Unraveling the biology of human pathogens is fundamental toward understanding mechanisms of pathogenesis and identifying genes essential for survival in the host. 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). The overall goal of the proposed work here is to identify the mechanism by which RBP6 activates developmental progression to infectious metacyclics and to characterize the RNA binding protein network that regulates the developmental program leading to epimastigotes and infectious metacyclics. To accomplish these goals, we will build on our major findings in the previous funding period, namely the identification of initial targets of RBP6 function, the depiction of the molecular characteristics of metacyclics, and the identification of a large compendium of molecules linked to the developmental program leading to infective metacyclic parasites. We will use innovative approaches involving a screen for RBP6 function with deep mutational scanning by combining metacyclic selection and high-throughput DNA sequencing and analyze gene expression at the single cell level with RNA-Seq. Taken together our research plan provides unique opportunities to illuminate the developmental program leading from non-infective procyclics to infectious metacyclics, a crucial process in the T. brucei life cycle.

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. Our proposed studies will provide an insight into the developmental program that leads to the establishment of infectious trypanosomes, which has the potential to expose new strategies to combat the diseases caused by these deadly parasites.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI110325-08
Application #
10112153
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Mcgugan, Glen C
Project Start
2014-03-01
Project End
2024-02-29
Budget Start
2021-03-01
Budget End
2022-02-28
Support Year
8
Fiscal Year
2021
Total Cost
Indirect Cost
Name
Yale University
Department
Public Health & Prev 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
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
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
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