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Tag: Roberto Docampo

Drug Target Validation of the Protein Kinase AEK1, Essential for Proliferation, Host Cell Invasion, and Intracellular Replication of the Human Pathogen Trypanosoma cruzi

Donna Huber on September 29, 2021

Protein phosphorylation is involved in several key biological roles in the complex life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease, and protein kinases are potential drug targets. Here, we report that the AGC essential kinase 1 (TcAEK1) exhibits a cytosolic localization and a higher level of expression in the replicative stages of the parasite. A CRISPR/Cas9 editing technique was used to generate ATP analog-sensitive TcAEK1 gatekeeper residue mutants that were selectively and acutely inhibited by bumped kinase inhibitors (BKIs). Analysis of a single allele deletion cell line (TcAEK1-SKO), and gatekeeper mutants upon treatment with inhibitor, showed that epimastigote forms exhibited a severe defect in cytokinesis. Moreover, we also demonstrated that TcAEK1 is essential for epimastigote proliferation, trypomastigote host cell invasion, and amastigote replication. We suggest that TcAEK1 is a pleiotropic player involved in cytokinesis regulation in T. cruzi and thus validate TcAEK1 as a drug target for further exploration. The gene editing strategy we applied to construct the ATP analog-sensitive enzyme could be appropriate for the study of other proteins of the T. cruzi kinome. IMPORTANCE Chagas disease affects 6 to 7 million people in the Americas, and its treatment has been limited to drugs with relatively high toxicity and low efficacy in the chronic phase of the infection. New validated targets are needed to combat this disease. In this work, we report the chemical and genetic validation of the protein kinase AEK1, which is essential for cytokinesis and infectivity, using a novel gene editing strategy.

Miguel A Chiurillo, Bryan C Jensen, Roberto Docampo. Microbiol Spectr. 2021 Sep 29;e0073821. doi: 10.1128/Spectrum.00738-21.

Calcium signaling in intracellular protist parasites

Donna Huber on September 24, 2021

Calcium ion (Ca2+) signaling is one of the most frequently employed mechanisms of signal transduction by eukaryotic cells, and starts with either Ca2+ release from intracellular stores or Ca2+ entry through the plasma membrane. In intracellular protist parasites Ca2+ signaling initiates a sequence of events that may facilitate their invasion of host cells, respond to environmental changes within the host, or regulate the function of their intracellular organelles. In this review we examine recent findings in Ca2+ signaling in two groups of intracellular protist parasites that have been studied in more detail, the apicomplexan and the trypanosomatid parasites.

Roberto Docampo, Silvia Nj Moreno. Current Opinion in Microbiology 2021, 64:33–40. https://doi.org/10.1016/j.mib.2021.09.002

Trainee Spotlight: Mayara Bertolini

Donna Huber on June 23, 2021

trainee Mayara Bertolini

Mayara Bertolini is a third year Ph.D. trainee in the laboratory of Dr. Roberto Docampo. She has recently been awarded a predoctoral fellowship from the American Heart Association.

Please tell us a little about yourself.

I am from São Paulo, Brazil and I have always been a very curious person that likes to discover unique things. Over time, I realized that biology was one of my favorite subjects, especially when it came to diseases. I decided to major in Biomedical Sciences at the Faculdade Anhanguera de Santa Bárbara D’Oeste (São Paulo, Brazil). After my graduation, I performed voluntary research training at the Laboratory of Bioenergetics of the Department of Clinical Pathology (School of Medical Sciences) of the State University of Campinas (Campinas, São Paulo, Brazil) under the supervision of Dr. Anibal Vercesi. Thereafter, I joined the Master’s program to continue my training as a scientist. There I met Dr. Roberto Docampo, who has collaborated with Dr. Vercesi for many years. Since then, I joined his research group, where Dr. Miguel Angel Chiurillo and Dr. Noelia Lander were also members of a very productive team, which has stimulated my fascination for research in parasitology. During my master’s, I was awarded a fellowship from the São Paulo Research Foundation (FAPESP) to perform a functional study of the regulatory subunits Mitochondrial Ca2+ Uptake 1 (MICU1) and 2 (MICU2) involved in calcium signaling in the parasite that causes Chagas disease, Trypanosoma cruzi. My master’s project elucidated some questions and opened doors to interesting new topics, which our group is very excited to explain.

Why did you choose UGA?

I wanted to continue working with the same model to improve my scientific thinking and to complete my laboratory training, and the Center for Tropical and Emerging Global Diseases (CTEGD) at UGA has a wide range of researchers working with trypanosomes. Pursuing my Ph.D. at UGA is an extraordinary opportunity because of CTEGD’s unique infrastructure, which consists of extremely qualified professionals and resources that facilitate the development of research projects.

What is your research focus? 

T. cruzi is one of the least well understood neglected tropical disease agents and current treatments remain inadequate partly due to a general lack of knowledge of this parasite’s basic biology. We are particularly interested in establishing the role and interaction between mitochondrial proteins involved in Ca2+ uptake in this organelle. Understanding the mechanisms of adaptation and survival of the parasite upon environmental challenges, as changes in concentration of free Ca2+, will lead to important insights into the biology of this parasite and the evolution of Ca2+ signaling in eukaryotic cells. Considering that disruption of Ca2+ homeostasis by toxic agents is related to the loss of cell viability, the identification of the possible differences in mitochondrial Ca2+ transport between these parasites and the host cells could be useful for the development of new chemotherapeutic agents against Chagas disease. The purpose of the AHA predoctoral fellowship is to enhance the training of students who intend to pursue careers as scientists aimed at improving global health and wellbeing, and I feel like I can contribute to this mission.

What are your future professional plans?

After my graduation from UGA, I hope to continue for a postdoctoral research position. In the future, I would like to establish a research group in Brazil using trypanosomatids as biological models for studying the structure and function of proteins.

Any advice for a student interested in this field?

Don’t be afraid to try new things and learn from it.

 

Support trainees like Mayara by giving today to the Center for Tropical & Emerging Global Diseases.

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Trypanosoma cruzi Letm1 is involved in mitochondrial Ca 2+ transport, and is essential for replication, differentiation, and host cell invasion

Donna Huber on June 7, 2021

Leucine zipper-EF-hand containing transmembrane protein 1 (Letm1) is a mitochondrial inner membrane protein involved in Ca2+ and K+ homeostasis in mammalian cells. Here, we demonstrate that the Letm1 orthologue of Trypanosoma cruzi, the etiologic agent of Chagas disease, is important for mitochondrial Ca2+ uptake and release. The results show that both mitochondrial Ca2+ influx and efflux are reduced in TcLetm1 knockdown (TcLetm1-KD) cells and increased in TcLetm1 overexpressing cells, without alterations in the mitochondrial membrane potential. Remarkably, TcLetm1 knockdown or overexpression increases or does not affect mitochondrial Ca2+ levels in epimastigotes, respectively. TcLetm1-KD epimastigotes have reduced growth, and both overexpression and knockdown of TcLetm1 cause a defect in metacyclogenesis. TcLetm1-KD also affected mitochondrial bioenergetics. Invasion of host cells by TcLetm1-KD trypomastigotes and their intracellular replication is greatly impaired. Taken together, our findings indicate that TcLetm1 is important for Ca2+ homeostasis and cell viability in T cruzi.

Guilherme Rodrigo Rm Dos Santos, Ana Catarina Rezende Leite, Noelia Lander, Miguel Angel Chiurillo, Aníbal Eugênio Vercesi, Roberto Docampo. FASEB J. 2021 Jul;35(7):e21685. doi: 10.1096/fj.202100120RR

TbVps41 regulates trafficking of endocytic but not biosynthetic cargo to lysosomes of bloodstream forms of Trypanosoma brucei

Donna Huber on May 27, 2021

The bloodstream stage of Trypanosoma brucei, the causative agent of African trypanosomiasis, is characterized by its high rate of endocytosis, which is involved in remodeling of its surface coat. Here we present evidence that RNAi-mediated expression down-regulation of vacuolar protein sorting 41 (Vps41), a component of the homotypic fusion and vacuole protein sorting (HOPS) complex, leads to a strong inhibition of endocytosis, vesicle accumulation, enlargement of the flagellar pocket (“big eye” phenotype), and dramatic effect on cell growth. Unexpectedly, other functions described for Vps41 in mammalian cells and yeasts, such as delivery of proteins to lysosomes, and lysosome-related organelles (acidocalcisomes) were unaffected, indicating that in trypanosomes post-Golgi trafficking is distinct from that of mammalian cells and yeasts. The essentiality of TbVps41 suggests that it is a potential drug target.

Srinivasan Ramakrishnan, Rodrigo P Baptista, Beejan Asady, Guozhong Huang, Roberto Docampo. FASEB J. 2021 Jun;35(6):e21641. doi: 10.1096/fj.202100487R

Mitochondrial Pyruvate Carrier Subunits Are Essential for Pyruvate-Driven Respiration, Infectivity, and Intracellular Replication of Trypanosoma cruzi

Donna Huber on April 6, 2021

Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of Trypanosoma cruzi, the etiologic agent of Chagas disease. Endogenous tagging of T. cruzi MPC1 (TcMPC1) and TcMPC2 with 3×c-Myc showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of TcMPC1 and TcMPC2 genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized TcMPC1-KO and TcMPC2-KO epimastigotes showed reduced O2 consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O2 consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca2+ uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration. TcMPC1-KO and TcMPC2-KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in TcMPC-KOs. Overall, T. cruzi MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes.

IMPORTANCE Trypanosoma cruzi is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Using the CRISPR/Cas9 technique, we generated individual T. cruzi MPC1 (TcMPC1) and TcMPC2 knockouts and demonstrated that they are essential for pyruvate-driven respiration. Interestingly, although glycolysis was reported as not an important source of energy for the infective stages, MPC was essential for normal host cell invasion and intracellular replication.

Raquel S. Negreiros, Noelia Lander, Miguel A. Chiurillo, Anibal E. Vercesi, Roberto Docampo. mBio Apr 2021, 12 (2) e00540-21; DOI: 10.1128/mBio.00540-21

Ca2+ entry at the plasma membrane and uptake by acidic stores is regulated by the activity of the V‐H+‐ATPase in Toxoplasma gondii

Donna Huber on April 1, 2021

Ca2+ is a universal intracellular signal that regulates many cellular functions. In Toxoplasma gondii, the controlled influx of extracellular and intracellular Ca2+ into the cytosol initiates a signaling cascade that promotes pathogenic processes like tissue destruction and dissemination. In this work we studied the role of proton transport in cytosolic Ca2+ homeostasis and the initiation of Ca2+ signaling. We used a T. gondii mutant of the V-ATPase, a pump previously shown to transport protons to the extracellular medium, control intracellular pH and membrane potential and we show that proton gradients are important for maintaining resting cytosolic Ca2+ at physiological levels and for Ca2+ influx. Proton transport was also important for Ca2+ storage by acidic stores and, unexpectedly, the endoplasmic reticulum. Proton transport impacted the amount of polyphosphate (polyP), a phosphate polymer that binds Ca2+ and concentrate in acidocalcisomes. This was supported by the co-localization of the vacuolar transporter chaperone 4 (VTC4), the catalytic subunit of the VTC complex that synthesizes polyP, with the V-ATPase in acidocalcisomes. Our work show that proton transport regulate plasma membrane Ca2+ transport and control acidocalcisome polyP and Ca2+ content impacting Ca2+ signaling and downstream stimulation of motility and egress in T. gondii.

Andrew J Stasic, Eric J Dykes, Ciro D Cordeiro, Stephen A Vella, Mojtaba S Fazli, Shannon Quinn, Roberto Docampo, Silvia N J Moreno. Mol Microbiol. 2021 Apr 1. doi: 10.1111/mmi.14722

Deletion of a Golgi protein in Trypanosoma cruzi reveals a critical role for Mn2+ in protein glycosylation needed for host cell invasion and intracellular replication

Donna Huber on March 15, 2021

Trypanosoma cruzi is a protist parasite and the causative agent of American trypanosomiasis or Chagas disease. The parasite life cycle in its mammalian host includes an intracellular stage, and glycosylated proteins play a key role in host-parasite interaction facilitating adhesion, invasion and immune evasion. Here, we report that a Golgi-localized Mn2+-Ca2+/H+ exchanger of T. cruzi (TcGDT1) is required for efficient protein glycosylation, host cell invasion, and intracellular replication. The Golgi localization was determined by immunofluorescence and electron microscopy assays. TcGDT1 was able to complement the growth defect of Saccharomyces cerevisiae null mutants of its ortholog ScGDT1 but ablation of TcGDT1 by CRISPR/Cas9 did not affect the growth of the insect stage of the parasite. The defect in protein glycosylation was rescued by Mn2+ supplementation to the growth medium, underscoring the importance of this transition metal for Golgi glycosylation of proteins.

Ramakrishnan S, Unger LM, Baptista RP, Cruz-Bustos T, Docampo R (2021) Deletion of a Golgi protein in Trypanosoma cruzi reveals a critical role for Mn2+ in protein glycosylation needed for host cell invasion and intracellular replication. PLoS Pathog 17(3): e1009399. https://doi.org/10.1371/journal.ppat.1009399

The Inositol Pyrophosphate Biosynthetic Pathway of Trypanosoma cruzi

Donna Huber on January 7, 2021

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