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.
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 2.7.6.1] 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.
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.
In contrast to animal cells, the inositol 1,4,5-trisphosphate receptor of Trypanosoma cruzi (TcIP3R) localizes to acidocalcisomes instead of the endoplasmic reticulum. Here, we present evidence that TcIP3R is a Ca2+ release channel gated by IP3 when expressed in DT40 cells knockout for all vertebrate IP3 receptors, and is required for Ca2+ uptake by T. cruzi mitochondria, regulating pyruvate dehydrogenase dephosphorylation and mitochondrial O2 consumption, and preventing autophagy. Localization studies revealed its co-localization with an acidocalcisome marker in all life cycle stages of the parasite. Ablation of TcIP3R by CRISPR/Cas9 genome editing caused: a) a reduction in O2 consumption rate and citrate synthase activity; b) decreased mitochondrial Ca2+ transport without affecting the membrane potential; c) increased ammonia production and AMP/ATP ratio; d) stimulation of autophagosome formation, and e) marked defects in growth of culture forms (epimastigotes) and invasion of host cells by infective stages (trypomastigotes). Moreover, TcIP3R overexpressing parasites showed decreased metacyclogenesis, trypomastigote host cell invasion and intracellular amastigote replication. In conclusion, the results suggest a modulatory activity of TcIP3R-mediated acidocalcisome Ca2+ release on cell bioenergetics in T. cruzi.
Lathosterol oxidase (LSO) catalyzes the formation of the C-5–C-6 double bond in the synthesis of various types of sterols in mammals, fungi, plants, and protozoa. In Leishmania parasites, mutations in LSO or other sterol biosynthetic genes are associated with amphotericin B resistance. To investigate the biological roles of sterol C-5–C-6 desaturation, we generated an LSO-null mutant line (lso−) in Leishmania major, the causative agent for cutaneous leishmaniasis. lso− parasites lacked the ergostane-based sterols commonly found in wild-type L. major and instead accumulated equivalent sterol species without the C-5–C-6 double bond. These mutant parasites were replicative in culture and displayed heightened resistance to amphotericin B. However, they survived poorly after reaching the maximal density and were highly vulnerable to the membrane-disrupting detergent Triton X-100. In addition, lso− mutants showed defects in regulating intracellular pH and were hypersensitive to acidic conditions. They also had potential alterations in the carbohydrate composition of lipophosphoglycan, a membrane-bound virulence factor in Leishmania. All these defects in lso− were corrected upon the restoration of LSO expression. Together, these findings suggest that the C-5–C-6 double bond is vital for the structure of the sterol core, and while the loss of LSO can lead to amphotericin B resistance, it also makes Leishmania parasites vulnerable to biologically relevant stress.
IMPORTANCE Sterols are essential membrane components in eukaryotes, and sterol synthesis inhibitors can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of sterols will facilitate the development of new drugs and counter drug resistance. LSO is required for the formation of the C-5–C-6 double bond in the sterol core structure in mammals, fungi, protozoans, plants, and algae. Functions of this C-5–C-6 double bond are not well understood. In this study, we generated and characterized a lathosterol oxidase-null mutant in Leishmania major. Our data suggest that LSO is vital for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to significant defects in stress response that could be exploited for drug development.
Yu Ning, Cheryl Frankfater, Fong-Fu Hsu, Rodrigo P Soares, Camila A Cardoso, Paula M Nogueira, Noelia Marina Lander, Roberto Docampo, Kai Zhang. mSphere. 2020 Jul 1;5(4):e00380-20. doi: 10.1128/mSphere.00380-20.
Docampo is Barbara and Sanford Orkin/GRA Eminent Scholar in Emerging Diseases and Cellular Biology and professor in the Franklin College of Arts and Sciences. (Photo by Billy Howard)
Roberto Docampo and colleagues at the University of Georgia’s Center for Tropical and Emerging Global Diseases have joined with 53 other lab groups to develop tools to genetically manipulate marine protists, a microscopic single-cell organism that plays an important ecological role in marine ecosystems. Their results were recently published in Nature Methods.
Protists aid in sequestering carbon dioxide, serve as a food source for many organisms (including humans), and cause the toxic red tides that have plagued Florida beaches in recent years. However, little is known about their cellular biology or evolutionary history, and no model organisms exist for this group. Protists are a highly diverse collection of species, and the inability to genetically modify a large majority of them has been a major hurdle to their study. A few protists, such as some parasitic protists which have an impact on human or animal health, have protocols, but they are not highly representative of the broader kingdom.
Funded by a $8 million grant from the Gordon and Betty Moore Foundation, researchers for the first time were able to develop protocols for transfection, or the introduction of foreign DNA, and gene expression in 13 species. They were also able to build on the tools already developed for eight other species. While they could not develop a universal protocol for genetic transfection for all protists due to their vast diversity, they were able to provide what the researchers are calling a synthetic “Transformation Roadmap.”
Docampo collaborated with Virginia Edgcomb and her lab at the Woods Hole Oceanographic Institute to develop genetic tools that would allow successful transfection of genes into Bodo saltans.
Bodo saltans is a unicellular organism found in marine and freshwater habitats. It belongs in the Discoba group which also includes the clinically significant parasitic protists Trypanosoma cruzi, Trypanosoma brucei, and Leishmania . Docampo and his team of researchers have been at the forefront of developing the genetic modification tool CRISPR/Cas9 for Trypanosoma cruzi, the causative agent of Chagas Disease.
“The development of tools to genetically modify [B. saltans] will be essential for the study of its biology and for the understanding of the evolution of the adaptions of trypanosomatids to parasitism,” said Docampo, Barbara and Sanford Orkin – GRA Eminent Scholar in Emerging Diseases and Cellular and professor in the Franklin College of Arts and Sciences department of cellular biology.
B. saltans, like the other protists in this study, will serve as a model organism for related protists that may be difficult to culture in the laboratory or in which protocols are unsuccessful. This study is not only a step toward closing the knowledge gap in the biology and evolution of this diverse kingdom of organisms but will also aid in the advancement of protisan biotechnology. Marine protists are an untapped resource and their study could reveal mechanisms and drug therapies to treat human and animal diseases.
The study is available online: Faktorová, D., Nisbet, R.E.R., Fernández Robledo, J.A. et al. Genetic tool development in marine protists: emerging model organisms for experimental cell biology. Nat Methods (2020). https://doi.org/10.1038/s41592-020-0796-x
Noelia Lander, a cellular biologist and postdoctoral researcher in Roberto Docampo‘s laboratory, has received the 2020 Postdoctoral Award from the UGA Research Foundation.
Lander has used her research to advance understanding of a dangerous parasite affecting millions of people worldwide. She adapted the CRISPR/Cas9 genome-editing system for the study of Trypanosoma cruzi, a human parasite that causes Chagas disease. In widely cited research, she proved the usefulness of this new gene-editing system and its range of applications in T. cruzi, which historically had been difficult to manipulate. Dozens of Chagas molecular biology labs worldwide use her CRISPR/Cas9 strategy to study the parasite’s proteins, characterize its metabolic pathways, understand its biology and search for new chemotherapeutic targets. More recently, she has used her system to study protein function and calcium signaling in T. cruzi. She has trained laboratory personnel and students in scientific research and is currently conducting the mentored phase of an NIH Pathway to Independence Award.
Created in 2011, Postdoctoral Research Awards recognize the remarkable contributions of postdoctoral research scholars to the UGA research enterprise. The UGA Research Foundation funds up to two awards a year to current scholars.
Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.
Roberto Docampo and several of his lab members are co-authors of this study.
Faktorová, D., Nisbet, R.E.R., Fernández Robledo, J.A. et al. Genetic tool development in marine protists: emerging model organisms for experimental cell biology. Nat Methods (2020). https://doi.org/10.1038/s41592-020-0796-x
Bisphosphonates are the most commonly prescribed drugs for the treatment of osteoporosis and other bone illnesses. Some of them have also shown antiparasitic activity. In search of improving the pharmacological profile of commercial bisphosphonates, our group had previously developed first row transition metal complexes with N-containing bisphosphonates (NBPs). In this work, we extended our studies to heteroleptic palladium–NBP complexes including DNA intercalating polypyridyl co-ligands (NN) with the aim of obtaining potential multi-target species. Complexes of the formula [Pd(NBP)2(NN)]·2NaCl·xH2O with NBP = alendronate (ale) or pamidronate (pam) and NN = 1,10 phenanthroline (phen) or 2,2′-bipyridine (bpy) were synthesized and fully characterized. All the obtained compounds were much more active in vitro against T. cruzi (amastigote form) than the corresponding NBP ligands. In addition, complexes were nontoxic to mammalian cells up to 50–100 µM. Compounds with phen as ligand were 15 times more active than their bpy analogous. Related to the potential mechanism of action, all complexes were potent inhibitors of two parasitic enzymes of the isoprenoid biosynthetic pathway. No correlation between the anti-T. cruzi activity and the enzymatic inhibition results was observed. On the contrary, the high antiparasitic activity of phen-containing complexes could be related to their ability to interact with DNA in an intercalative-like mode. These rationally designed compounds are good candidates for further studies and good leaders for future drug developments.
