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Author: Donna Huber

Two CTEGD trainees receive AHA fellowships

Photos of Graduate student Baihetiya “Barna” Baierna and postdoctoral fellow Mayara Bertolini
Graduate student Baihetiya “Barna” Baierna and postdoctoral fellow Mayara Bertolini received fellowships from the American Heart Association, supporting their research and education. Both are studying parasites in the University of Georgia’s Center for Tropical and Emerging Global Diseases. (Photos courtesy of CTEGD)

 

Baihetiya “Barna” Baierna, a cellular biology graduate student in Silvia Moreno’s laboratory, received an American Heart Association Pre-doctoral Fellowship. It will fund her training for the next two years as she studies the mitochondrion of Toxoplasma gondii.

Baierna grew up wanting to follow in her mother’s footsteps as a scientist.

“My mom worked for the regional CDC in China and I was interested in science since a young age,” Baierna said.

After completing her undergraduate degree in biochemistry, she was sure she wanted to continue her training in graduate school. After being accepted into the Department of Cellular Biology program, she joined the Moreno Laboratory.

Toxoplasma gondii infects approximately one third of the world human population. The infection can cause serious complications in people with a suppressed immune system. Baierna’s research aims at validating novel T. gondii mitochondrial proteins as novel chemotherapeutic targets for improved chemotherapy of toxoplasmosis. This is important because the present drugs are not effective against the chronic stages of the infection. She has developed novel strategies for the discovery of new mitochondrial proteins and already found a novel enzymatic activity highly divergent from the mammalian counterpart. The outcome of this project will expand the knowledge of the T. gondii mitochondrion, as well as helping with the identification of viable drug targets.

“An AHA Fellowship is a very competitive award, but Barna deserves it and we are very proud of her,” said Moreno.

“Preparing the grant proposal was a great learning experience and it will help me with my career development,” said Baierna, “I’m very happy that it was funded.”

Mayara Bertolini, a post-doctoral fellow in Roberto Docampo’s laboratory, received an American Heart Association Post-doctoral Fellowship. It will support her training for one year.

After receiving her bachelor’s degree, Bertolini obtained her master’s degree in a lab that Docampo had set up in Brazil working on T. cruzi. From there she decided to pursue her Ph.D. at the University of Georgia. She completed her Ph.D. in 2023.

Trypanosoma cruzi is the parasite that causes Chagas disease. At least 6 million people, mostly in South America, are infected with the parasite. T. cruzi is transmitted to humans through the feces of an insect commonly referred to as the kissing bug. While Chagas disease was first discovered in 1909, there is still a lot that is unknown about the biology of T. cruzi. This lack of knowledge has hindered drug development. Bertolini’s project is focused on the role of polyphosphate during the Trypanosoma cruzi life cycle.

“This is the second fellowship from the AHA that Mayara has received. She got a two-year pre-doctoral fellowship before and has done outstanding work,” said Docampo.

“AHA Fellowships are very competitive and I’m thrilled my proposal was selected,” said Bertolini. “In addition to supporting my training, there is support for career development and networking opportunities.”

 

The story originally appeared at https://research.uga.edu/news/two-ctegd-trainees-receive-aha-fellowships/

Genomic and virulence analysis of in vitro cultured Cryptosporidium parvum

Fig 1. Diagramatic section through the hollow fiber bioreactor.
Fig 1. Diagramatic section through the hollow fiber bioreactor.

 

Recent advances in the in vitro cultivation of Cryptosporidium parvum using hollow fiber bioreactor technology (HFB) have permitted continuous growth of parasites that complete all life cycle stages. The method provides access to all stages of the parasite and provides a method for non-animal production of oocysts for use in clinical trials. Here we examined the effect of long-term (>20 months) in vitro culture on virulence-factors, genome conservation, and in vivo pathogenicity of the host by in vitro cultured parasites. We find low-level sequence variation that is consistent with that observed in calf-passaged parasites. Further using a calf model infection, oocysts obtained from the HFB caused diarrhea of the same volume, duration and oocyst shedding intensity as in vivo passaged parasites.

Nigel Yarlett, Mary Morada, Deborah A Schaefer, Kevin Ackman, Elizabeth Carranza, Rodrigo de Paula Baptista, Michael W Riggs, Jessica Kissinger. PLoS Pathog. 2024 Feb 28;20(2):e1011992. doi: 10.1371/journal.ppat.1011992.

Regulation of Calcium entry by cyclic GMP signaling in Toxoplasma gondii

Figure 1. Calcium entry through the plasma membrane of extracellular T. gondii tachyzoites.
Figure 1. Calcium entry through the plasma membrane of extracellular T. gondii tachyzoites.

 

Ca2+ signaling impacts almost every aspect of cellular life. Ca2+ signals are generated through the opening of ion channels that permit the flow of Ca2+ down an electrochemical gradient. Cytosolic Ca2+ fluctuations can be generated through Ca2+ entry from the extracellular milieu or release from intracellular stores. In Toxoplasma gondii, Ca2+ ions play critical roles in several essential functions for the parasite like invasion of host cells, motility and egress. Plasma membrane Ca2+ entry in T. gondii was previously shown to be activated by cytosolic calcium and inhibited by the voltage-operated Ca2+ channel blocker nifedipine. However, Ca2+ entry in T. gondii did not show the classical characteristics of store regulation. In this work, we characterized the mechanism by which cytosolic Ca2+ regulates plasma membrane Ca2+ entry in extracellular T. gondii tachyzoites loaded with the Ca2+ indicator Fura 2. We compared the inhibition by nifedipine with the effect of the broad spectrum TRP channel inhibitor, anthranilic acid or ACA and we find that both inhibitors act on different Ca2+ entry activities. We demonstrate, using pharmacological and genetic tools, that an intracellular signaling pathway engaging cyclicGMP (cGMP), protein kinase G (PKG), Ca2+ and the phosphatidyl inositol phospholipase C (PI-PLC) affects Ca2+ entry and we present a model for crosstalk between cGMP and cytosolic Ca2+ for the activation of T. gondii‘s lytic cycle traits.

Miryam A Hortua Triana, Karla M Márquez-Nogueras, Mojtaba Sedigh Fazli, Shannon Quinn, Silvia N J Moreno. J Biol Chem. 2024 Feb 19:105771. doi: 10.1016/j.jbc.2024.105771

Trainee Spotlight: Corey Rennolds

Corey Rennolds

 

My name is Corey Rennolds, and I’ve been a postdoctoral researcher in Tania Rozario’s lab at UGA since August 2022. I’m originally from Cobb County, GA, where I went to grade school, received my B.S. in Biology from Georgia Tech in 2013, and completed my PhD at the University of Maryland, College Park in 2022.

What made you want to study science?
The big bucks, baby!! More honestly, I enjoy learning how things work for its own sake, and I liked the idea of a career spent always learning more about how things in the world work. I started as an undergraduate in engineering but quickly switched to biology when I realized that I was more interested in natural systems than artificial ones (and that I wasn’t very good at calculus). I have other interests of course, but science translates the most smoothly of those into a stable and rewarding way to make a living.

Why did you choose UGA?
I’m originally from the Atlanta area and spent a lot of time in Athens when I was an undergraduate, even though I went to Tech. Now living and working here feels like coming home for me. I finished my PhD and wanted to continue in a research-oriented direction as a postdoc in an academic setting, and UGA is a big, well-funded institution with a strong biology contingent and several faculty in the ballpark of my more narrow expertise. Altogether, it seemed like a good fit.

What is your project/research focus and why did you choose this research focus?
Dr. Rozario learned during her own postdoctoral work that the rat tapeworm Hymenolepis diminuta requires a population of stem cells maintained in the adult worm in order to grow and regenerate, but there was little information on how these cells are activated, how many different varieties there are, their plasticity, and how they differentiate into mature tissue types. Dr. Rozario wanted to hire a postdoc with experience in transcriptomics and regenerative biology in non-model organisms, which is fortunately my background. I thought the project was really interesting with opportunity to do novel work that would stand out. It also gives me the chance to learn a lot of cutting-edge techniques that can be valuable for my research in the long term.

Have you received any awards or honors?
Aside from the T32 postdoctoral fellowship through the CTEGD, I received a few scholarships, fellowships, and other awards during graduate school, including small research grants from Sigma Xi, the Cosmos Club, and Washington Biologists’ Field Club. I would also be remiss not to mention my first-place finish in the most recent CTEGD chili cook-off.

What are your career goals?
I spent most of graduate school as a TA (tip for prospective graduate students: ask your PI about funding!) and so racked up plenty of experience in teaching and discovered that I really enjoy doing it. I want teaching to be a significant part of my career activities going forward, as opposed to just full-time research. Research-wise, though, I am interested in building an independent research program focused on bridging evolutionary-developmental biology with comparative and ecological physiology. To put it simply, I want to study how living things grow, develop, and repair themselves, where and how they get the resources to do these things, and how those processes are affected by environmental factors, including over evolutionary timescales. Working with intestinal parasites is definitely an interesting and challenging context for thinking about these sorts of broad questions.

What is your favorite thing about UGA and Athens?
Athens is close enough to Atlanta to access its amenities but far enough away to be its own ecosystem free of the sprawl. It’s big enough to have a little of everything, including a vibrant and diverse arts scene, but small enough to get to know most of the people in whatever sphere you want to be involved in. The university offers plenty of opportunities for both intellectual stimulation and less-intellectual partying. The traffic isn’t too bad.

Any advice for a student interested in this field?
Don’t settle too much. It is perfectly fine to have standards during your education and assert yourself when called for. You should study what you enjoy, attend school somewhere you want to be, and work with people you get along with. Not everything will be perfect and you should learn when to compromise, of course, but it’s your life and your career. If something isn’t working out, make a change, and be open to alternative paths—if I didn’t take the initiative to change course when I did, I wouldn’t be a biologist now. Think carefully about what is in your best interest personally and professionally in both the short and long term. Also, learn when to identify opportunities to learn something useful or gain valuable experience. In CTEGD, there are a lot of different technical resources, training and professional development opportunities, and diverse faculty expertise; make use of all these things, it’s what they’re there for!

 

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

In Vitro Antimalarial Activity of Trichothecenes against Liver and Blood Stages of Plasmodium Species

graphical representation of abstract

Trichothecenes (TCNs) are a large group of tricyclic sesquiterpenoid mycotoxins that have intriguing structural features and remarkable biological activities. Herein, we focused on three TCNs (anguidine, verrucarin A, and verrucarol) and their ability to target both the blood and liver stages of Plasmodium species, the parasite responsible for malaria. Anguidine and verrucarin A were found to be highly effective against the blood and liver stages of malaria, while verrucarol had no effect at the highest concentration tested. However, these compounds were also found to be cytotoxic and, thus, not selective, making them unsuitable for drug development. Nonetheless, they could be useful as chemical probes for protein synthesis inhibitors due to their direct impact on parasite synthesis processes.

Prakash T Parvatkar, Steven P Maher, Yingzhao Zhao, Caitlin A Cooper, Sagan T de Castro, Julie Péneau, Amélie Vantaux, Benoît Witkowski, Dennis E Kyle, Roman Manetsch. J Nat Prod. 2024 Jan 23. doi: 10.1021/acs.jnatprod.3c01019.

Diego Huet zeroes in on parasite that affects thousands each year

Diego Huet
Diego Huet, assistant professor in the College of Pharmacy and the Center for Tropical & Emerging Global Diseases, studies parasites that cause disease in both humans and animals. His lab has ramped up a project to better understand the biology of Toxoplasma gondii , an organism carried by cats that is related to the parasite that causes malaria. (Photo by Lauren Corcino)

 

From an early age, Diego Huet has been interested in the unusual and fascinating found in the natural world.

His early encounters with animals, plants and insects nurtured his curiosity about nature. Their striking colors and sometimes strange shapes drew his interest, and even today he continues to capture them through macro photography. It was this fascination that led him to the parasite he studies today.

“I was always drawn to ‘unconventional’ or ‘weird’ science,” said Huet, an assistant professor in the College of Pharmacy’s Department of Pharmaceutical and Biomedical Sciences and member of the Center for Tropical and Emerging Global Diseases. “But I also wanted to be hands on, which is what led me to molecular and cellular biology.”

As a doctoral student, Huet began studying Trypanosoma brucei, a parasite commonly transmitted by the tsetse fly. Wanting to study a different parasite as a postdoctoral researcher, he was torn between studying Plasmodium, which is the causative agent of malaria, and Toxoplasma gondii, a related parasite that is carried by cats. Both parasites, which belong to a group of organisms called apicomplexans, cause diseases in humans and animals, and there remain large knowledge gaps in our basic understanding of them. Ultimately, he chose the latter.

Plasmodium is difficult to manipulate,” Huet said. “Toxoplasma is related to Plasmodium, but is easier to work with because it isn’t as complex, and what we learn about Toxo could also increase our knowledge of Plasmodium.”

Just as yeast and fruit flies are used as model organisms to study human biology, Toxoplasma can be used as a model for shared features of apicomplexan biology.

Besides aiding in the understanding of other parasites of human and veterinary concern, including parasites that cause malaria in tropical and subtropical regions of the world, Toxoplasma gondii also causes human and animal disease. More than 40 million people in the U.S. are estimated to carry T. gondii. Although most never show symptoms, it poses a major health threat to immunocompromised individuals and pregnant women as it can lead to miscarriage and birth defects. Toxoplasmosis, the disease caused by Toxoplasma, is considered a leading cause of death among foodborne illnesses though it can also be transmitted through contact with cat feces.

The Centers for Disease Control and Prevention has it listed as a neglected parasitic infection in the United States and a target for public health action.

Huet joined the faculty at the University of Georgia in 2019 and has developed a robust research program to expand knowledge of the basic biology of Toxoplasma.

Madelaine Usey
Madelaine Usey is a cellular biology graduate student in the Huet Laboratory.

In a recently published study in “mBio”, cellular biology doctoral candidate and Huet Lab member Madelaine Usey looked at proteins critical for mitochondrial function in T. gondii. The mitochondrion is considered the “powerhouse of the cell,” but it is an enzyme called ATP synthase that generates the cellular energy.

“Our findings are really exciting for drug discovery,” Usey said. “Many of the proteins that make up the ATP synthase are different in Toxoplasma compared to other organisms. In this study, we were able to figure out what two of those novel subunits are doing—they act as scaffolding for this enormous ATP synthase complex.”

These proteins are unique to Toxoplasma and could be used in drug discovery as targets since they are important for mitochondrial functioning.

Another project in Huet’s laboratory, which recently received funding through a grant from the National Institute of General Medical Sciences, investigates how organelles within the parasite communicate.

“Traditionally, we thought organelles send and receive calcium and other metabolites in much the same way we receive a package through the mail,” Huet said. “Cells form vesicles to transport materials to specific locations within the cell. The vesicles are labeled with proteins that act like a postal address, telling the vesicle where to go.”

However, cells can also exchange material through another process.

“When the organelles’ membranes get close together, they form what is called a membrane contact site,” Huet said. “In this case it is more like one organelle hand delivers the package to another.”

A membrane contact site is a specialized protein structure that organelles use for intracellular communication. However, it is not a well understood structure in apicomplexans. In addition, these parasites have additional organelles not found in traditional models like humans and yeast, so Huet is trying to understand how the organellar communication is happening in apicomplexans using Toxoplasma as a model.

Identifying such proteins and their functions could lead to better drug targets and better drug treatments, which all the neglected parasitic diseases need.

“Toxo’s genome isn’t well annotated,” Huet said. “Finding membrane contact site proteins is an arduous task—it’s a goal of my lab to identify some of them and their involvement in Toxoplasma membrane contact sites.”

 

This article was first published at https://research.uga.edu/news/diego-huet-zeroes-in-on-parasite-that-affects-thousands-each-year/

Genetic crosses within and between species of Cryptosporidium

Figure 1 PheRS can be used as a selection marker for stable transgenesis.
PheRS can be used as a selection marker for stable transgenesis.

Parasites and their hosts are engaged in reciprocal coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium, genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach, multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing Cryptosporidium parvum, a parasite of cattle and humans, and Cryptosporidium tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity.

Sebastian Shaw, Ian S Cohn, Rodrigo P Baptista, Guoqin Xia, Bruno Melillo, Fiifi Agyabeng-Dadzie, Jessica C Kissinger, Boris Striepen. Proc Natl Acad Sci USA. 2024 Jan 2;121(1):e2313210120. doi: 10.1073/pnas.2313210120.

Aptamer-Based Imaging of Polyisoprenoids in the Malaria Parasite

Figure 1. Schemes of the positive and negative selection cycles are illustrated.
Figure 1. Schemes of the positive and negative selection cycles are illustrated.

 

Dolichols are isoprenoid end-products of the mevalonate and 2C-methyl-D-erythritol-4-phosphate pathways. The synthesis of dolichols is initiated with the addition of several molecules of isopentenyl diphosphate to farnesyl diphosphate. This reaction is catalyzed by a cis-prenyltransferase and leads to the formation of polyprenyl diphosphate. Subsequent steps involve the dephosphorylation and reduction of the α-isoprene unit by a polyprenol reductase, resulting in the generation of dolichol. The size of the dolichol varies, depending on the number of isoprene units incorporated. In eukaryotes, dolichols are synthesized as a mixture of four or more different lengths. Their biosynthesis is predicted to occur in the endoplasmic reticulum, where dolichols play an essential role in protein glycosylation. In this study, we have developed a selection of aptamers targeting dolichols and enhanced their specificity by incorporating fatty acids for negative selection. One aptamer showed high enrichment and specificity for linear polyisoprenoids containing at least one oxygen atom, such as an alcohol or aldehyde, in the α-isoprene unit. The selected aptamer proved to be a valuable tool for the subcellular localization of polyisoprenoids in the malaria parasite. To the best of our knowledge, this is the first time that polyisoprenoids have been localized within a cell using aptamer-based imaging techniques.

Flavia M Zimbres, Emilio F Merino, Grant J Butschek, Joshua H Butler, Frédéric Ducongé, Maria B Cassera. Molecules. 2023 Dec 28;29(1):178. doi: 10.3390/molecules29010178.

Inherently Reduced Expression of ASC Restricts Caspase-1 Processing in Hepatocytes and Promotes Plasmodium Infection

Fig. 1 Inherently reduced expression of pro–caspase-1 and ASC in hepatocytes.
Inherently reduced expression of pro–caspase-1 and ASC in hepatocytes.

 

Inflammasome-mediated caspase-1 activation facilitates innate immune control of Plasmodium in the liver, thereby limiting the incidence and severity of clinical malaria. However, caspase-1 processing occurs incompletely in both mouse and human hepatocytes and precludes the generation of mature IL-1β or IL-18, unlike in other cells. Why this is so or how it impacts Plasmodium control in the liver has remained unknown. We show that an inherently reduced expression of the inflammasome adaptor molecule apoptosis-associated specklike protein containing CARD (ASC) is responsible for the incomplete proteolytic processing of caspase-1 in murine hepatocytes. Transgenically enhancing ASC expression in hepatocytes enabled complete caspase-1 processing, enhanced pyroptotic cell death, maturation of the proinflammatory cytokines IL-1β and IL-18 that was otherwise absent, and better overall control of Plasmodium infection in the liver of mice. This, however, impeded the protection offered by live attenuated antimalarial vaccination. Tempering ASC expression in mouse macrophages, on the other hand, resulted in incomplete processing of caspase-1. Our work shows how caspase-1 activation and function in host cells are fundamentally defined by ASC expression and offers a potential new pathway to create better disease and vaccination outcomes by modifying the latter.

Camila Marques-da-Silva, Clyde Schmidt-Silva, Rodrigo P Baptista, Samarchith P Kurup. J Immunol. 2023 Dec 27:ji2300440. doi: 10.4049/jimmunol.2300440. Online ahead of print.

On the origin and evolution of the mosquito male-determining factor Nix

Background and workflow.

The mosquito family Culicidae is divided into two subfamilies named the Culicinae and Anophelinae. Nix, the dominant male-determining factor, has only been found in the culicines Aedes aegypti and Ae. albopictus, two important arboviral vectors that belong to the subgenus Stegomyia. Here we performed sex-specific whole-genome sequencing and RNAseq of divergent mosquito species and explored additional male-inclusive datasets to investigate the distribution of Nix. Except for the Culex genus, Nix homologs were found in all species surveyed from the Culicinae subfamily, including 12 additional species from three highly divergent tribes comprising 4 genera, suggesting Nix originated at least 133-165 MYA. Heterologous expression of one of three divergent Nix ORFs in Ae. aegypti resulted in partial masculinization of genetic females as evidenced by morphology and doublesex splicing. Phylogenetic analysis suggests Nix is related to femaleless (fle), a recently described intermediate sex-determining factor found exclusively in anopheline mosquitoes. Nix from all species has a conserved structure, including three RNA-recognition motifs (RRMs), as does fle. However, Nix has evolved at a much faster rate than fle. The RRM3 of both Nix and fle are distantly related to the single RRM of a widely distributed and conserved splicing factor transformer-2 (tra2). RRM3-based phylogenetic analysis suggests this domain in Nix and fle may have evolved from tra2 or a tra2-related gene in a common ancestor of mosquitoes. Our results provide insights into the evolution of sex-determination in mosquitoes and will inform broad applications of mosquito-control strategies based on manipulating sex ratios towards the non-biting males.

James K Biedler, Azadeh Aryan, Yumin Qi, Aihua Wang, Ellen O Martinson, Daniel A Hartman, Fan Yang, Atashi Sharma, Katherine S Morton, Mark Potters, Chujia Chen, Stephen L Dobson, Gregory D Ebel, Rebekah C Kading, Sally Paulson, Rui-De Xue, Michael R Strand, Zhijian Tu. Mol Biol Evol. 2023 Dec 21:msad276. doi: 10.1093/molbev/msad276. Online ahead of print.