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Category: publications

EAT-18 is an essential auxiliary protein interacting with the non-alpha nAChR subunit EAT-2 to form a functional receptor

Nematode parasites infect approximately 1.5 billion people globally and are a significant public health concern. There is an accepted need for new, more effective anthelmintic drugs. Nicotinic acetylcholine receptors on parasite nerve and somatic muscle are targets of the cholinomimetic anthelmintics, while glutamate-gated chloride channels in the pharynx of the nematode are affected by the avermectins. Here we describe a novel nicotinic acetylcholine receptor on the nematode pharynx that is a potential new drug target. This homomeric receptor is comprised of five non-α EAT-2 subunits and is not sensitive to existing cholinomimetic anthelmintics. We found that EAT-18, a novel auxiliary subunit protein, is essential for functional expression of the receptor. EAT-18 directly interacts with the mature receptor, and different homologs alter the pharmacological properties. Thus we have described not only a novel potential drug target but also a new type of obligate auxiliary protein for nAChRs.

Shivani Choudhary, Samuel K. Buxton, Sreekanth Puttachary, Saurabh Verma, Gunnar R. Mair, Ciaran J. McCoy, Barbara J. Reaves, Adrian J. Wolstenholme, Richard J. Martin, Alan P. Robertson. PLoS Pathog. 2020 Apr 3;16(4):e1008396. doi: 10.1371/journal.ppat.1008396.

Galtonosides A-E: Antiproliferative and Antiplasmodial Cholestane Glycosides from Galtonia regalis

An extract of Galtonia regalis from the Natural Products Discovery Institute showed moderate antiplasmodial activity, with an IC50 value less than 1.25 μg/mL. The two known cholestane glycosides 1 and 2 and the five new cholestane glycosides galtonosides A–E (37) were isolated after bioassay-directed fractionation. The structures of the new compounds were determined by interpretation of their NMR and mass spectra. Among these compounds, galtonoside B (4) displayed the most potent antiplasmodial activity, with an IC50 value of 0.214 μM against the drug-resistant Dd2 strain of Plasmodium falciparum.

Yongle Du, Brooke A. Martin, Ana Lisa Valenciano, Jason A. Clement, Michael Goetz, Maria B. Cassera, David G. I. Kingston. J Nat Prod. 2020 Mar 31. doi: 10.1021/acs.jnatprod.9b01064.

Multi-target heteroleptic palladium bisphosphonate complexes

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.

Micaella Cipriani, Santiago Rostán, Ignacio León, Zhu-Hong Li, Jorge S. Gancheff, Ulrike Kemmerling, Claudio Olea Azar, Susana Etcheverry, Roberto Docampo, Dinorah Gambino & Lucía Otero. J Biol Inorg Chem. 2020 Mar 30. doi: 10.1007/s00775-020-01779-y.

CRISPR/Cas9 Technology Applied to the Study of Proteins Involved in Calcium Signaling in Trypanosoma cruzi

Chagas disease is a vector-borne tropical disease affecting millions of people worldwide, for which there is no vaccine or satisfactory treatment available. It is caused by the protozoan parasite Trypanosoma cruzi and considered endemic from North to South America. This parasite has unique metabolic and structural characteristics that make it an attractive organism for basic research. The genetic manipulation of T. cruzi has been historically challenging, as compared to other pathogenic protozoans. However, the use of the prokaryotic CRISPR/Cas9 system for genome editing has significantly improved the ability to generate genetically modified T. cruzi cell lines, becoming a powerful tool for the functional study of proteins in different stages of this parasite’s life cycle, including infective trypomastigotes and intracellular amastigotes. Using the CRISPR/Cas9 method that we adapted to T. cruzi, it has been possible to perform knockout, complementation and in situ tagging of T. cruzi genes. In our system we cotransfect T. cruzi epimastigotes with an expression vector containing the Cas9 sequence and a single guide RNA, together with a donor DNA template to promote DNA break repair by homologous recombination. As a result, we have obtained homogeneous populations of mutant epimastigotes using a single resistance marker to modify both alleles of the gene. Mitochondrial Ca2+ transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca2+ increases, for the bioenergetics of the cells, and for viability and infectivity. In this chapter we describe the most effective methods to achieve genome editing in T. cruzi using as example the generation of mutant cell lines to study proteins involved in calcium homeostasis. Specifically, we describe the methods we have used for the study of three proteins involved in the calcium signaling cascade of T. cruzi: the inositol 1,4,5-trisphosphate receptor (TcIP3R), the mitochondrial calcium uniporter (TcMCU) and the calcium-sensitive pyruvate dehydrogenase phosphatase (TcPDP), using CRISPR/Cas9 technology as an approach to establish their role in the regulation of energy metabolism.

Noelia Lander, Miguel A. Chiurillo, Roberto Docampo. Methods Mol Biol. 2020;2116:177-197. doi: 10.1007/978-1-0716-0294-2_13.

Isolation and Characterization of Acidocalcisomes from Trypanosomatids

Acidocalcisomes are membrane-bounded, electron-dense, acidic organelles, rich in calcium and polyphosphate. These organelles were first described in trypanosomatids and later found from bacteria to human cells. Some of the functions of the acidocalcisome are the storage of cations and phosphorus, participation in pyrophosphate (PPi) and polyphosphate (polyP) metabolism, calcium signaling, maintenance of intracellular pH homeostasis, autophagy, and osmoregulation. Isolation of acidocalcisomes is an important technique for understanding their composition and function. Here, we provide detailed subcellular fractionation protocols using iodixanol gradient centrifugations to isolate high-quality acidocalcisomes from Trypanosoma brucei, which are subsequently validated by electron microscopy, and enzymatic and immunoblot assays with organellar markers.

Guozhong Huang, Silvia N. J. Moreno, Roberto Docampo. Methods Mol Biol. 2020;2116:673-688. doi: 10.1007/978-1-0716-0294-2_40.

Plasmodium vivax Liver and Blood Stages Recruit the Druggable Host Membrane Channel Aquaporin-3

Plasmodium vivax infects hepatocytes to form schizonts that cause blood infection, or dormant hypnozoites that can persist for months in the liver before leading to relapsing blood infections. The molecular processes that drive Pvivax schizont and hypnozoite survival remain largely unknown, but they likely involve a rich network of host-pathogen interactions, including those occurring at the host-parasite interface, the parasitophorous vacuole membrane (PVM). Using a recently developed Pvivax liver-stage model system we demonstrate that host aquaporin-3 (AQP3) localizes to the PVM of schizonts and hypnozoites within 5 days after invasion. This recruitment is also observed in Pvivax-infected reticulocytes. Chemical treatment with the AQP3 inhibitor auphen reduces Pvivax liver hypnozoite and schizont burden, and inhibits Pvivax asexual blood-stage growth. These findings reveal a role for AQP3 in Pvivax liver and blood stages and suggest that the protein may be targeted for therapeutic treatment.

Dora Posfai, Steven P. Maher, Camille Roesch, Amélie Vantaux, Kayla Sylvester, Julie Péneau, Jean Popovici, Dennis E. Kyle, Benoît Witkowski, Emily R. Derbyshire. Cell Chem Biol. 2020 Mar 24. pii: S2451-9456(20)30083-0. doi: 10.1016/j.chembiol.2020.03.009.

Acute Plasmodium Infection Promotes Interferon-Gamma-Dependent Resistance to Ebola Virus Infection

During the 2013-2016 Ebola virus (EBOV) epidemic, a significant number of patients admitted to Ebola treatment units were co-infected with Plasmodium falciparum, a predominant agent of malaria. However, there is no consensus on how malaria impacts EBOV infection. The effect of acute Plasmodium infection on EBOV challenge was investigated using mouse-adapted EBOV and a biosafety level 2 (BSL-2) model virus. We demonstrate that acute Plasmodium infection protects from lethal viral challenge, dependent upon interferon gamma (IFN-γ) elicited as a result of parasite infection. Plasmodium-infected mice lacking the IFN-γ receptor are not protected. Ex vivo incubation of naive human or mouse macrophages with sera from acutely parasitemic rodents or macaques programs a proinflammatory phenotype dependent on IFN-γ and renders cells resistant to EBOV infection. We conclude that acute Plasmodium infection can safeguard against EBOV by the production of protective IFN-γ. These findings have implications for anti-malaria therapies administered during episodic EBOV outbreaks in Africa.

Kai J Rogers, Olena Shtanko, Rahul Vijay, Laura N Mallinger, Chester J Joyner, Mary R Galinski, Noah S Butler, Wendy Maury. Cell Rep. 2020 Mar 24;30(12):4041-4051.e4. doi: 10.1016/j.celrep.2020.02.104.

The Mitochondrial Calcium Uniporter Interacts with Subunit c of the ATP Synthase of Trypanosomes and Humans

Mitochondrial Ca2+ transport mediated by the uniporter complex (MCUC) plays a key role in the regulation of cell bioenergetics in both trypanosomes and mammals. Here we report that Trypanosoma brucei MCU (TbMCU) subunits interact with subunit c of the mitochondrial ATP synthase (ATPc), as determined by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Mutagenesis analysis in combination with MYTH assays suggested that transmembrane helices (TMHs) are determinants of this specific interaction. In situ tagging, followed by immunoprecipitation and immunofluorescence microscopy, revealed that T. brucei ATPc (TbATPc) coimmunoprecipitates with TbMCUC subunits and colocalizes with them to the mitochondria. Blue native PAGE and immunodetection analyses indicated that the TbMCUC is present together with the ATP synthase in a large protein complex with a molecular weight of approximately 900 kDa. Ablation of the TbMCUC subunits by RNA interference (RNAi) significantly increased the AMP/ATP ratio, revealing the downregulation of ATP production in the cells. Interestingly, the direct physical MCU-ATPc interaction is conserved in Trypanosoma cruzi and human cells. Specific interaction between human MCU (HsMCU) and human ATPc (HsATPc) was confirmed in vitro by mutagenesis and MYTH assays and in vivo by coimmunoprecipitation. In summary, our study has identified that MCU complex physically interacts with mitochondrial ATP synthase, possibly forming an MCUC-ATP megacomplex that couples ADP and Pi transport with ATP synthesis, a process that is stimulated by Ca2+ in trypanosomes and human cells.

IMPORTANCE The mitochondrial calcium uniporter (MCU) is essential for the regulation of oxidative phosphorylation in mammalian cells, and we have shown that in Trypanosoma brucei, the etiologic agent of sleeping sickness, this channel is essential for its survival and infectivity. Here we reveal that that Trypanosoma brucei MCU subunits interact with subunit c of the mitochondrial ATP synthase (ATPc). Interestingly, the direct physical MCU-ATPc interaction is conserved in T. cruzi and human cells.

Guozhong HuangRoberto Docampo. mBio. 2020 Mar 17;11(2). pii: e00268-20. doi: 10.1128/mBio.00268-20.

Update on Cryptosporidium spp.: highlights from the Seventh International Giardia and Cryptosporidium Conference

While cryptosporidiosis is recognized as being among the most common causes of human parasitic diarrhea in the world, there is currently limited knowledge on Cryptosporidium infection mechanisms, incomplete codification of diagnostic methods, and a need for additional therapeutic options. In response, the Seventh International Giardia and Cryptosporidium Conference (IGCC 2019) was hosted from 23 to 26 June 2019, at the Rouen Normandy University, France. This trusted event brought together an international delegation of researchers to synthesize recent advances and identify key research questions and knowledge gaps. The program of the interdisciplinary conference included all aspects of host-parasite relationships from basic research to applications to human and veterinary medicine, and environmental issues associated with waterborne parasites and their epidemiological consequences. In relation to Cryptosporidium and cryptosporidiosis, the primary research areas for which novel findings and the most impressive communications were presented and discussed included: Cryptosporidium in environmental waters, seafood, and fresh produce; Animal epidemiology; Human cryptosporidiosis and epidemiology; Genomes and genomic evolution encompassing: Comparative genomics of Cryptosporidium spp., Genomic insights into biology, Acquiring and utilizing genome sequences, Genetic manipulation; Host-parasite interaction (immunology, microbiome); and Diagnosis and treatment. High quality presentations discussed at the conference reflected decisive progress and identified new opportunities that will engage investigators and funding agencies to spur future research in a “one health” approach to improve basic knowledge and the clinical and public health management of zoonotic cryptosporidiosis.

Giovanni Widmer, David Carmena, Martin Kváč, Rachel M. Chalmers, Jessica C. Kissinger, Lihua Xiao, Adam Sateriale, Boris Striepen, Fabrice Laurent, Sonia Lacroix-Lamandé, Gilles Gargala and Loïc Favennec. Parasite. 2020;27:14. doi: 10.1051/parasite/2020011.

Lexis and Grammar of Mitochondrial RNA Processing in Trypanosomes

Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3′ extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3′ adenylation, uridine insertion/deletion editing, and 3′ A/U-tailing. The rRNAs and gRNAs are 3′ uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.

Inna Aphasizheva, Juan Alfonzo, Jason Carnes, Igor Cestari, Jorge Cruz-Reyes, H. Ulrich Göringer, Stephen Hajduk, Julius Lukeš, Susan Madison-Antenucci, Dmitri A. Maslov, Suzanne M. McDermott, Torsten Ochsenreiter, Laurie K. Read, Reza Salavati, Achim Schnaufer, André Schneider, Larry Simpson, Kenneth Stuart, Vyacheslav Yurchenko, Z. Hong Zhou, Alena Zíková, Liye Zhang, Sara Zimmer, Ruslan Aphasizhev. Trends Parasitol. 2020 Apr;36(4):337-355. doi: 10.1016/ Epub 2020 Feb 28.