The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including transmembrane proteins, across multiple membranes to the RBC. However, the process by which these exported membrane proteins are extracted from the parasite plasma membrane for export remains unknown. To address this question, we fused the exported membrane protein, skeleton binding protein 1 (SBP1), with TurboID, a rapid, efficient, and promiscuous biotin ligase (SBP1TbID). Using time-resolved, proximity biotinylation, and label-free quantitative proteomics, we identified two groups of SBP1TbID interactors: early interactors (pre-export) and late interactors (post-export). Notably, two promising membrane-associated proteins were identified as pre-export interactors, one of which possesses a predicted translocon domain, that could facilitate the export of membrane proteins. Further investigation using conditional mutants of these candidate proteins showed that these proteins were essential for asexual growth and localize to the host-parasite interface during early stages of the intraerythrocytic cycle. These data suggest that they may play a role in ushering membrane proteins from the PPM for export to the host RBC.
David Anaguano, Watcharatip Dedkhad, Carrie F Brooks, David W Cobb, Vasant Muralidharan. J Cell Sci. 2023 Sep 29;jcs.260506. doi: 10.1242/jcs.260506
Super-resolution microscopy showing malaria parasites infecting human red blood cells. credit: Muthugapatti Kandasamy, Biomedical Microscopy Core
They say what doesn’t kill you makes you stronger. Whoever coined that adage had probably never heard of Plasmodium.
It’s a microscopic parasite, invisible to the naked eye but common in tropical and subtropical regions throughout the world. Each year, millions of people are infected by Plasmodium and exposed to an even more debilitating—and often deadly—disease: malaria.
Malaria is one of the deadliest diseases known to man. It can lead to extreme illness, marked by fever, chills, headaches and fatigue. More than half the world’s population is at risk of contracting the disease, and those who develop relapsing infections suffer a host of associated costs.
Limited educational opportunities and wage loss lead to an often unbreakable cycle of poverty. Vulnerable populations are most at risk.
“When I’m teaching in an endemic area like Africa, it isn’t unusual to find a student who needs to sleep during part of the workshop because they have malaria,” researcher Jessica Kissinger said.
It’s a challenge she and her collaborators in the University of Georgia’s Center for Tropical and Emerging Global Diseases (CTEGD) are trying to combat.
When the Center was established in 1998, there were only a couple of faculty members studying Plasmodium. Now, 25 years later, it has become a world-class powerhouse of multidisciplinary malaria research. Scientists examine various species of the dangerous parasite, studying its life cycle and the mosquito that transmits it.
While Plasmodium seems to have superpowers that allow it to evade detection and resist treatment, CTEGD researchers are working together to innovate and transfer science from the lab to interventions on the ground.
A 50,000-piece puzzle with no edges
Plasmodium is a complex organism, and studying it is like putting together a jigsaw puzzle. Some researchers contribute pieces related to the blood or liver stages of the parasite’s lifecycle, while others provide insights about hosts interactions. One way UGA’s research connects with the global effort to eradicate malaria is PlasmoDb—a resource derived in part from Kissinger’s research that is now part of a host of databases under the umbrella of The Eukaryotic Pathogen, Vector and Host information Resource (VEuPathDB).
“Our group has been able to help many others when their research question crosses into an –omic,” Kissinger said, referring to in-house shorthand for domains like genomics, proteomics and metabolomics.
Kissinger, Distinguished Research Professor of genetics in the Franklin College of Arts & Sciences, became interested in malaria and Plasmodium during her postdoctoral training at the National Institutes of Health (NIH). Working from an evolutionary biology perspective, she’s interested in how the parasite has changed over time.
PlasmoDb, a database of Plasmodium informatics resources, is a tool developed in part by the work of Distinguished Research Professor Jessica Kissinger, who became interested in malaria during her postdoctoral training at the National Institutes of Health.
“I see it as an arms race,” Kissinger said. “I want to understand what moves they have and can make.”
To understand the parasite, you must dive deep into its genetic code.
Kissinger paired her work in Plasmodium genomics with her interest in computing by helping create the database with information from the Plasmodium genome project completed in 2002. The Malaria Host-Pathogen Interaction Center, one of her projects at UGA, was a seven-year, multi-institutional effort funded, in part, by NIH to create data sets that could be used in systems biology of the host-pathogen interaction during the development of disease.
“Wouldn’t it be neat if, from the beginning of infection all the way to cure, you knew everything that was going on in the organism all the time?” Kissinger said, noting the project’s goal.
They generated terabytes of data that, along with data from the global research community, are publicly accessible and reusable through PlasmoDB and other resources.
Being part of a group that is studying so many different aspects of malaria helps put Kissinger’s research into perspective. Now, in addition to understanding the parasite, she also thinks about tools needed to facilitate research from peers.
High-tech solutions rely on basic research
David Peterson, professor of infectious diseases in the College of Veterinary Medicine, noted that low-tech solutions have mitigated malaria’s human costs. He acknowledged, however, that their long-term goals required more.
“We have to acknowledge that low-tech solutions, such as mosquito nets, have saved lives,” Peterson said. “But to develop the high-tech solutions that will one day end malaria, we need basic research.”
Pregnant women are particularly vulnerable to malaria because their existing immunity to malaria fails to protect them during pregnancy. Placental malaria often results in premature birth and low birth weight.
Peterson is interested in a binding protein that allows the parasite to adhere to the placenta. While many P. falciparum parasites have only one gene copy that encodes the placental binding protein, Peterson is investigating Plasmodiumisolates with two or more slightly different copies.
But why isn’t one copy enough?
Professor David Peterson of the College of Veterinary Medicine acknowledges the importance of low-tech solutions like mosquito nets but said to mitigate its effects required better understanding at the genetic level.
That is the primary question Peterson is focused on. He wants to understand how Plasmodium uses extra copies to evade the immune system, distinguishing the role of each requires tools that Vasant Muralidharan, associate professor of cellular biology, has.
Muralidharan’s interest began when he contracted malaria himself. Through access to good health care, he made a full recovery, but the pain he endured remained. He wanted to understand this parasite. Even more, he wanted to make an impact with research.
His graduate training focused on biophysics, but soon his interest in Plasmodium resurfaced. He discovered there was a lack of tools to study the parasite on a genetic level.
“It’s like a house of cards, and each card is a gene,” Muralidharan said. “You can remove one and see what happens—does the house fall or remain standing?”
This is an illustration of the life cycle of the parasites of the genus, Plasmodium, that are causal agents of malaria.(Illustration by CDC/ Alexander J. da Silva, PhD; Melanie Moser)
In the days before CRISPR/Cas9, there wasn’t a precise way to remove genes. Muralidharan is among the pioneers of gene-editing techniques in Plasmodium.
Like Peterson, Muralidharan focuses on proteins secreted by the parasite. He studies the largely unknown process that allows the parasite to invade a red blood cell (RBC), replicate and escape. The lack of tools was a major hindrance, so Muralidharan created new ones.
These tools have been used by Muralidharan’s CTEGD and CDC colleagues to see how drugs might fail. Muralidharan’s laboratory can create mutant Plasmodium parasites that become resistant to a particular drug, and genome sequence databases allow researchers to check if that mutant is already circulating in malaria endemic regions.
Building a research bridge to endemic regions
Plasmodium vivax is the predominant malaria parasite in Southeast Asia. It causes “relapsing malaria” during which some parasites go “dormant” after entering the liver instead of reproducing. This phase is a major obstacle for current treatments.
CTEGD Director Dennis Kyle, GRA Eminent Scholar Chair in Antiparasitic Drug Discovery and head of the Department of Cellular Biology, became fascinated with the Plasmodium parasite early in his career, spending time living in Thailand and working in refugee camps where malaria is prevalent.
CTEGD Director Dennis Kyle was moved to follow through with his work as a researcher on a trip to a refugee camp in Thailand. Upon seeing the challenges residents faced, he thought perhaps he should have become a physician. Instead, a local leader impressed upon him the impact you could have in generating new treatments that could benefit everyone. (Photo by Andrew Davis Tucker/UGA)
“When I first got to the refugee camp and saw the situation people were living in, I questioned my decision to become a scientist in the lab instead of becoming a physician,” Kyle said, recalling a camp he worked in that housed about 1,300 kids between the ages of 2 and 15. “There was a guy who was a leader in the group who probably had no more than an early high school education. He said, ‘Look at what you can do—you might generate something that would benefit all of us. The physicians we have in the camp can only work on a few people at a time.’”
Kyle’s laboratory is looking to repurpose medications that have antimalarial properties, a safe way to reduce the development time from lab to clinical use. He’s optimistic we will see a drug treatment that eliminates vivax malaria.
“That’s where UGA is playing a major role,” he said. “The Gates Foundation funded us to develop tools to study the dormant parasite in the liver. And we’ve been successful.”
“We use mouse models to delve into the fundamental host-parasite interactions, which you cannot do practicallyin humans,” Kurup said. “Our understanding of these fundamental processes gives rise to newer and better vaccination approaches and drugs.”
Like other Plasmodium researchers, Joyner became interested in parasites at an early age. During an undergraduate parasitology class, he discovered how little was known about P. vivax. He was already interested in how diseases develop, so for graduate school he focused on the liver stage of vivax malaria. However, it was a difficult task.
Samarchith Kurup is an assistant professor of cellular biology studying the human immune response to Plasmodium infection. (photo credit: Lauren Corcino)
Assistant Professor Chet Joyner discovered how little was known about Plasmodium vivax as an undergraduate student.
“At the time, the technologies weren’t there,” Joyner said. “Dennis was working on his system, but it wasn’t on the scene yet. I changed from studying the parasite to studying the animal model to understand pathogenesis and immunology in humans.”
Joyner joined UGA after completing his postdoctoral training at Emory University, where he developed a non-mouse animal model to study vivax malaria.
“We have to go to [Thailand] where people are infected and collect blood samples and then feed mosquitoes these samples to do the necessary studies,” Kyle said. “That’s been very impactful. We’ve gotten a lot of data out of it, and now with Chet’s model it all can be done under one roof.”
Joyner wants to understand the human immune response with a focus on vaccine development. Building on Muralidharan’s and other researchers’ findings of how the parasite interacts with the RBCs, Joyner’s vaccine program targets a specific protein in the parasite that inhibits the development of immunity.
“My colleagues have shown that if you knock this protein out in the parasite, the immune response in mice is actually great, and we are now working together to evaluate this in non-mouse models.” Joyner said.
Joyner also has collaborated with Belen Cassera, professor of biochemistry, to screen drug compounds. Cassera’s training focused on metabolism to find drug targets. She is particularly interested in how a drug functions.
“If we understand how the drug works, it will help us predict potential side effects in humans,” Cassera said. “We can’t predict everything, but knowing how it works gives you some confidence in whether it will work in humans.”
Cassera is focused on finding drugs that will treat the more lethal Plasmodium falciparum, the predominant species in Africa, which is rapidly becoming resistant to current treatments. Her work is complementary to Kyle’s.
“They run certain assays for the liver-stage infection, and our lab benefits because we want to know if the drug we are developing is specific for the blood stage or can tackle all stages,” Cassera said.
Professor Belen Cassera is identifying drugs that will treat the lethal Plasmodium falciparum, a predominant species of the parasite in Africa that has become resistant to many current treatments.
Don’t forget the mosquito
“Malaria is a vector-borne disease transmitted by a mosquito. You need to tackle not only the parasite in the human but also stop its transmission,” Cassera said. “CTEGD is unique because we can study the whole life cycle, including the mosquito.”
Michael Strand, H.M. Pulliam Chair of Entomology in the College of Agricultural and Environmental Sciences and a National Academy of Sciences Fellow, is an expert on parasite-host interactions. Instead of the human host, he is interested in mosquitoes. Recent work indicates blood feeding behavior of mosquitoes strongly affects malaria parasite development while the gut microbiota of mosquitos could lead to new ways to control populations. Having the SporoCore insectory on campus aids his research.
Michael Strand is an expert on parasite-host interactions. His research focuses on mosquitoes and their effects on malaria parasite development.
Established in 2020, SporoCore, under the management of Ash Pathak, assistant research scientist in the Department of Infectious Diseases, provides both uninfected and Plasmodium-infected Anopheles stephensi mosquitoes to researchers at UGA and other institutions. Like Joyner’s animal model, the insectory allows for research to be done in the U.S. that would otherwise require field work in an endemic country.
Old-school interventions like mosquito nets, combined with new drug therapies, have reduced the number of malaria deaths, which declined over the last 30 years before rising slightly during the COVID-19 pandemic. Great strides have been made to control and treat malaria—but not enough. New tools, like the ones being developed at CTEGD, are needed to keep pushing malaria’s morbidity and mortality rates in the right direction.
“The hard part—what can’t be done easily with the tools we already have—is being done,” Kyle said. “We just need new tools, which is one of the things that our center is really a leader in.”
UGA’s Lọla Fagbami, winner of a Burroughs Wellcome Fund 2022 Postdoctoral Diversity Enrichment Program fellowship, is a native of Lagos, Nigeria, who relocated to the United States with her family in the late 1990s. She is passionate about expanding scientific literacy through outreach and mentoring as well as refuting chemophobia—the fear of or aversion to chemicals and chemistry. (Photo by Lauren Corcino)
Fagbami, UGA’s first PDEP Fellow, conducts research on the human malaria parasite Plasmodium falciparum at the Center for Tropical and Emerging Global Diseases. She works with Vasant Muralidharan, associate professor of cellular biology in the Franklin College of Arts and Sciences, who nominated her for the award.
“Dr. Fagbami has excellent training in metabolomics, mass spectrometry and Plasmodium drug discovery. Her exceptional work as a graduate student has shown how human malaria-causing parasites use metabolic adaptation to induce antimalarial drug resistance. Dr. Fagbami is a fearless, highly intelligent, accomplished and outstanding scientist who will be a leader in our field,” Muralidharan wrote in his nomination letter.
“Her research project addresses a major gap in the field that has enormous implications for malaria elimination and eradication efforts,” he added.
The PDEP award provides $60,000 over three years to support career-development activities for historically excluded minority postdoctoral fellows pursuing academic careers in biomedical or medical research, according to the Burroughs Wellcome Fund.
“This award is an investment in me as a scientist and leader and will help advance my career to the next level,” Fagbami said. “I am excited to join the extraordinary community of PDEP scholars and also connect with program alumni who have successfully made the transition to research independence.”
Fagbami earned a B.S. in chemistry at Emory University, an M.B.S. and an M.P.H. in health policy at Rutgers University, and a Ph.D. in chemical biology at Harvard University.
Malaria remains a major public health issue, infecting nearly 220 million people every year. The spread of drug-resistant strains of Plasmodium falciparum around the world threatens the progress made against this disease. Therefore, identifying druggable and essential pathways in P. falciparum parasites remains a major area of research. One poorly understood area of parasite biology is the formation of disulfide bonds, which is an essential requirement for the folding of numerous proteins. Specialized chaperones with thioredoxin (Trx) domains catalyze the redox functions necessary for breaking incorrect and forming correct disulfide bonds in proteins. Defining the substrates of these redox chaperones is difficult and immunoprecipitation based assays cannot distinguish between substrates and interacting partners. Further, the substrate or client interactions with the redox chaperones are usually transient in nature. Activity based crosslinkers that rely on the nucleophilic cysteines on Trx domains and the disulfide bond forming cysteines on clients provide an easily scalable method to trap and identify the substrates of Trx-domain containing chaperones. The cell permeable crosslinker divinyl sulfone (DVSF) is active only in the presence of nucleophilic cysteines in proteins and, therefore, traps Trx domains with their substrates, as they form mixed disulfide bonds during the course of their catalytic activity. This allows the identification of substrates that rely on Trx activity for their folding, as well as discovering small molecules that interfere with Trx domain activity. Graphic abstract: Identification of thioredoxin domain substrates via divinylsulfone crosslinking and immunoprecipitation-mass spectrometry.
Vasant Muralidharan, associate professor of cellular biology and a member of CTEGD, was recently interviewed for The Athens Frontline podcast episode The Next Pandemic. Listen as he discusses mRNA technology, the pandemic, and other emerging diseases.
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.
Fig 1. Conditional protein knockdown used throughout the Plasmodium falciparum life cycle.
Malaria, caused by infection with Plasmodium parasites, remains a significant global health concern. For decades, genetic intractability and limited tools hindered our ability to study essential proteins and pathways in Plasmodium falciparum, the parasite associated with the most severe malaria cases. However, recent years have seen major leaps forward in the ability to genetically manipulate P. falciparum parasites and conditionally control protein expression/function. The conditional knockdown systems used in P. falciparum target all 3 components of the central dogma, allowing researchers to conditionally control gene expression, translation, and protein function. Here, we review some of the common knockdown systems that have been adapted or developed for use in P. falciparum. Much of the work done using conditional knockdown approaches has been performed in asexual, blood-stage parasites, but we also highlight their uses in other parts of the life cycle and discuss new ways of applying these systems outside of the intraerythrocytic stages. With the use of these tools, the field’s understanding of parasite biology is ever increasing, and promising new pathways for antimalarial drug development are being discovered.
Malaria remains a major global health problem, creating a constant need for research to identify druggable weaknesses in P. falciparum biology. As important components of cellular redox biology, members of the Thioredoxin (Trx) superfamily of proteins have received interest as potential drug targets in Apicomplexans. However, the function and essentiality of endoplasmic reticulum (ER)-localized Trx-domain proteins within P. falciparum has not been investigated. We generated conditional mutants of the protein PfJ2-an ER chaperone and member of the Trx superfamily-and show that it is essential for asexual parasite survival. Using a crosslinker specific for redox-active cysteines, we identified PfJ2 substrates as PfPDI8 and PfPDI11, both members of the Trx superfamily as well, which suggests a redox-regulatory role for PfJ2. Knockdown of these PDIs in PfJ2 conditional mutants show that PfPDI11 may not be essential. However, PfPDI8 is required for asexual growth and our data suggest it may work in a complex with PfJ2 and other ER chaperones. Finally, we show that the redox interactions between these Trx-domain proteins in the parasite ER and their substrates are sensitive to small molecule inhibition. Together these data build a model for how Trx-domain proteins in the P. falciparum ER work together to assist protein folding and demonstrate the suitability of ER-localized Trx-domain proteins for antimalarial drug development.
David W. Cobb, Heather M. Kudyba, Alejandra Villegas, Michael R. Hoopmann, Rodrigo P. Baptista, Baylee Bruton, Michelle Krakowiak, Robert L. Moritz, Vasant Muralidharan. PLoS Pathog. 2021 Feb 3;17(2):e1009293. doi: 10.1371/journal.ppat.1009293.
The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, co-occurrence of polyprenols and dolichols, i.e. detection of a dolichol along with significant levels of its precursor polyprenol, are unusual in eukaryotic cells. Our metabolomics studies revealed that cis-polyisoprenoids are more diverse in the malaria parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite’s development. Moreover, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite.
Flavia M Zimbres, Ana Lisa Valenciano, Emilio F Merino, Anat Florentin, Nicole R Holderman, Guijuan He, Katarzyna Gawarecka, Karolina Skorupinska-Tudek, Maria L Fernández-Murga, Ewa Swiezewska, Xiaofeng Wang, Vasant Muralidharan, Maria Belen Cassera. Sci Rep. 2020 Aug 6;10(1):13264. doi: 10.1038/s41598-020-70246-0.
