T. brucei is a parasite that causes sleeping sickness in humans and Nagana in cattle in sub-Saharan Africa. When growing in mammalian host, T. brucei cells regularly switch their major surface glycoprotein, to evade the host immune system - a phenomenon called antigenic variation. These surface glycoproteins are exclusively expressed from one of ~ 20 loci located next to chromosome ends - telomeres. Telomeres in yeasts form a specialized structure that influences transcription of genes located close by. A similar phenomenon has also been observed in T. brucei, and telomeres may play an important role in antigenic variation. The ultimate goal of telomere studies in Trypanosoma brucei is to understand telomere functions in antigenic variation, an essential aspect of T. brucei pathogenesis. The fundamental step would be to identify telomere components and characterize their functions, which is the primary goal of this proposal.
Specific Aim 1 : To purify tbTRF (a double-stranded telomere DMA binding factor in T. brucei) protein complex, using two approaches: 1) Sequential immunoprecipitate pull-down of FLAG-HA-HA tagged tbTRF using anti-FLAG and anti-HA monoclonal antibodies. Proteins co-immunoprecipitated will be identified by mass spectrometry. 2) Yeast 2-hybrid screen using lexA-tbTRF as bait and a T. brucei GAD-fusion cDNA library. Candidates for tbTRF-interaction factors will be first confirmed for their interaction with tbTRF in vivo by co-immunoprecipitation. Positive clones will be characterized for their roles in antigenic variation and telomere functions by examination of phenotypes in knockout or RNAi knockdown cell lines.
Specific Aim 2 : Select non-lethal interaction-deficient tbTRF mutations in the tbTRFH domain to better characterize tbTRF's function. Random point mutations or small deletions will be generated in the tbTRFH domain, using PCR cloning. Mutants that lose the interaction with wild-type tbTRF but remain the ability to interact with themselves will be screened using both conventional and reverse 2-hybrid analysis. Cell lines with knock-in or overexpression of these mutant alleles will be characterized for their phenotypes in antigenic variation. The identification of telomere complex components and subsequent characterization of their functions would be a good starting point for studying telomere functions in antigenic variation, an essential aspect of T. brucei pathogenesis.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Pathogenic Eukaryotes Study Section (PTHE)
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Joy, Deirdre A
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Cleveland State University
Schools of Arts and Sciences
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Sandhu, Ranjodh; Li, Bibo (2017) Telomerase activity is required for the telomere G-overhang structure in Trypanosoma brucei. Sci Rep 7:15983
Nanavaty, Vishal; Sandhu, Ranjodh; Jehi, Sanaa E et al. (2017) Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids. Nucleic Acids Res 45:5785-5796
Jehi, Sanaa E; Nanavaty, Vishal; Li, Bibo (2016) Trypanosoma brucei TIF2 and TRF Suppress VSG Switching Using Overlapping and Independent Mechanisms. PLoS One 11:e0156746
Li, Bibo (2015) DNA double-strand breaks and telomeres play important roles in trypanosoma brucei antigenic variation. Eukaryot Cell 14:196-205
Jehi, Sanaa E; Li, Xiaohua; Sandhu, Ranjodh et al. (2014) Suppression of subtelomeric VSG switching by Trypanosoma brucei TRF requires its TTAGGG repeat-binding activity. Nucleic Acids Res 42:12899-911
Jehi, Sanaa E; Wu, Fan; Li, Bibo (2014) Trypanosoma brucei TIF2 suppresses VSG switching by maintaining subtelomere integrity. Cell Res 24:870-85
Benmerzouga, Imaan; ConcepciĆ³n-Acevedo, Jeniffer; Kim, Hee-Sook et al. (2013) Trypanosoma brucei Orc1 is essential for nuclear DNA replication and affects both VSG silencing and VSG switching. Mol Microbiol 87:196-210
Sandhu, Ranjodh; Sanford, Samantha; Basu, Shrabani et al. (2013) A trans-spliced telomerase RNA dictates telomere synthesis in Trypanosoma brucei. Cell Res 23:537-51
Mazumdar, Tapati; Sandhu, Ranjodh; Qadan, Maha et al. (2013) Hedgehog signaling regulates telomerase reverse transcriptase in human cancer cells. PLoS One 8:e75253
Pandya, Unnati M; Sandhu, Ranjodh; Li, Bibo (2013) Silencing subtelomeric VSGs by Trypanosoma brucei RAP1 at the insect stage involves chromatin structure changes. Nucleic Acids Res 41:7673-82

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