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 #
2R01AI028798-26
Application #
8999550
Study Section
Special Emphasis Panel (ZRG1-IDM-P (02))
Program Officer
Mcgugan, Glen C
Project Start
1989-12-01
Project End
2020-10-31
Budget Start
2015-11-15
Budget End
2016-10-31
Support Year
26
Fiscal Year
2016
Total Cost
$835,872
Indirect Cost
$287,582
Name
Yale University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06511
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
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
Kolev, Nikolay G; Ullu, Elisabetta; Tschudi, Christian (2015) Construction of Trypanosoma brucei Illumina RNA-Seq libraries enriched for transcript ends. Methods Mol Biol 1201:165-75
Ericson, Megan; Janes, Michael A; Butter, Falk et al. (2014) On the extent and role of the small proteome in the parasitic eukaryote Trypanosoma brucei. BMC Biol 12:14
Shi, Huafang; Barnes, Rebecca L; Carriero, Nicholas et al. (2014) Role of the Trypanosoma brucei HEN1 family methyltransferase in small interfering RNA modification. Eukaryot Cell 13:77-86
Kolev, Nikolay G; Ullu, Elisabetta; Tschudi, Christian (2014) The emerging role of RNA-binding proteins in the life cycle of Trypanosoma brucei. Cell Microbiol 16:482-9
Atayde, Vanessa D; Shi, Huafang; Franklin, Joseph B et al. (2013) The structure and repertoire of small interfering RNAs in Leishmania (Viannia) braziliensis reveal diversification in the trypanosomatid RNAi pathway. Mol Microbiol 87:580-93
Barnes, Rebecca L; Shi, Huafang; Kolev, Nikolay G et al. (2012) Comparative genomics reveals two novel RNAi factors in Trypanosoma brucei and provides insight into the core machinery. PLoS Pathog 8:e1002678
Tschudi, Christian; Shi, Huafang; Franklin, Joseph B et al. (2012) Small interfering RNA-producing loci in the ancient parasitic eukaryote Trypanosoma brucei. BMC Genomics 13:427
Kolev, Nikolay G; Ramey-Butler, Kiantra; Cross, George A M et al. (2012) Developmental progression to infectivity in Trypanosoma brucei triggered by an RNA-binding protein. Science 338:1352-3

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