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.
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.
CTEGD member Chet Joyner and CTEGD director Dennis Kyle receive a grant from the Bill & Melinda Gates Foundation for malaria drug development and testing
Two UGA researchers are working to make it easier to develop effective treatments for malaria, a disease that sickens millions worldwide and kills hundreds of thousands each year.
In tropical climates around the globe, malaria poses a grave risk to already vulnerable populations. In 2019, the World Health Organization estimated that there were 229 million clinical cases of malaria worldwide and 409,000 deaths, usually in children below the age of five.
Currently, developing and testing drugs for malaria requires scientists to work in areas where the disease is prevalent or to work with expensive, hard-to-source equipment. Chester Joyner, an Assistant Professor in the Center for Vaccines and Immunology, and Dennis Kyle, Professor of Infectious Diseases and Cellular Biology, are working to reduce those barriers to malaria drug testing and development.
Joyner and Kyle aim to establish systems that rely on equipment most researchers can obtain: a petri dish. If successful, Joyner says this new culture system will reduce costs and be distributed more easily to advance drug and vaccine research. The University of Georgia College of Veterinary Medicine received a grant for malaria drug development and testing from the Bill & Melinda Gates Foundation.
Worldwide, there are many malaria-causing parasites that result in varying degrees of illness. Joyner and Kyle’s research focuses on defeating one of the most challenging: Plasmodium vivax. Unlike many other malaria parasites, P. vivax can lie dormant in the livers of its hosts—allowing the infected to travel abroad completely unaware that they’re carrying a potentially deadly passenger.
“Most infections with P. vivax are not due to new infections,” says Joyner. “These infections come from this parasite activating and potentially causing disease and sustaining transmission.”
Malaria disproportionately affects the poorest communities in the world, creating a cycle of disease and poverty that current treatments have improved but been unable to stop. However, treating the dormant forms of P. vivax has been particularly challenging because they can cause more harm than good in at-risk populations like pregnant women and people with certain blood conditions.
“We want researchers to have access to technologies to study P. vivax and develop new approaches to control and eliminate this parasite,” Joyner explains.
Background: Evidence indicates that whereas repeated rounds of mass drug administration (MDA) programs have reduced schistosomiasis prevalence to appreciable levels in some communities referred to here as responding villages (R). However, prevalence has remained high or less than anticipated in other areas referred to here as persistent hotspot villages (PHS). Using a cross-sectional quantitative approach, this study investigated the factors associated with sustained high Schistosoma mansoni prevalence in some villages despite repeated high annual treatment coverage in western Kenya.
Method: Water contact sites selected based on observation of points where people consistently go to collect water, wash clothes, bathe, swim or play (young children), wash cars and harvest sand were mapped using hand-held smart phones on the Commcare platform. Quantitative cross-sectional surveys on behavioral characteristics were conducted using interviewer-based semi-structured questionnaires administered to assess water usage/contact patterns and open defecation. Questionnaires were administered to 15 households per village, 50 pupils per school and 1 head teacher per school. One stool and urine sample was collected from 50 school children aged 9-12 year old and 50 adults from both responding (R) and persistent hotspot (PHS) villages. Stool was analyzed by the Kato-Katz method for eggs of S. mansoni and soil-transmitted helminths. Urine samples were tested using the point-of-care circulating cathodic antigen (POC-CCA) test for detection of S. mansoni antigen.
Results: There was higher latrine coverage in R (n = 6) relative to PHS villages (n = 6) with only 33% of schools in the PHS villages meeting the WHO threshold for boy: latrine coverage ratio versus 83.3% in R, while no villages met the girl: latrine ratio requirement. A higher proportion of individuals accessed unprotected water sources for both bathing and drinking (68.5% for children and 89% for adults) in PHS relative to R villages. In addition, frequency of accessing water sources was higher in PHS villages, with swimming being the most frequent activity. As expected based upon selection criteria, both prevalence and intensity of S. mansoni were higher in the PHS relative to R villages (prevalence: 43.7% vs 20.2%; P < 0.001; intensity: 73.8 ± 200.6 vs 22.2 ± 96.0, P < 0.0001), respectively.
Conclusion: Unprotected water sources and low latrine coverage are contributing factors to PHS for schistosomiasis in western Kenya. Efforts to increase provision of potable water and improvement in latrine infrastructure is recommended to augment control efforts in the PHS areas.
Musuva RM, Odiere MR, Mwinzi PNM, Omondi IO, Rawago FO, Matendechero SH, Kittur N, Campbell Jr CH, Colley DG. (2021) Unprotected water sources and low latrine coverage are contributing factors to persistent hotspots for schistosomiasis in western Kenya. PLoS ONE 16(9): e0253115. https://doi.org/10.1371/journal.pone.0253115
Belen Cassera is leading a research team that will test two new drugs for the treatment of malaria. The team’s work will be funded by a $3.7 million grant from the National Institutes of Health. (Photo credit: Amy Ware)
Though malaria was eliminated from the U.S. 70 years ago, the mosquito-borne disease caused by the Plasmodium parasite is still rampant in many parts of the world – nearly 40% of the world’s population is at risk of contracting it, and nearly 450,000 people die each year from it. With the rise of drug resistance, the current medical treatments aren’t enough to end this disease.
“Every drug treatment currently in use for malaria is showing resistance or reduced efficacy,” said Belen Cassera, a member of the University of Georgia’s Center for Tropical and Emerging Global Diseases. “Furthermore, there are very limited treatments for the most vulnerable – children and pregnant women. Over 60% of deaths are children under the age of 5.”
Cassera is co-leading the research team that recently received a $3.7 million grant from the National Institutes of Health to test two new drug candidates.
“These compounds are really promising as they are easy to synthesize, cheap, reliable, have a low toxicity profile, and kill the parasites fast,” said Cassera, associate professor in the Department of Biochemistry and Molecular Biology, part of the Franklin College of Arts and Sciences.
What’s unique about these compounds is that they can kill the parasite in three development stages in humans. Current treatments only target the blood stage, which is when clinical symptoms appear.
The life cycle of the Plasmodium parasite is complex. When an infected mosquito bites a person, just a small number of parasites – usually less than a hundred – are injected into the bite site and then travel to the liver, where they multiply in number to thousands. Once their numbers are sufficient enough, they invade the bloodstream and infect red blood cells.
When the number of parasites reaches 100 million, symptoms occur and some of the parasites develop into a sexual form, also known as the gametocyte stage. This is when symptoms occur. The sexual form is then transmitted back to the mosquito when the person is bitten again.
This complex life cycle makes it difficult to find a treatment that will eradicate the disease. Breaking the cycle of transmission between humans and mosquitos is key to accomplishing that goal. That’s why the team is excited about discovering compounds that can attack the parasite on multiple fronts.
“We are really a powerhouse team,” said Cassera. “We have a leading medicinal chemistry expert in Paul Carlier, the robust parasitology resources of UGA, and Max Totrov brings the machine-learning expertise to tie it all together.”
Cassera is a UGA Innovation Fellow, and she also credits the knowledge gained at UGA’s 2019 Innovation Bootcamp with helping her prepare a grant proposal that would be of particular interest to drug manufacturers.
Cassera has been working for several years to identify new drug candidates, along with Carlier, a professor in the Virginia Tech College of Science’s Department of Chemistry and director of the Virginia Tech Center for Drug Discovery, and Max Totrov, a computational chemist at Molsoft.
“We started working with the Malaria Box from Medicines for Malaria Venture, and the discoveries we made in basic malaria biochemistry and medicinal chemistry really springboarded us to a new level and led us in this new direction,” Cassera said.
Cassera is leading the testing of the new chemical variations of the antimalarial compounds prepared by Carlier for effectiveness in cellular and animal models.
“My lab will be looking at levels of toxicity, the potential for resistance, and how well they work both directly on the parasite and in infected mice,” she said. “We’ll be performing the studies for making the go/no-go decision for these compounds.”
A joint patent application for both drug candidates was recently filed, and the team is optimistic that their research will yield fast-acting candidates for advanced pre-clinical evaluation.
Associate professor Belen Cassera is one step closer to introducing her research to the marketplace. Having spent the summer as UGA’s newest Innovation Fellow, Cassera has learned a lot about how to bring parasitic disease therapeutics arising from her research to market.
“In fall 2019, I was among the 18 chosen women from UGA who participated in the inaugural Innovation Bootcamp, where we learned about the Innovation Fellow program, among several other opportunities designed to guide faculty seeking to commercialize their discoveries,” said Cassera, an associate professor in biochemistry and molecular biology and member of the Center for Tropical and Emerging Global Diseases. “The bootcamp was the ‘switch on’ I needed to refocus my research, and being chosen as an Innovation Fellow is the ‘takeoff’ of this new journey for me.”
Cassera’s research focuses primarily on the discovery and development of novel anti-parasitic drugs, aiming to understand how therapeutics work at the biochemical and cellular levels. A month into her fellowship, Cassera is already gaining new insight into the commercialization process and how it can inform her approach to research.
“I have experienced a great transformation in my research goals,” she said. “In every aspect that we have addressed, I see a translation back to my lab—everything is connected. For instance, I now understand how to utilize knowledge and resources that we already have to expand and grow into other areas that will bring in more funding, new knowledge and potentially new products.”
Launched in 2019 as part of UGA’s Innovation District initiative, the Innovation Fellows program encourages faculty and staff to pursue commercialization and development of their research through Innovation Gateway. Fellows are trained in how to successfully translate their research projects into a marketable products, receive mentorship from a fellow faculty and/or industry partner, and receive up $10,000 to support their activities.
“Belen is a very technical person with a very precise end goal in mind,” said Ian Biggs, director of programming for the Innovation District and director of Innovation Gateway’s startup program. “The goal of the Gateway team is to provide her with the tools, expertise and guidance she needs to turn her vision into a commercialized reality.”
Thanks to the Innovation Fellows program, the future is not only bright for Cassera’s research, but also for the rest of her academic career as well.
“The insights and knowledge I’ve gained from this fellowship will help me substantially improve my teaching, training and mentoring of students pursuing their careers in the biotech and pharmaceutical industries,” she said.
Applications for the 2021 fall cohort are now open. The deadline to apply is Aug. 15.
PhD Candidate Ale Villegas and Advisor Dr. Vasant Muralidharan (Photo Courtesy of Vasant Muralidharan)
Malaria’s connection to Georgia goes back to the colonial period. The Southeastern United States provided prime conditions for a thriving mosquito population which ensured the spread of the disease. The state capital moved from Louisville to Milledgeville in 1806 in part because of malaria outbreaks among the state’s General Assembly.
Later, the federal Office of Malaria Control in War Areas was established in Atlanta instead of Washington D.C. because of its proximity to malaria. The center was succeeded in 1946 by the Communicable Disease Center which is now the Centers for Disease Control. While Malaria was mostly eliminated in the U.S. by 1951, it still impacts millions of people around the globe. Cue Ale Villegas, a doctoral candidate in Cellular Biology.
Villegas and her advisor, Dr. Vasant Muralidharan, were recently awarded a Gilliam Graduate Fellowship from the Howard Hughes Medical Institute. The goal of the fellowship is to increase the diversity among scientists who are prepared to assume leadership roles in science. The program selects pairs of students and their dissertation advisers based on their scientific leadership and commitment to advance diversity and inclusion in the sciences.
Villegas’s research is on the edge of the unknown. She works with Muralidharan in UGA’s Center for Tropical and Emerging Global Diseases where they aim to understand the parasite that causes malaria.
“I’m exploring the mechanisms by which malaria parasites develop in human red blood cells,” said Villegas. “I am studying Plasmodium falciparum, the most common and deadly species that infects humans. These studies can inform therapeutic treatments in the future.”
PhD Candidate Ale Villegas. Villegas is in the cellular biology department. (Photo Courtesy of Ale Villegas)
Villegas specifically studies a malaria parasite glycosyltransferase or an enzyme that adds sugar molecules to other biomolecules. These enzymes may be needed by the parasite to survive and resist the immune response. There are few experts or studies in this area, but Villegas saw beyond those challenges to the critical importance of understanding malaria immune response.
“She is a very talented young scientist who has undertaken a challenging and high-impact research project,” said Muralidharan. “Her initial work was fraught with technical difficulties and setbacks, most of which are attributable to the difficulties in working with the hard-to-study malaria parasite. I am very impressed by her toughness and intellectual capacity as she solved one technical issue after another. She is now poised to move the field forward in a meaningful way.”
Villegas has also worked with Dr. Robert Haltiwanger and his graduate students in the Complex-Carbohydrate Research Center at UGA to advance her research. Haltiwanger is a leading expert on fringe-like glycosyltransferases like the enzyme she studies.
“Having Dr. Haltiwanger on campus is amazingly lucky,” said Villegas. “He and his graduate students go above and beyond when I need help or need to try out experiments. I’m glad to have access to his knowledge, experienced grad students, and sometimes his reagents!”
“What these parasite-derived sugar modifications are and how they form could inform a better vaccine or other drug therapies for malaria,” said Villegas.
Rings of P. falciparum in a thick blood smear. (Photo Courtesy of CDC)
Malaria still kills around 450,000 people each year. Most of these victims are children under the age of five. There are no effective vaccines and the parasite has gained resistance to all antimalarials currently in clinical use. Villegas’ research on this parasite sugar-adding enzyme could have important implications for future treatments and vaccine development.
The Gilliam Fellowship allows Villegas to pursue other passions in addition to science. She is a leader in student advocacy and devoted to helping students gain access to resources to advocate for themselves.
“I practice and promote student and self-advocacy by serving on the UGA Graduate Student Association and the student science policy group (SPEAR),” said Villegas. “With fellow SPEAR members, I have organized advocacy days workshops to empower students to advocate for themselves and issues they are passionate about.”
“I have found that those who are most successful understand failure very well,” said Muralidharan. “We need to normalize this. We are working to figure out the unknown. Failure in science is normal, and it is critical for discovery.”
Dr. Vasant Muralidharan’s lab utilizes molecular genetics, cell biology, and biochemistry to study the biological mechanisms driving the disease.
The award also provides funding for Muralidharan to develop mentoring skills and to share those skills with other faculty members at UGA. He has served as a mentor for many either first-generation or underrepresented students in STEM. He explains that scientists need strong support systems, especially when they experience failure in the lab. The people around them help the most.
When Villegas graduates, she hopes to continue working on and learning about science policy and advocacy. Her ideal job would allow her to be a scientist in addition to being an advocate for graduate students and a creator of equitable graduate education policies.
The Gilliam Graduate Fellowship provides Villegas an opportunity to move closer to her goals and to contribute to potentially life-saving research that could reduce the global threat of malaria.
African trypanosomes utilize glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) to evade the host immune system. VSG turnover is thought to be mediated via cleavage of the GPI anchor by endogenous GPI-specific phospholipase C (GPI-PLC). However, GPI-PLC is topologically sequestered from VSG substrates in intact cells. Recently, A. J. Szempruch, S. E. Sykes, R. Kieft, L. Dennison, et al. (Cell 164:246-257, 2016, https://doi.org/10.1016/j.cell.2015.11.051) demonstrated the release of nanotubes that septate to form free VSG+ extracellular vesicles (EVs). Here, we evaluated the relative contributions of GPI hydrolysis and EV formation to VSG turnover in wild-type (WT) and GPI-PLC null cells. The turnover rate of VSG was consistent with prior measurements (half-life [t1/2] of ∼26 h) but dropped significantly in the absence of GPI-PLC (t1/2 of ∼36 h). Ectopic complementation restored normal turnover rates, confirming the role of GPI-PLC in turnover. However, physical characterization of shed VSG in WT cells indicated that at least 50% is released directly from cell membranes with intact GPI anchors. Shedding of EVs plays an insignificant role in total VSG turnover in both WT and null cells. In additional studies, GPI-PLC was found to have no role in biosynthetic and endocytic trafficking to the lysosome but did influence the rate of receptor-mediated endocytosis. These results indicate that VSG turnover is a bimodal process involving both direct shedding and GPI hydrolysis. IMPORTANCE African trypanosomes, the protozoan agent of human African trypanosomaisis, avoid the host immune system by switching expression of the variant surface glycoprotein (VSG). VSG is a long-lived protein that has long been thought to be turned over by hydrolysis of its glycolipid membrane anchor. Recent work demonstrating the shedding of VSG-containing extracellular vesicles has led us to reinvestigate the mode of VSG turnover. We found that VSG is shed in part by glycolipid hydrolysis but also in approximately equal part by direct shedding of protein with intact lipid anchors. Shedding of exocytic vesicles made a very minor contribution to overall VSG turnover. These results indicate that VSG turnover is a bimodal process and significantly alter our understanding of the “life cycle” of this critical virulence factor.
Paige Garrison, Umaer Khan, Michael Cipriano, Peter J Bush, Jacquelyn McDonald, Aakash Sur, Peter J Myler, Terry K Smith, Stephen L Hajduk, James D Bangs. mBio. 2021 Jul 27;e0172521. doi: 10.1128/mBio.01725-21.
