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Tag: Silvia Moreno

An Endoplasmic Reticulum CREC Family Protein Regulates the Egress Proteolytic Cascade in Malaria Parasites

The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be required for biogenesis and secretion of egress-related organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparum endoplasmic reticulum-resident calcium-binding protein (PfERC), a member of the CREC family. Knockdown of the PfERC gene showed that this gene is essential for asexual growth of P. falciparum Analysis of the intraerythrocytic life cycle revealed that PfERC is essential for parasite egress but is not required for protein trafficking or calcium storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease SUB1. Using double mutant parasites, we showed that PfERC is required for the proteolytic maturation of the essential aspartic protease plasmepsin X, which is required for SUB1 cleavage. Further, we showed that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish that the ER-resident CREC family protein PfERC is a key early regulator of the egress proteolytic cascade of malaria parasites.

IMPORTANCE The divergent eukaryotic parasites that cause malaria grow and divide within a vacuole inside a host cell, which they have to break open once they finish cell division. The egress of daughter parasites requires the activation of a proteolytic cascade, and a subtilisin-like protease initiates a proteolytic cascade to break down the membranes blocking egress. It is assumed that the parasite endoplasmic reticulum plays a role in this process, but the proteins in this organelle required for egress remain unknown. We have identified an early ER-resident regulator essential for the maturation of the recently discovered aspartic protease in the egress proteolytic cascade, plasmepsin X, which is required for maturation of the subtilisin-like protease. Conditional loss of PfERC results in the formation of immature and inactive egress proteases that are unable to breakdown the vacuolar membrane barring release of daughter parasites.

Manuel A. Fierro, Beejan Asady, Carrie F. Brooks, David W. Cobb, Alejandra Villegas, Silvia N. J. Moreno, Vasant Muralidharan. mBio. 2020 Feb 25;11(1). pii: e03078-19. doi: 10.1128/mBio.03078-19.

Genetic Indicators for Calcium Signaling Studies in Toxoplasma gondii

Fluctuations of the cytosolic calcium ion (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. Cells express a sophisticated set of mechanisms to balance the cytosolic Ca2+ levels and the signals that elevate Ca2+ in the cytosol are compensated by mechanisms that reduce it. Alterations in Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, such as heart failure or neuronal death.

The genetic tractability of Toxoplasma gondii and the availability of genetic tools enabled the use of Genetically Encoded Calcium Indicators (GECIs) expressed in the cytoplasm, which started a new era in the studies of Toxoplasma calcium signaling. It was finally possible to see Ca2+ oscillations prior to exit of the parasite from host cells. Years after Endo et al showed that ionophores triggered egress, the assumption that oscillations occur prior to egress from host cells has been validated by experiments using GECIs. GECIs allowed the visualization of specific Ca2+ signals in live intracellular parasites and to distinguish these signals from host cell calcium fluctuations. In this chapter we present an overview describing “tried and true” methods of our lab who pioneered the first use of GECI’s in Toxoplasma, including GECI choice, methodology for transfection and selection of ideal clones, their characterization, and the use of GECI-expressing parasites for fluorometric and microscopic analysis.

Stephen A. Vella, Abigail Calixto, Beejan Asady, Zhu-Hong Li, Silvia N. J. Moreno. Methods Mol Biol. 2020;2071:187-207. doi: 10.1007/978-1-4939-9857-9_11.

Synthesis and biological evaluation of 1-alkylaminomethyl-1,1-bisphosphonic acids against Trypanosoma cruzi and Toxoplasma gondii

As an extension of our project aimed at the search for new chemotherapeutic agents against Chagas disease and toxoplasmosis, several 1,1-bisphosphonates were designed, synthesized and biologically evaluated against Trypanosoma cruzi and Toxoplasma gondii, the etiologic agents of these diseases, respectively. In particular, and based on the antiparasitic activity exhibited by 2-alkylaminoethyl-1,1-bisphosphonates targeting farnesyl diphosphate synthase, a series of linear 2-alkylaminomethyl-1,1-bisphosphonic acids (compounds 2133), that is, the position of the amino group was one carbon closer to the gem-phosphonate moiety, were evaluated as growth inhibitors against the clinically more relevant dividing form (amastigotes) of T. cruzi. Although all of these compounds resulted to be devoid of antiparasitic activity, these results were valuable for a rigorous SAR study. In addition, unexpectedly, the synthetic designed 2-cycloalkylaminoethyl-1,1-bisphosphonic acids 4749 were free of antiparasitic activity. Moreover, long chain sulfur-containing 1,1-bisphosphonic acids, such as compounds 545659, turned out to be nanomolar growth inhibitors of tachyzoites of T. gondii. As many bisphosphonate-containing molecules are FDA-approved drugs for the treatment of bone resorption disorders, their potential nontoxicity makes them good candidates to control American trypanosomiasis and toxoplasmosis.

Tamila Galaka, Bruno N. Falcone, Catherine Li, Sergio H. Szajnman, Silvia N.J. Moreno, Roberto Docampo, Juan B.Rodriguez. Bioorg Med Chem. 2019 Jul 4. pii: S0968-0896(19)30740-0. doi: 10.1016/j.bmc.2019.07.004.

The Vacuolar Zinc Transporter TgZnT Protects Toxoplasma gondii from Zinc Toxicity

Zinc (Zn2+) is the most abundant biological metal ion aside from iron and is an essential element in numerous biological systems, acting as a cofactor for a large number of enzymes and regulatory proteins. Zn2+ must be tightly regulated, as both the deficiency and overabundance of intracellular free Zn2+ are harmful to cells. Zn2+ transporters (ZnTs) play important functions in cells by reducing intracellular Zn2+ levels by transporting the ion out of the cytoplasm. We characterized a Toxoplasma gondii gene (TgGT1_251630, TgZnT), which is annotated as the only ZnT family Zn2+ transporter in T. gondii. TgZnT localizes to novel vesicles that fuse with the plant-like vacuole (PLV), an endosome-like organelle. Mutant parasites lacking TgZnT exhibit reduced viability in in vitro assays. This phenotype was exacerbated by increasing zinc concentrations in the extracellular media and was rescued by media with reduced zinc. Heterologous expression of TgZnT in a Zn2+-sensitive Saccharomyces cerevisiae yeast strain partially restored growth in media with higher Zn2+ concentrations. These results suggest that TgZnT transports Zn2+ into the PLV and plays an important role in the Zn2+tolerance of T. gondii extracellular tachyzoites.

IMPORTANCE Toxoplasma gondii is an intracellular pathogen of human and animals. T. gondii pathogenesis is associated with its lytic cycle, which involves invasion, replication, egress out of the host cell, and invasion of a new one. T. gondii must be able to tolerate abrupt changes in the composition of the surrounding milieu as it progresses through its lytic cycle. We report the characterization of a Zn2+ transporter of T. gondii (TgZnT) that is important for parasite growth. TgZnT restored Zn2+ tolerance in yeast mutants that were unable to grow in media with high concentrations of Zn2+. We propose that TgZnT plays a role in Zn2+ homeostasis during the T. gondii lytic cycle.

Nathan M. Chasen, Andrew J. Stasic, Beejan Asady, Isabelle Coppens, Silvia N. J. Moreno. 2019. mSphere.; 4(3). pii: e00086-19. doi: 10.1128/mSphere.00086-19.

The Toxoplasma Vacuolar H+-ATPase Regulates Intracellular pH and Impacts the Maturation of Essential Secretory Proteins

Vacuolar-proton ATPases (V-ATPases) are conserved complexes that couple the hydrolysis of ATP to the pumping of protons across membranes. V-ATPases are known to play diverse roles in cellular physiology. We studied the Toxoplasma gondiiV-ATPase complex and discovered a dual role of the pump in protecting parasites against ionic stress and in the maturation of secretory proteins in endosomal-like compartments. Toxoplasma V-ATPase subunits localize to the plasma membrane and to acidic vesicles, and characterization of conditional mutants of the a1 subunit highlighted the functionality of the complex at both locations. Microneme and rhoptry proteins are required for invasion and modulation of host cells, and they traffic via endosome-like compartments in which proteolytic maturation occurs. We show that the V-ATPase supports the maturation of rhoptry and microneme proteins, and their maturases, during their traffic to their corresponding organelles. This work underscores a role for V-ATPases in regulating virulence pathways.

Andrew J.Stasic, Nathan M.Chasen, Eric J.Dykes, Stephen A.Vella, Beejan Asady, Vincent J. Starai, Silvia N.J. Moreno. 2019. Cell Rep.;27(7):2132-2146.e7. doi: 10.1016/j.celrep.2019.04.038.

Trainee Spotlight: Stephen Vella

Stephen Vella is a Ph.D. trainee in Silvia Moreno’s laboratory. He is originally from Indiana where he received his B.S. in microbiology at Indiana University. In his first year at UGA, he was awarded an Excellence in Graduate Recruitment Award and a Provost’s Scholars of Excellence Award Fellowship. He has also been awarded an Outstanding Poster Presentation at the Molecular Parasitology Meeting in 2016. And in 2017, he was awarded a T32 fellowship from CTEGD.

Silvia Moreno named Corresponding Member of the Latin American Academy of Sciences

Silvia Moreno

Silvia Moreno was recently named Corresponding Member of the Latin American Academy of Sciences. She is a distinguished research professor in the department of cellular biology and also serves as director of CTEGD’s NIH-funded Training Grant in Interdisciplinary Parasitology, Vector Biology, Emerging Diseases. Her research team works with Toxoplasma gondii, an apicomplexan parasite that infects almost one-third of the world population.

The Academia de Ciencias de América Latina, created in 1982 under the sponsorship of the Pontifical Academy of Sciences, promotes and contributes to the advancement of mathematical, physical, chemical, earth, and life sciences, and to their application to the development and integration of Latin America and the Caribbean. The Academy promotes cooperation among scientific institutions and the exchange of persons and scientific knowledge for the integration of Latin American and the Caribbean; studies of sciences policy that contribute to the stable and continuous development of the countries of Latin American and the Caribbean; science at different educational levels and among the entire population.

 

Silvia Moreno named Distinguished Research Professor

Silvia J. Morenoa professor in the cellular biology department and director for the NIH Training Grant in Tropical and Emerging Global Diseases, is recognized for her studies on calcium signaling in parasitic protozoa.

Her work defined the link between calcium signaling and pathogenesis of infectious organisms. Her research focuses on Toxoplasma gondii, a pathogen that infects one-third of the world population. She and her team discovered mechanisms of calcium signaling in parasites and novel compartments that store calcium that are different from those present in mammalian cells. Her laboratory developed new genetic tools to study calcium that could be used for high-throughput assays to find new pharmacological agents for the potential treatment of parasitic diseases.

Based on another fundamental discovery from her lab, that Toxoplasma takes specific nutrients from its host, she proposed the development of therapeutics that combine host-encoded and parasite-encoded functions as a novel approach for chemotherapy.

Trainee Spotlight: Karla Márquez Nogueras

trainee Karla Márquez Nogueras

NIH T32 Trainee Karla M. Márquez Nogueras is in her 4th year of graduate training in Silvia Moreno‘s laboratory. Before entering the Ph.D. program at UGA, she taught for a semester at Turabo University in Puerto Rico, teaching undergraduate courses like Introduction to Microbiology and Human Anatomy. She has a Bachelor’s degree in Industrial Microbiology and a Master’s in Science where she focused on generating renewable energy systems using methane generated by anaerobic microbial communities.

Karla’s research focus

Karla’s project focuses on calcium signaling in Toxoplasma gondii. Calcium is a universal signal molecule and very little is known about calcium signaling in T. gondii, even considering that all steps of the parasite’s lytic cycle are regulated by calcium. Calcium is highly regulated by Toxoplasma, specially upon exit from host cells and the surrounding calcium changes from very low levels inside the host cell to the high concentration found in the extracellular environment. In order to shed light into the mechanisms involved and to discover the molecules involved they are studying two key aspects: the calcium channels that could be responsible for calcium entry into the cytosol and the calcium binding proteins that could regulate them.

“When I first entered grad school my research goals were different,” said Karla. “During my rotation in Dr. Moreno’s lab, I became fascinated by the biology of Toxoplasma and by how little is known about calcium signaling in T. gondii. As a scientist, I became very curious and interested in finding more about these signaling pathways and I decided to change my research focus.”

Trainee capstone experience

Each T32 trainee is provided with the opportunity to complete a capstone experience at the end of their fellowship. This experience allows for an extended visit to a collaborator’s laboratory or travel to a scientific meeting where they present their research and interact with colleagues.

“I was invited to the University of Puerto Rico to present my research project and discuss graduate and fellowship opportunities available at UGA. I would be presenting at an undergraduate event organized by the University.”

In addition, she would like to visit the laboratory of Dr. Ivana Kuo at Northwestern University to study the function of two TRP channels that Karla is characterizing. Dr. Kuo uses lipid layers and regular patch-clamp to characterize intracellular and plasma membrane channels. Using this system Karla hopes to understand the physiology of these channels that are important for calcium signaling in T. gondii.

T32 Fellowship helps trainees achieve their goals

“The fellowship will provide me with the necessary experience and opportunities for me to develop the skills to become a better scientist.”

Karla would like to go back to Puerto Rico and establish her own research lab. She would like to have the opportunity to train and give students the same opportunities that were given to her during her Ph.D. training.

“All the skills gained throughout this two years will prepare me for my ultimate goal which is to have my own research lab.”

Learn more about our trainee fellowship programs.