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Author: Donna Huber

Analysis of Long Non-Coding RNA in Cryptosporidium parvum Reveals Significant Stage-Specific Antisense Transcription

Cryptosporidium is a protist parasite that has been identified as the second leading cause of moderate to severe diarrhea in children younger than two and a significant cause of mortality worldwide. Cryptosporidium has a complex, obligate, intracellular but extra cytoplasmic lifecycle in a single host. How genes are regulated in this parasite remains largely unknown. Long non-coding RNAs (lncRNAs) play critical regulatory roles, including gene expression across a broad range of organisms. Cryptosporidium lncRNAs have been reported to enter the host cell nucleus and affect the host response. However, no systematic study of lncRNAs in Cryptosporidium has been conducted to identify additional lncRNAs. In this study, we analyzed a C. parvum in vitro strand-specific RNA-seq developmental time series covering both asexual and sexual stages to identify lncRNAs associated with parasite development. In total, we identified 396 novel lncRNAs, mostly antisense, with 86% being differentially expressed. Surprisingly, nearly 10% of annotated mRNAs have an antisense transcript. lncRNAs occur most often at the 3′ end of their corresponding sense mRNA. Putative lncRNA regulatory regions were identified and many appear to encode bidirectional promoters. A positive correlation between lncRNA and upstream mRNA expression was observed. Evolutionary conservation and expression of lncRNA candidates was observed between C. parvumC. hominis and C. baileyi. Ten C. parvum protein-encoding genes with antisense transcripts have P. falciparum orthologs that also have antisense transcripts. Three C. parvum lncRNAs with exceptional properties (e.g., intron splicing) were experimentally validated using RT-PCR and RT-qPCR. This initial characterization of the C. parvum non-coding transcriptome facilitates further investigations into the roles of lncRNAs in parasite development and host-pathogen interactions.

Yiran Li, Rodrigo P. Baptista, Adam Sateriale, Boris Striepen and Jessica C. Kissinger. Front Cell Infect Microbiol. 2021 Jan 14;10:608298. doi: 10.3389/fcimb.2020.608298. eCollection 2020.

The Inositol Pyrophosphate Biosynthetic Pathway of Trypanosoma cruzi

Inositol phosphates (IPs) are phosphorylated derivatives of myo-inositol involved in the regulation of several cellular processes through their interaction with specific proteins. Their synthesis relies on the activity of specific kinases that use ATP as phosphate donor. Here, we combined reverse genetics and liquid chromatography coupled to mass spectrometry (LC-MS) to dissect the inositol phosphate biosynthetic pathway and its metabolic intermediates in the main life cycle stages (epimastigotes, cell-derived trypomastigotes, and amastigotes) of Trypanosoma cruzi, the etiologic agent of Chagas disease. We found evidence of the presence of highly phosphorylated IPs, like inositol hexakisphosphate (IP6), inositol heptakisphosphate (IP7), and inositol octakisphosphate (IP8), that were not detected before by HPLC analyses of the products of radiolabeled exogenous inositol. The kinases involved in their synthesis (inositol polyphosphate multikinase (TcIPMK), inositol 5-phosphate kinase (TcIP5K), and inositol 6-phosphate kinase (TcIP6K)) were also identified. TcIPMK is dispensable in epimastigotes, important for the synthesis of polyphosphate, and critical for the virulence of the infective stages. TcIP5K is essential for normal epimastigote growth, while TcIP6K mutants displayed defects in epimastigote motility and growth. Our results demonstrate the relevance of highly phosphorylated IPs in the life cycle of T. cruzi.

Brian S Mantilla, Leticia D Do Amaral, Henning J Jessen, Roberto Docampo. ACS Chem Biol. 2021 Jan 7. doi: 10.1021/acschembio.0c00759

The IP 3 receptor and Ca 2+ signaling in trypanosomes

Trypanosoma cruzi, and the T. brucei group of parasites cause neglected diseases that affect millions of people around the world. These unicellular microorganisms have complex life cycles involving an insect vector and a mammalian host. Both groups of pathogens possess an inositol 1,4,5-trisphosphate (IP3)/diacylglycerol (DAG) signaling pathway, and an IP3 receptor, but with lineage-specific adaptations that make them different from their mammalian counterparts. The phospholipase C (PLC), which hydrolyzes phosphatidyl inositol 4,5-bisphosphate (PIP2) to IP3 is N-terminally myristoylated and palmitoylated. Acidocalcisomes, which are lysosome-related organelles rich in polyphosphate, are the main intracellular Ca2+ stores. The inositol 1,4,5-trisphosphate receptor (IP3R) localizes to acidocalcisomes instead of the endoplasmic reticulum. The trypanosome IP3R is stimulated by luminal phosphate and pyrophosphate, which are hydrolysis products of polyphosphate (polyP), and inhibited by tripolyphosphate (polyP3), which is the most abundant polyP in acidocalcisomes. Ca2+ signaling is important for host cell invasion and differentiation and to maintain cellular bioenergetics.

Roberto Docampo, Guozhong Huang. Biochim Biophys Acta Mol Cell Res. 2021 Jan 6;118947. doi: 10.1016/j.bbamcr.2021.118947

Affinity‐based proteomics reveals novel targets of inositol pyrophosphate (5‐IP7)‐dependent phosphorylation and binding in Trypanosoma cruzi replicative stages

Diphosphoinositol-5-pentakisphosphate (5-PP-IP5 ), also known as inositol heptakisphosphate (5-IP7 ), has been described as a high-energy phosphate metabolite that participates in the regulation of multiple cellular processes through protein binding or serine pyrophosphorylation, a post-translational modification involving a β-phosphoryl transfer. In this study, utilizing an immobilized 5-IP7 affinity reagent, we performed pull-down experiments coupled with mass spectrometry identification, and bioinformatic analysis, to reveal 5-IP7 -regulated processes in the two proliferative stages of the unicellular parasite Trypanosoma cruzi. Our protein screen clearly defined two cohorts of putative targets either in the presence of magnesium ions or in metal-free conditions. We endogenously tagged four protein candidates and immunopurified them to assess whether 5-IP7 -driven phosphorylation is conserved in T. cruzi. Among the most interesting targets, we identified a choline/o-acetyltransferase domain-containing phosphoprotein that undergoes 5-IP7 -mediated phosphorylation events at a polyserine tract (Ser578-580 ). We also identified a novel SPX domain-containing phosphoribosyl transferase [EC] herein termed as TcPRPPS4. Our data revealed new possible functional roles of 5-IP7 in this divergent eukaryote, and provided potential new targets for chemotherapy.

Brian S Mantilla, Karunakaran Kalesh, Nathaniel W Brown Jr, Dorothea Fiedler, Roberto Docampo. Mol Microbiol. 2020 Dec 22. doi: 10.1111/mmi.14672.

Evolution of the monitoring and evaluation strategies to support the World Health Organization’s Global Programme to Eliminate Lymphatic Filariasis

The Global Programme to Eliminate Lymphatic Filariasis (GPELF) was established with the ambitious goal of eliminating LF as a public health problem. The remarkable success of the GPELF over the past 2 decades in carrying out its principal strategy of scaling up and scaling down mass drug administration has relied first on the development of a rigorous monitoring and evaluation (M&E) framework and then the willingness of the World Health Organization and its community of partners to modify this framework in response to the practical experiences of national programmes. This flexibility was facilitated by the strong partnership that developed among researchers, LF programme managers and donors willing to support the necessary research agenda. This brief review summarizes the historical evolution of the GPELF M&E strategies and highlights current research needed to achieve the elimination goal.

Patrick J Lammie, Katherine M Gass, Jonathan King, Michael S Deming, David G Addiss, Gautam Biswas, Eric A Ottesen, Ralph Henderson. Int Health. 2020 Dec 22;13(Supplement_1):S65-S70. doi: 10.1093/inthealth/ihaa084.

Fall 2020 Newsletter

Learn about our research activities, faculty honors, new grants, and trainees from the summer and fall.

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Different Sensitivity of Control and MICU1- and MICU2-Ablated Trypanosoma cruzi Mitochondrial Calcium Uniporter Complex to Ruthenium-Based Inhibitors

The mitochondrial Ca2+ uptake in trypanosomatids shares biochemical characteristics with that of animals. However, the composition of the mitochondrial Ca2+ uniporter complex (MCUC) in these parasites is quite peculiar, suggesting lineage-specific adaptations. In this work, we compared the inhibitory activity of ruthenium red (RuRed) and Ru360, the most commonly used MCUC inhibitors, with that of the recently described inhibitor Ru265, on Trypanosoma cruzi, the agent of Chagas disease. Ru265 was more potent than Ru360 and RuRed in inhibiting mitochondrial Ca2+ transport in permeabilized cells. When dose-response effects were investigated, an increase in sensitivity for Ru360 and Ru265 was observed in TcMICU1-KO and TcMICU2-KO cells as compared with control cells. In the presence of RuRed, a significant increase in sensitivity was observed only in TcMICU2-KO cells. However, application of Ru265 to intact cells did not affect growth and respiration of epimastigotes, mitochondrial Ca2+ uptake in Rhod-2-labeled intact cells, or attachment to host cells and infection by trypomastigotes, suggesting a low permeability for this compound in trypanosomes.

Mayara S Bertolini, Roberto Docampo. Int J Mol Sci. 2020 Dec 7;21(23):E9316. doi: 10.3390/ijms21239316.

Rick Tarleton elected AAAS Fellow

Photo credit: Andrew Tucker

University of Georgia researcher Rick Tarleton has been elected as a 2020 American Association for the Advancement of Science (AAAS) Fellow by the AAAS Council “for distinguished contributions to the field of biological sciences, particularly for his research contributions and leadership to control Chagas Disease.”

Tarleton is a Regents Professor in the Department of Cellular Biology and UGA Athletic Association Distinguished Professor in Biological Sciences.

“It is indeed an honor to be acknowledged in this way – it reflects the strong efforts of many past and present members of the lab,” stated Tarleton, founder of the Center for Tropical and Emerging Global Diseases.

Since his undergraduate days, Tarleton’s research has focused on Trypanosoma cruzi infection, which causes the potentially fatal illness Chagas Disease. Historically, Tarleton’s research has attempted to answer broad questions such as how is immune control initiated and maintained during the infection, how does T. cruzi manage to avoid immune clearance and maintain an infection of decades in host, and what is the relationship between immunity, parasite persistence, and disease development. In an effort to answer these questions and more, Tarleton’s research group has developed tools to better study T. cruzi. They pioneered the use of the gene editing tool CRISPR in T. cruzi. Recently, they applied light sheet fluorescent microscopy to view infection in whole mouse organs. The Tarleton Research Group is also actively pursuing drug discovery for T. cruzi infection in a number of animal models including rodent, dog, and nonhuman primates. Their recent discovery of a dormancy stage in T. cruzi infections has revolutionized their drug treatment research, bringing them one step closer to finding a cure for this infection that affects at least 6 million people.

Tarleton’s work has largely been funded by the National Institutes of Health, the Wellcome Trust, the Burroughs Wellcome Fund, and partnerships with several pharmaceutical groups.

In addition to establishing the Center for Tropical and Emerging Global Diseases at UGA, he has been instrumental in organizing the Chagas Drug Discovery Consortium, which brings together U.S.-based laboratories with international groups. Tarleton is also the founder and current president of The Chagas Disease Foundation. He has been honored with a number of awards, including the Lamar Dodd Outstanding Researcher Award and being named a Burroughs Wellcome Fund Scholar in Molecular Parasitology. In 2017, he was elected as a Fellow of the American Academy of Microbiology.

“Rick’s election as a Fellow of AAAS is recognition of his immense contributions to the study of T. cruzi,” said Dennis Kyle, director of the Center for Tropical and Emerging Global Diseases. “His research has advanced our understanding of immune response to the pathogen, has developed new molecular approaches to study the parasite, and has accelerated drug discovery for Chagas Disease.”