Toxoplasma gondii Archives - Page 3 of 4 - Center for Tropical and Emerging Global Diseases

A terminal α3-galactose modification regulates an E3 ubiquitin ligase subunit in Toxoplasma gondii

Donna Huber on May 15, 2020

Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is modified by a prolyl hydroxylase that mediates O₂-regulation of the social amoeba Dictyostelium and the parasite Toxoplasma gondii. The full effect of hydroxylation requires modification of the hydroxyproline by a pentasaccharide that, in Dictyostelium, influences Skp1 structure to favor assembly of Skp1/F-box protein subcomplexes. In Toxoplasma, the presence of a contrasting penultimate sugar assembled by a different glycosyltransferase enables testing of the conformational control model. To define the final sugar and its linkage, here we identified the glycosyltransferase that completes the glycan and found that it is closely related to glycogenin, an enzyme that may prime glycogen synthesis in yeast and animals. However, the Toxoplasma enzyme catalyzes formation of a Galα1,3Glcα- rather than the Glcα1,4Glcα- linkage formed by glycogenin. Kinetic and crystallographic experiments showed that the glycosyltransferase Gat1 is specific for Skp1 in Toxoplasma and also in another protist, the crop pathogen Pythium ultimum. The fifth sugar is important for glycan function as indicated by the slow-growth phenotype of gat1Δ parasites. Computational analyses indicated that, despite the sequence difference, the Toxoplasma glycan still assumes an ordered conformation that controls Skp1 structure and revealed the importance of non-polar packing interactions of the fifth sugar. The substitution of glycosyltransferases in Toxoplasma and Pythium by an unrelated bifunctional enzyme that assembles a distinct but structurally compatible glycan in Dictyostelium is a remarkable case of convergent evolution, that emphasizes the importance of the terminal α-galactose and establishes the phylogenetic breadth of Skp1 glycoregulation.

Msano Mandalasi, Hyun W. Kim, David Thieker, M. Osman Sheikh, Elisabet Gas-Pascual, Kazi Rahman, Peng Zhao, Nitin G. Daniel, Hanke van der Wel, H. Travis Ichikawa, John N. Glushka, Lance Wells, Robert J. Woods, Zachary A. Wood, and Christopher M. West. J Biol Chem. 2020 May 15. pii: jbc.RA120.013792. doi: 10.1074/jbc.RA120.013792.

Trainee Spotlight: Edwin Pierre Louis

Donna Huber on March 23, 2020

Edwin Pierre Louis is a pre-doctoral trainee in the laboratory of Dr. Drew Etheridge. Originally from Haiti, he immigrated to the US to attend the University of Florida (UF), where he graduated with a Bachelor of Science in Biochemistry Molecular Biology. After earning his degree at UF, Edwin accepted a position as a biological scientist in the UF Center of Excellence for Regenerative Health Biotechnology, with a focus on gene and cell based therapeutic development, where he worked for three years. There, he first discovered his love of host-pathogen interactions as a biological scientist working under the supervision of Dr. Richard Snyder for the component Florida Biologix at this center and later merged to create Brammer Bio which was subsequently acquired by Thermo Fisher Scientific. During this time in industry, he realized that to improve his scientific capacities he would need to continue his studies by pursuing a graduate degree. As part of his preparations to apply to a graduate program, he joined the UGA post-baccalaureate PREP program whose mission is to prepare students interested in a graduate degree for the application process. During this time, he was granted the opportunity to join Dr. Michael Terns’ laboratory for a year where he investigated the molecular mechanism of CRISPR-Cas based viral defense in Streptococcus thermophilus as well as prime adaptation events in the type II-A CRISPR-Cas system.

Since attending UGA, Edwin has been awarded both the Gateway to Graduate School Bridge Program and the Graduate Scholars Leadership, Engagement and Development Program (GS LEAD) scholarships sponsored by the National Science Foundation (NSF).

What is your research focus and why are you interested in the topic?

Broadly, my key research interests center around how organisms like viruses and parasites manipulate their host cell in order to grow and propagate. My current project is focused on elucidating how the protozoan pathogen Toxoplasma gondii is able to use secreted protein effectors to manipulate its host cells functions.

Why did you choose UGA?

I chose to study at the University of Georgia, in part, because of my excellent post-baccalaureate experience in the PREP program. It was evident from my interactions that UGA excels at fostering a productive relationship between students and faculty. Regardless of any faculty member’s relationship to the students, there was a sustained willingness for faculty to give of their time in order to see the students succeed. I also decided to pursue my PhD at UGA because of the cutting-edge research and in particular the collection of outstanding parasitologists that is uniquely found in the Center for Tropical and Emerging Global Diseases (CTEGD).

What are your future professional plans?

As I continue my graduate studies on host pathogen interaction, I plan to do some post-doctoral trainings to augment my apprenticeship and ultimately become an independent scientist to lead my own research group. I also hope to be able to give back to the local community that has contributed so much to my own personal success by donating my time and knowledge to mentor young budding scientists especially those from underprivileged homes and/or underdeveloped countries.

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Genetic Indicators for Calcium Signaling Studies in Toxoplasma gondii

Donna Huber on November 23, 2019

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.

The Vacuolar Zinc Transporter TgZnT Protects Toxoplasma gondii from Zinc Toxicity

Donna Huber on May 22, 2019

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.

Video: Distinguished Research Professor Silvia Moreno

Donna Huber on March 28, 2019

Silvia Moreno, a Distinguished Research Professor in the Department of Cellular Biology and a member of CTEGD, studies Toxoplasma gondii, an apicomplexan parasite that infects almost one-third of the world population.

Trainee Spotlight: Stephen Vella

Donna Huber on September 23, 2018

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.

Trainee Spotlight: Karla Márquez Nogueras

Donna Huber on January 22, 2018

trainee Karla Márquez Nogueras

NIH T32 Trainee Karla M. Márquez Nogueras is in her 4^th 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

“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.

Trainee Spotlight: Msano Mandalasi

Donna Huber on October 31, 2017

trainee Msano Mandalasi
Msano Mandalasi, a post-doctoral trainee in Chris West‘s laboratory, is originally from Malawi, (located in southeastern Africa) and obtained her bachelor’s degree in Chemistry from the University of Malawi. After graduation, she worked briefly for the University of Malawi and then came to the US to obtain a Master’s degree in Chemistry. Later, she enrolled in a doctoral graduate program at the University of Maryland Eastern Shore where she graduated in 2012. She spent two years teaching undergraduate Chemistry before deciding to get back into research. She joined Dr. West’s group while he was at the University of Oklahoma and moved with the lab to the University of Georgia.

Msano’s research focus

The focus of Msano’s project is on the role of prolyl hydroxylation and glycosylation of E3 Ubiquitin ligase on Toxoplasma growth.

With a research background mostly in chemistry and biochemistry, her graduate research introduced her to some aspect of parasitology working on Schistosome glycobiology. However, she did not have a strong background in molecular biology prior to joining the West lab. This current project merges glycobiology and molecular biology and also extends some parasitology studies, thus giving her the opportunity to learn molecular biology and parasitology to complement her chemistry background. A combination of this expert knowledge will benefit her to address the research objectives on her Toxoplasma project.

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. Msano plans to use her capstone experience to give oral presentations at scientific meetings, to publish some of the studies conducted within this time period, and interact with other trainees in the program.

T32 fellowship helps trainees achieve their goals

“Through the funding provided by the T32 Training Grant, I will be able to address research questions that should lead to launching my own area of research,” said Msano.

Msano hopes to run her own independent research program in academia one day.

Support trainees like Msano Mandalasi by giving to the Center for Tropical & Emerging Global Diseases

A Double Hit Strategy May Provide Better Treatment for Toxoplasmosis

Donna Huber on October 23, 2017

Zhu Hong Li is the lead author on the study.

Silvia Moreno and her research team at the University of Georgia’s Center for Tropical & Emerging Global Diseases and Department of Cellular Biology provided evidence that it is possible to develop a drug combination that acts synergistically by inhibiting host and parasite enzymes in a recently published article in Antimicrobial Agents and Chemotherapy.

What is toxoplasmosis?

Toxoplasmosis is caused by the pervasive intracellular Apicomplexan parasite Toxoplasma gondii. The parasite is found throughout the world and can infect humans and a number of animal species. In the U.S., people may contract it by consuming undercooked meats, especially pork, lamb, venison, or through contact with contaminated cat feces.

Human infections are usually asymptomatic but the parasite can persist in the form of tissue cysts. It has been estimated that 30–50% of the global population may be chronically infected with Toxoplasma. The immune system of a healthy individual can control the infection, but it can reactivate when there is immunosuppression due to organ transplant, cancer chemotherapy, or in people infected with HIV.

Toxoplasmosis is especially dangerous to the unborn fetus when the mother becomes infected during pregnancy as it can result in miscarriage or stillbirth. Surviving infants can suffer from visual, hearing, motor, cognitive, and other problems.

Some strains of T. gondii can cause severe ocular disease in people with a healthy immune system. Current drug therapies do not prevent disease progression that leads to blindness in ocular toxoplasmosis patients.

Toxoplasmosis represents a serious public health problem and no preventative or therapeutic vaccine is available for humans.

Need for better drug treatments for toxoplasmosis

Drugs presently used against toxoplasmosis do not eradicate chronic infection and as many as half of treated patients do not respond to the therapy. Additionally, a large number of people have an allergic reaction to the current treatment option. Furthermore, some the current drugs have recently become very expensive.

There is a need for safe and effective treatment.

Moreno and her team study the isoprenoid pathway to identify new drug targets. Isoprenoids are lipid compounds with many important functions. One particular step in this pathway has been identified as essential in T. gondii. A drug targeting this pathway could kill the parasite.

Drug combination may provide more effective and less expensive treatment

Moreno’s group proposes a double hit strategy of combining inhibitors of host and parasite pathways as a novel approach against toxoplasmosis. They have found a synergistic effect by combining new and potent sulfur-containing bisphosphonates, as well as other commercially available bisphosphonates, with several statins against a lethal infection of T. gondii using a virulence mouse model.

Bisphosphonates are widely used for the treatment of bone disorders. Previous studies by Moreno and her colleagues have shown that bisphosphonates inhibit the growth of a variety of protozoan parasites like T. gondii. There are a number of commercially available bisphosphonate drugs.

Statins are a class of drugs typically prescribed to lower cholesterol. They work by blocking a particular enzyme known as 3-hydroxy-methylglutaryl-coenzyme A reductase. As with the bisphosphonates, there are already a number of commercially available statins.

Bisphosphonates alone have been very effective when treating a lethal infection of T. gondii in mice. However, Moreno’s team found that combining bisphosphonates with the statin atorvastatin (Lipitor) was almost 3 times more effective under similar conditions of infection and treatment. Additionally, they found very low doses of both drugs could be used for treatment, which would significantly decrease the potential for adverse side effects.

This double hit strategy may be the key to effective treatment because the parasite not only makes its own isoprenoids, but it can also import them from the host. The ability to manipulate the host cell for its own benefit poses a challenge for drug development against toxoplasmosis. Therefore, inhibiting the host from producing this material along with inhibiting the parasite’s ability to create isoprenoids is an interesting and novel strategy for drug development.

Further studies for this novel therapeutic approach needed

This study demonstrates that early treatment is key to the cure of infection with a particular strain of T. gondii for acute infection. Since current treatments often fail to cure chronic infection Moreno and her group will next test this combination strategy in an established chronic infection mouse model.

Furthermore, Moreno predicts that this double-hit strategy of inhibiting both host and parasite pathways will work for other intracellular Apicomplexan parasites, such as the malaria-causing Plasmodium parasite. Additional studies will be needed to test this hypothesis.

An online version of this study is available: Li Z-H, Li C, Szajnman SH, Rodriguez JB, Moreno SNJ. 2017. Synergistic activity between statins and bisphosphonates against acute experimental toxoplasmosis. Antimicrob Agents Chemother 61:e02628-16. https://doi.org/10.1128/AAC.02628-16

Keep Up the Good (Bench) Work!

Donna Huber on August 1, 2016

Tag: Toxoplasma gondii