This application focuses on the protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animals in sub-Saharan Africa. There are no vaccines, and therapeutic drugs have serious side effects and decreasing efficacy. Thus, there is a pressing need for research to better understand the biology of these human pathogens and the mechanisms they use to survive within their hosts. T. brucei undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector, which among others involves changes in cell morphology, surface coat composition, metabolism, signaling pathways and gene expression. Consequently, these parasites have evolved adaptations to allow for their survival in both the gut and salivary glands of the tsetse fly, as well as in the bloodstream of their mammalian host. By overexpressing a single RNA-binding protein (RBP6) in non- infectious trypanosomes, we recapitulated in vitro the events leading to acquisition of infectivity in the insect vector, including the expression of metacyclic variant surface glycoproteins (mVSGs). At present, little is known how mVSG gene expression is activated and how the expression is switched to bloodstream-form VSGs, once the parasite enters a mammalian host. One major goal of this application will be to examine how trypanosomes receive instructions to begin synthesizing the mVSG coat, how each cell expresses a single mVSG, and how mVSG expression is repressed and switched to the expression of bloodstream-form VSGs. We will apply a number of high-throughput approaches to monitor the chromatin structure of mVSG genes during developmental progression and test whether a second RNA-binding protein (RBP10) is a facilitator of [m]VSG expression. Primary transcripts and mature mRNAs will be monitored using RNA-Seq, the position, amount, and orientation of transcriptionally engaged RNA polymerase I will be surveyed by global run-on-sequencing (GRO-Seq) and transcription factor binding will be gauged by chromatin immunoprecipitation (ChIP) coupled with Illumina sequencing (ChIP-Seq). A second emphasis will be on RNA interference (RNAi). Since our discovery of RNAi in T. brucei in 1998, this pathway has been a focus of our investigations, which have led to the finding that RNAi functions both in the nucleus and in the cytoplasm and to the identification of five core RNAi genes. We will employ different approaches to address the question what defines the RNA-induced silencing complex (RISC), i.e. what other cellular factors functionally interact with the RNAi machinery, and how the levels of Argonaute are regulated. Finally, we will further address the biological function of RNAi by determining the RNAi targets during the T. brucei developmental cycle, which is now possible with our newly developed differentiation system.

Public Health Relevance

Parasitic protozoa are a major cause of global infectious diseases and thus, represent one of the most serious threats to public health. Among these are African trypanosomes, the causative agents of African trypanosomiasis or sleeping sickness in humans and a wasting and fatal disease (Nagana) in cattle, domestic pigs and other farm animals causing a profound effect on the economy of much of the continent.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI028798-29
Application #
9589451
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mcgugan, Glen C
Project Start
1989-12-01
Project End
2020-10-31
Budget Start
2018-11-01
Budget End
2019-10-31
Support Year
29
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Yale University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Shi, Huafang; Butler, Kiantra; Tschudi, Christian (2018) A single-point mutation in the RNA-binding protein 6 generates Trypanosoma brucei metacyclics that are able to progress to bloodstream forms in vitro. Mol Biochem Parasitol 224:50-56
Kolev, Nikolay G; Ramsdell, Trisha K; Tschudi, Christian (2018) Temperature shift activates bloodstream VSG expression site promoters in Trypanosoma brucei. Mol Biochem Parasitol 226:20-23
Shi, Huafang; Butler, Kiantra; Tschudi, Christian (2018) Differential expression analysis of transcriptome data of Trypanosoma brucei RBP6 induction in procyclics leading to infectious metacyclics and bloodstream forms in vitro. Data Brief 20:978-980
Srivastava, Ankita; Badjatia, Nitika; Lee, Ju Huck et al. (2018) An RNA polymerase II-associated TFIIF-like complex is indispensable for SL RNA gene transcription in Trypanosoma brucei. Nucleic Acids Res 46:1695-1709
Kolev, Nikolay G; G├╝nzl, Arthur; Tschudi, Christian (2017) Metacyclic VSG expression site promoters are recognized by the same general transcription factor that is required for RNA polymerase I transcription of bloodstream expression sites. Mol Biochem Parasitol 216:52-55
Christiano, Romain; Kolev, Nikolay G; Shi, Huafang et al. (2017) The proteome and transcriptome of the infectious metacyclic form of Trypanosoma brucei define quiescent cells primed for mammalian invasion. Mol Microbiol 106:74-92
Damasceno, Jeziel D; Silva, Gabriel LA; Tschudi, Christian et al. (2017) Evidence for regulated expression of Telomeric Repeat-containing RNAs (TERRA) in parasitic trypanosomatids. Mem Inst Oswaldo Cruz 112:572-576
Alves e Silva, Thiago Luiz; Savage, Amy F; Aksoy, Serap (2016) Transcript Abundance of Putative Lipid Phosphate Phosphatases During Development of Trypanosoma brucei in the Tsetse Fly. Am J Trop Med Hyg 94:890-3
Savage, Amy F; Kolev, Nikolay G; Franklin, Joseph B et al. (2016) Transcriptome Profiling of Trypanosoma brucei Development in the Tsetse Fly Vector Glossina morsitans. PLoS One 11:e0168877
Ramey-Butler, Kiantra; Ullu, Elisabetta; Kolev, Nikolay G et al. (2015) Synchronous expression of individual metacyclic variant surface glycoprotein genes in Trypanosoma brucei. Mol Biochem Parasitol 200:1-4

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