Trypanosoma brucei is a protozoan parasite that causes African trypanosomiasis in humans and nagana in cattle. In mammalian hosts, bloodstream form (BF) T. brucei undergoes antigenic variation and regularly switches its surface antigen, variant surface glycoprotein (VSG), to evade the host's immune attack. Although T. brucei has more than a thousand VSG genes and pseudogenes, VSG is expressed exclusively from subtelomeric loci in a strictly monoallelic manner, which ensures effectiveness of VSG switching and maximizes its efficiency. Therefore, VSG switching and monoallelic VSG expression are essential for T. brucei pathogenesis. Telomeres, being adjacent to the expression sites of VSGs, have long been proposed to play an important role in VSG expression regulation. Indeed, it has been shown that in yeast, human, and T. brucei cells, telomeres form a heterochromatic structure that affects the transcription of reporter genes targeted to subtelomeres. Particularly in yeast, this telomeric silencing has been shown to depend on telomere protein RAP1. To explore telomere functions in VSG expression and switching regulation, we have been focusing on identification of T. brucei telomere-specific proteins and characterization of their functions. We have cloned the first T. brucei telomere-binding protein, tbTRF. More importantly, we have recently identified T. brucei RAP1 in a yeast 2-hybrid screen using tbTRF as bait, confirmed that tbRAP1 is an intrinsic component of the telomere complex, and demonstrated that it is essential for silencing subtelomeric VSGs in BF T. brucei cells. This discovery reveals T. brucei telomere as a key player in surface antigen expression control and identifies tbRAP1 as a potential target for anti-parasite drugs. Our finding also shows that T. brucei is similar to a couple of other pathogens including P. falciparum and C. glabrata, in which telomeric silencing plays an important role in regulation of virulence gene expression. We plan to further study how telomere proteins, particular tbRAP1 and tbTRF, regulate VSG expression and switching. This study would be helpful for eventual eradication of T. brucei and other similar microbial pathogens. We propose several approaches to further study the functions of tbRAP1 and tbTRF. First, our observations suggest that localization of tbRAP1 to the telomere is crucial for its VSG silencing function. We therefore hypothesize that if tbRAP1 binds telomere DNA directly, this activity would be critical for VSG silencing. However, if tbRAP1 lacks any DNA binding activity, the interaction between tbRAP1 and tbTRF and the telomere binding function of tbTRF would be essential for anchoring tbRAP1 to the telomere. We will test these hypotheses in Specific Aim 1 using in vitro approaches. These studies will reveal a key mechanism for tbRAP1-mediated silencing and elucidate similar and unique features of the DNA binding and protein-protein interaction functions of tbRAP1 and other RAP1 homologs. Our work would therefore help to identify potential targets for anti-parasite drugs.
In Specific Aim 2, we aim to carry out a series of in vivo genetic analyses to further understand tbRAP1's function. First, we hypothesize that tbRAP1 not only participates in VSG-silencing control but also influences VSG switching rates, which will be tested in Aim 2.1. Second, we will elucidate the relationship between different functions of tbRAP1 and to determine which domains of tbRAP1 are essential for VSG expression and/or switching regulation using systematic genetic approaches in Aim 2.2. These studies will help to reveal the underlying mechanisms of tbRAP1's function in antigenic variation. Third, we hypothesize that tbRAP1 applies its silencing effect by modulating the chromatin structure. We will therefore examine whether tbRAP1 preferentially associates with the silent chromatin and whether loss of tbRAP1 causes any changes in the chromatin structure of the derepressed ESs. Last, we hypothesize that tbRAP1 works with other unknown co-factors to establish/maintain the silencing structure at subtelomeric loci. In order to search for tbRAP1-interacting factors that also play important roles in antigenic variation, we aim to identify components of tbRAP1 protein complex by sequential immunoprecipitation. Studying functions of other factors in the same tbRAP1-mediated silencing pathway will help us to better understand the underlying mechanisms. Among the tbRAP1-interacting candidates, we may identify downstream effectors involved in VSG silencing/switching and proteins involved in the regulation of the expression, stability, or activity of tbRAP1. Identification of novel factors involved in antigenic variation also provides more potential targets for anti-parasite drugs and helps for eventual elimination of this parasite.

Public Health Relevance

Trypanosoma brucei is a protozoan parasite that causes African trypanosomiasis in humans. This disease is inevitably fatal without treatment and the number one cause of mortality in several central African countries. T. brucei also infects domestic livestock, particularly cattle, which is a major obstacle to the economic development in the affected rural area in sub-Sahara Africa. The main reason for persistent infection of T. brucei is that in mammalian hosts, T. brucei undergoes antigenic variation and regularly switches its surface antigen coat to evade the host's immune attack. To ensure the effectiveness of antigenic variation and maximize its efficiency, at any time T. brucei only expresses a single type of surface antigen from regions next to the telomeres, which are nucleoprotein complexes at the end of linear chromosomes. Therefore, the monoallelic expression of T. brucei surface antigen and antigenic variation are essential for T. brucei pathogenesis. We have recently demonstrated that a protein associated with the telomere, T. brucei RAP1, plays a critical role in the monoallelic expression of T. brucei surface antigen. TbRAP1 interacts with another telomere protein, T. brucei TRF, which binds directly to the telomere DNA. In this proposal, we aim to extend our characterization of tbRAP1 and tbTRF's functions in surface antigen expression regulation and antigenic variation and their underlying mechanisms. Our findings have revealed T. brucei telomere as an important player in surface antigen expression control. This is similar to a couple of other microbial pathogens, Plasmodium falciparum that causes malaria and Candida glabrata that causes opportunistic urogenital tract and bloodstream infections, in which telomeres have been shown to play a similar role in regulation of virulence gene expression. In addition, a closely related parasite, T. cruzi, causes Chagas Disease, which inflicts ~300,000 people in the US. The incidence of this disease is increasing in the US and has raised great concerns recently. The telomere complexes in T. brucei and T. cruzi are very similar. Our proposed studies will help to better understand T. brucei pathogenesis and develop anti-parasite drugs in the future that target essential telomere proteins, which will contribute to eventual elimination of T. brucei and other similar microbial pathogens.

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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
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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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