres. However, VSGs are expressed exclusively from subtelomeric polycistronic VSG expression
sites (ESs). VSG is the last gene in any ES and within 2 kb from the telomere repeats. T. brucei has multiple
ESs with very similar sequences, but only one ES is fully active at any time, resulting in a single type of VSG
being expressed on the cells surface. DNA recombination has been shown to be a major pathway for VSG
switching. Most VSG genes are flanked by two common sequences, which provide sequence homology for
recombination between the active and a silent VSG gene in DNA recombination-mediated VSG switching
events. First, all VSG 3’UTR has a common 14 nt sequences. Long telomere sequences are also found
downstream of ES-linked VSGs and VSGs at minichromosome subtelomeres. Second, upstream of most VSG
genes are 70 bp repeats. The 70 bp repeats in ESs can be several kb to several tens kb long. It has been
shown that introducing a DNA double strand break in the 70 bp repeats immediately upstream of the active
VSG gene increases the VSG switching rate for ~ 250 fold. In addition, DNA breaks are found in the 70 bp
repeats in WT cells. Therefore, 70 bp repeat integrity is expected to significantly affect VSG switching
frequency. However, it is unknown whether any proteins specifically associate with the 70 bp repeats,
even though their sequences are highly conserved, and their associated proteins are expected to help
maintain their integrity and to participate in the regulation of VSG switching. In this small project, we aim
to use an improved “end-targeting proteomics of isolated chromatin segments” (ePICh) approach to isolate the
70 bp repeat chromatin and identify proteins associated with the 70 bp repeats by mass spectrometry. We will
validate candidates identified in the 70 bp repeat ePICh and initiate their functional analysis. Our work will
reveal clearly whether any proteins specifically associate with the 70 bp repeats, which will allow us to
investigate how 70 bp repeats integrity is maintained. Our findings will open up new avenues for better
understanding of the T. brucei pathogenesis and contribute to eradicate this parasite eventually.
