Christopher West, head of the Department of Biochemistry and Molecular Biology, a researcher in the Complex Carbohydrate Research Center and a member of CTEGD, belongs to a small group of internationally recognized parasite glycobiologists. His rigorous, transformative research explores cellular processes involving various structures, enzymes and roles of glycans, or sugar chains. His studies have identified fundamental cell-to-cell mechanisms of environmental sensing and signaling in glycobiology. Some of his seminal discoveries involve the biosynthesis and roles of novel glycan molecules in the model organism, Dictyostelium discoideum. One of his crucial contributions to glycobiology has been to describe at molecular resolution that organism’s biochemical response pathway to altered oxygen levels, allowing it to respond to its environment’s available oxygen. Since arriving at UGA, he has translated these findings to an opportunistic human pathogen, Toxoplasma gondii, which can grow and infect cells in low-oxygen environments. His research with collaborators at UGA and internationally has opened a new field of oxygen-sensing in protists, exploring how this environmental factor can control the behavior and virulence of pathogenic parasites.
Trachoma, caused by ocular Chlamydia trachomatis infection, is targeted for global elimination as a public health problem by 2030. To provide evidence for use of antibodies to monitor C. trachomatis transmission, we collated IgG responses to Pgp3 antigen, PCR positivity, and clinical observations from 19,811 children aged 1-9 years in 14 populations. We demonstrate that age-seroprevalence curves consistently shift along a gradient of transmission intensity: rising steeply in populations with high levels of infection and active trachoma and becoming flat in populations near elimination. Seroprevalence (range: 0-54%) and seroconversion rates (range: 0-15 per 100 person-years) correlate with PCR prevalence (r: 0.87, 95% CI: 0.57, 0.97). A seroprevalence threshold of 13.5% (seroconversion rate 2.75 per 100 person-years) identifies clusters with any PCR-identified infection at high sensitivity ( >90%) and moderate specificity (69-75%). Antibody responses in young children provide a robust, generalizable approach to monitor population progress toward and beyond trachoma elimination.
Christine Tedijanto, Anthony W Solomon, Diana L Martin, Scott D Nash, Jeremy D Keenan, Thomas M Lietman, Patrick J Lammie, Kristen Aiemjoy, Abdou Amza, Solomon Aragie, Ahmed M Arzika, E Kelly Callahan, Sydney Carolan, Adisu Abebe Dawed, E Brook Goodhew, Sarah Gwyn, Jaouad Hammou, Boubacar Kadri, Khumbo Kalua, Ramatou Maliki, Beido Nassirou, Fikre Seife, Zerihun Tadesse, Sheila K West, Dionna M Wittberg, Taye Zeru Tadege, Benjamin F Arnold. Nat Commun. 2023 Jun 5;14(1):3269. doi: 10.1038/s41467-023-38940-5.
Biomolecular condensation underlies the biogenesis of an expanding array of membraneless assemblies, including stress granules (SGs), which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar of a few scaffold proteins that make up these phases, but how the partitioning of hundreds of SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, an SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14 aa sequence that acts as a condensation switch and is conserved across the eukaryote lineage. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal an unexpected molecular function for ancient poly(A)-binding proteins as regulators of biomolecular condensate proteins. These findings may inspire approaches to therapeutically target aberrant phases in disease.
Steven Boeynaems, Yanniv Dorone, Yanrong Zhuang, Victoria Shabardina, Guozhong Huang, Anca Marian, Garam Kim, Anushka Sanyal, Nesli-Ece Şen, Daniel Griffith, Roberto Docampo, Keren Lasker, Iñaki Ruiz-Trillo, Georg Auburger, Alex S Holehouse, Edor Kabashi, Yi Lin, Aaron D Gitler. Mol Cell. 2023 Jun 1;S1097-2765(23)00381-7. doi: 10.1016/j.molcel.2023.05.025.
The genomes of kinetoplastids are organized into polycistronic transcription units that are flanked by a modified DNA base (base J, beta-D-glucosyl-hydroxymethyluracil). Previous work established a role of base J in promoting RNA polymerase II (Pol II) termination in Leishmania major and Trypanosoma brucei. We recently identified a PJW/PP1 complex in Leishmania containing a J-binding protein (JBP3), PP1 phosphatase 1, PP1 interactive-regulatory protein (PNUTS) and Wdr82. Analyses suggested the complex regulates transcription termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of proteins, including Pol II, by PP1. However, we never addressed the role of PP1, the sole catalytic component, in Pol II transcription termination. We now demonstrate that deletion of the PP1 component of the PJW/PP1 complex in L. major, PP1-8e, leads to readthrough transcription at the 3′-end of polycistronic gene arrays. We show PP1-8e has in vitro phosphatase activity that is lost upon mutation of a key catalytic residue and associates with PNUTS via the conserved RVxF motif. Additionally, purified PJW complex with associated PP1-8e, but not complex lacking PP1-8e, led to dephosphorylation of Pol II, suggesting a direct role of PNUTS/PP1 holoenzymes in regulating transcription termination via dephosphorylating Pol II in the nucleus.
Rudo Kieft, Yang Zhang, Haidong Yan, Robert J Schmitz, Robert Sabatini. Nucleic Acids Res. 2023 May 17;gkad394. doi: 10.1093/nar/gkad394. Online ahead of print.
The Chagas field has gone >50 years without tangible progress toward new therapies. My colleagues and I have recently reported on a benzoxaborole compound that achieves consistent parasitological cure in experimentally infected mice and in naturally infected non-human primates (NHPs). While these results do not assure success in human clinical trials, they significantly de-risk this process and form a strong justification for such trials. Highly effective drug discovery depends on a solid understanding of host and parasite biology and excellent knowledge in designing and validating chemical entities. This opinion piece seeks to provide perspectives on the process that led to the discovery of AN15368, with the hope that this will facilitate the discovery of additional clinical candidates for Chagas disease.
Acanthamoeba species, Naegleria fowleri, and Balamuthia mandrillaris are opportunistic pathogens that cause a range of brain, skin, eye, and disseminated diseases in humans and animals. These pathogenic free-living amoebae (pFLA) are commonly misdiagnosed and have sub-optimal treatment regimens which contribute to the extremely high mortality rates (>90%) when they infect the central nervous system. To address the unmet medical need for effective therapeutics, we screened kinase inhibitor chemotypes against three pFLA using phenotypic drug assays involving CellTiter-Glo 2.0. Herein, we report the activity of the compounds against the trophozoite stage of each of the three amoebae, ranging from nanomolar to low micromolar potency. The most potent compounds that were identified from this screening effort were: 2d (A. castellanii EC50: 0.92 ± 0.3 μM; and N. fowleri EC50: 0.43 ± 0.13 μM), 1c and 2b (N. fowleri EC50s: <0.63 μM, and 0.3 ± 0.21 μM), and 4b and 7b (B. mandrillaris EC50s: 1.0 ± 0.12 μM, and 1.4 ± 0.17 μM, respectively). With several of these pharmacophores already possessing blood-brain barrier (BBB) permeability properties, or are predicted to penetrate the BBB, these hits present novel starting points for optimization as future treatments for pFLA-caused diseases.
Lori Ferrins, Melissa J Buskes, Madison M Kapteyn, Hannah N Engels, Suzanne E Enos, Chenyang Lu, Dana M Klug, Baljinder Singh, Antonio Quotadamo, Kelly Bachovchin, Westley F Tear, Andrew E Spaulding, Katherine C Forbes, Seema Bag, Mitch Rivers, Catherine LeBlanc, Erin Burchfield, Jeremy R Armand, Rosario Diaz-Gonzalez, Gloria Ceballos-Perez, Raquel García-Hernández, Guiomar Pérez-Moreno, Cristina Bosch-Navarrete, Luis Miguel Ruiz-Pérez, Francisco Gamarro, Dolores González-Pacanowska, Miguel Navarro, Kojo Mensa-Wilmot, Michael P Pollastri, Dennis E Kyle, Christopher A Rice. Front Microbiol. 2023 May 10;14:1149145. doi: 10.3389/fmicb.2023.1149145. eCollection 2023.
Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.
Background: Mosquitoes serve as the vector of canine heartworm (Dirofilaria immitis), which represents a significant and persistent threat to canine health. A reduction in the longevity and/or reproductive success of mosquitoes that take a blood meal from fluralaner-treated dogs may consequently reduce the local transmission of heartworm and prevent new infections. A novel secondary effect of an oral formulation of the ectoparasiticide fluralaner (Bravecto®) against a laboratory strain of the mosquito Aedes aegypti, a potential major vector of canine heartworm, was investigated in this study.
Methods: Six dogs were administered a single dose of fluralaner orally in the form of Bravecto® Chews (at the labeled fluralaner dose of 25 mg/kg body weight), while six control dogs received no treatment. Mosquitoes were fed on blood that was collected from each dog prior to treatment and weekly for 15 weeks post-treatment to assess the continued effects of fluralaner as its serum level decreased. Mosquito fitness was assessed by three parameters: rate of successful blood-feeding, survival, and egg laying.
Results: Successful blood-feeding rate was similar between control and treatment groups. In the fluralaner treatment, mosquito survival was significantly reduced within the first 24 h after blood-feeding, for the first 12 weeks post-treatment of the dogs (efficacy range = 33.2-73.3%). Survival of mosquitoes up until a potentially heartworm-infective timepoint (14 days post-blood-feeding) was significantly reduced in the fluralaner-treated group at several timepoints (1, 2, 5, 11, 12, 13, 14, and 15 weeks post-treatment; efficacy range = 49.4-91.4%), but was less consistently reduced at the other timepoints. Egg laying by mosquitoes was almost completely suppressed for the first 13 weeks following treatment of the dogs with fluralaner (treatment efficacy ≥ 99.8%).
Conclusions: Mosquitoes fed blood from fluralaner-treated dogs experienced a significant reduction in survival and fecundity. These findings support the potential for a reduction in heartworm transmission directly by lethal effects on the vector and indirectly through a reduction of the local vector population when mosquitoes are exposed to animals treated with fluralaner.
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.”
Trypanosoma cruzi naturally infects a broad range of mammalian species and frequently results in the pathology that has been most extensively characterized in human Chagas disease. Currently employed treatment regimens fail to achieve parasitological cure of T. cruzi infection in the majority of cases. In this study, we have extended our previous investigations of more effective, higher dose, intermittent administration protocols using the FDA-approved drug benznidazole (BNZ), in experimentally infected mice and in naturally infected dogs and nonhuman primates (NHP). Collectively, these studies demonstrate that twice-weekly administration of BNZ for more than 4 months at doses that are ~2.5-fold that of previously used daily dosing protocols, provided the best chance to obtain parasitological cure. Dosing less frequently or for shorter time periods was less dependable in all species. Prior treatment using an ineffective dosing regimen in NHPs did not prevent the attainment of parasitological cure with an intensified BNZ dosing protocol. Furthermore, parasites isolated after a failed BNZ treatment showed nearly identical susceptibility to BNZ as those obtained prior to treatment, confirming the low risk of induction of drug resistance with BNZ and the ability to adjust the treatment protocol when an initial regimen fails. These results provide guidance for the use of BNZ as an effective treatment for T. cruzi infection and encourage its wider use, minimally in high value dogs and at-risk NHP, but also potentially in humans, until better options are available.
Juan M Bustamante, Brooke E White, Gregory K Wilkerson, Carolyn L Hodo, Lisa D Auckland, Wei Wang, Stephanie McCain, Sarah A Hamer, Ashley B Saunders, Rick L Tarleton. Antimicrob Agents Chemother. 2023 Apr 11;e0013223. doi: 10.1128/aac.00132-23.
