Malaria is caused by the protozoan parasite Plasmodium, which undergoes a complex life cycle in a human host and a mosquito vector. The parasite’s cyclic GMP (cGMP)-dependent protein kinase (PKG) is essential at multiple steps of the life cycle. Phosphoproteomic studies in Plasmodium falciparum erythrocytic stages and Plasmodium berghei ookinetes have identified proteolysis as a major biological pathway dependent on PKG activity. To further understand PKG’s mechanism of action, we screened a yeast two-hybrid library for P. falciparum proteins that interact with P. falciparum PKG (PfPKG) and tested peptide libraries to identify its phosphorylation site preferences. Our data suggest that PfPKG has a distinct phosphorylation site and that PfPKG directly phosphorylates parasite RPT1, one of six AAA+ ATPases present in the 19S regulatory particle of the proteasome. PfPKG and RPT1 interact in vitro, and the interacting fragment of RPT1 carries a PfPKG consensus phosphorylation site; a peptide carrying this consensus site competes with the RPT1 fragment for binding to PfPKG and is efficiently phosphorylated by PfPKG. These data suggest that PfPKG’s phosphorylation of RPT1 could contribute to its regulation of parasite proteolysis. We demonstrate that proteolysis plays an important role in a biological process known to require Plasmodium PKG: invasion by sporozoites of hepatocytes. A small-molecule inhibitor of proteasomal activity blocks sporozoite invasion in an additive manner when combined with a Plasmodium PKG-specific inhibitor. Mining the previously described parasite PKG-dependent phosphoproteomes using the consensus phosphorylation motif identified additional proteins that are likely to be direct substrates of the enzyme.
Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.
Kudyba, H. M., Cobb, D. W., Florentin, A., Krakowiak, M., Muralidharan, V. 2018. J. Vis. Exp. (139), e57747, doi:10.3791/57747
Kerri Miazowicz is a 3rd year Ph.D. trainee in Courtney Murdock‘s laboratory. She grew up in southern Michigan where she received a B.S. in microbiology from Michigan State University in 2012. After graduation, she spent more than two years as a Postbaccalaureate IRTA Fellow at the Rocky Mountain Laboratories, NIH/NIAID in Montana with the Virus Ecology Unit within the Laboratory of Virology.
Choosing the University of Georgia
Kerri chose the University of Georgia for her graduate training because she wanted to conduct interdisciplinary research related to disease ecology and vector-borne disease transmission.
“UGA hosts many experts across several scientific disciplines allowing me to link molecular biology and individual level phenotypes to population-level dynamics,” said Kerri. ” UGA is also home to the Center for Tropical and Emerging Global Diseases and the Center for Ecology of Infectious Diseases (CEID), which both provide valuable research resources and expertise.”
Research Focus
Kerri’s research focuses on environmental drivers of mosquito-borne disease transmission. Mainly, understanding how the environment affects the mosquito vector and modeling the consequences of these interactions on transmission dynamics.
“My current project revolves around temperature effects on Anopheles stephensi [the primary mosquito that transmits malaria in Asia] trait performance (longevity, biting frequency, and population growth), and mathematically exploring the implications these effects have on transmission.”
She will also investigate how Plasmodium, the parasite that causes malaria, exposure and infection modify these mosquito traits which are critical in transmission events.
“I find vector-borne diseases interesting due to the immense complexity that these systems contain,” said Kerri. “I also find it interesting to think about ‘scaling-up’ the outcome of molecular interactions and individual phenotypes to the context of population-level dynamics.”
Kerri has been able to conduct fieldwork within the Athens area to study how microclimate across an urban area can influence mosquito development along with adult mosquito traits with are important for mosquito-borne disease transmission.
“If I was able to travel for research purpose, it would be to India or Africa, where malaria is endemic, to study local mosquito populations.”
Trainee Earns Accolades
Kerri has received a number of awards recognizing her academic and research achievements. In 2009, she was named a 2008-2009 Regional Semifinalist for the Young Epidemiology Scholars (YES) Program. In 2010, Kerri was named a National Institutes of Health Undergraduate Scholar.
In 2014, she received an OITE Travel Award to attend the NIH Graduate and Professional School Fair, which allows NIH interns and postbacs to explore where the next step in their training will be.
Since coming to UGA, she has received an American Society of Virology Travel Award (2016) and a National Science Foundation Graduate Research Fellowship that funds 3 years of research training. In 2017, she received a travel award to attend the annual meeting of the Vector Behavior Ecology Research Coordination Network (VectorBITE RCN) at Imperial College in London.
What’s Next
While Kerri has a few more years of training at UGA, she hopes to continue conducting scientific research related to disease ecology and transmission dynamics in either an academic or government setting.
Your financial gift to CTEGD funds the Training Innovations in Parasitologic Studies (TIPS) Fellowships which allows trainees like Kerri Miazowicz to travel to international field sites for research. Give Today!
Elvis Ofori Ameyaw is a Fulbright Scholar visiting M. Belen Cassera‘s laboratory in the department of molecular biology and biochemistry. He is a senior lecturer, Head of the Department of Biomedical Sciences and the Vice-Dean of the School of Allied Health Sciences in the College of Health and Allied Sciences at the University of Cape Coast in Ghana.
Dr. Ameyaw holds a B. Pharm and Ph.D. in Pharmacology. His research focuses on natural product drug discovery for infectious, in particular, malaria and Leishmania, and inflammatory diseases. At the University of Georgia, he is using in vitro techniques to screen some natural products isolates from plants that are traditionally used to treat malaria in Ghana.
“UGA is globally known for excellent research and education and my host scientist, Prof. M. Belen Cassera has created an envious and reputable niche in natural product research,” said Dr. Ameyaw.
The availability of seminars and other opportunities to interact with leading scientists also factored into Dr. Ameyaw’s decision to come to UGA.
“The research staff at UGA are very supportive and willing to share ideas.” said Dr. Ameyaw.
Athens reminds him of the college town of Cape Coast where he resides and works in Ghana.
“The city makes me feel at home away from home.”
Read more about Dr. Cassera’s natural products research.
Super-resolution microscopy showing malaria parasites infecting human red blood cells. Image credit: Muthugapatti Kandasamy, Biomedical Microscopy Core
Vasant Muralidharan and his research team at the University of Georgia’s Center for Tropical and Emerging Global Diseases are making great strides in understanding how the malaria parasite hijacks red blood cells to cause disease but many of the parasite’s strategies remain elusive. A new $1.875 million grant from the National Institutes of Health will allow them to continue this research.
Malaria is a parasitic disease that infects nearly 220 million people and kills nearly half a million people every year. Almost all the deaths occur in young children and primarily in sub-Saharan Africa. The parasite Plasmodium falciparum invades human red blood cells which directly leads to malaria symptoms that include headaches, muscle pain, periodic fevers with shivering, severe anemia, trouble breathing, and kidney failure. The parasite can also cause the most severe forms of malaria, such as cerebral malaria which can lead to brain damage, coma and death, and placental malaria, which occurs in pregnancy and can be life-threatening to both the mother and fetus.
Complete control of the infected red blood cell is required for parasites to grow and spread. The malaria parasite remodels the host cell by exporting hundreds of parasite proteins across numerous membranes that transform all aspects of infected red blood cells to suit its needs. The export of these proteins by P. falciparum to the host red blood cells is a unique parasite-driven process that is associated with many of the clinical manifestations of malaria, including death. The mechanisms which these proteins are exported are unknown.
“Exported proteins, many of them absolutely essential for the growth of the parasite, are recognized and sorted throughout the trafficking process by dedicated machinery that we have only now begun to understand,” said Muralidharan, assistant professor in the department of cellular biology.
His lab hopes to reveal unique protein trafficking mechanisms of P. falciparum that may be targets for antimalarial drug development.
“We expect that this project will significantly advance our understanding of the protein export pathway in P. falciparum and how key decisions are made within the parasite that usher exported proteins to their site of action in the infected red blood cells,” concluded Muralidharan.
National Institutes of Health Award R01 AI130139 “Elucidating the trafficking mechanisms of effector proteins to the Plasmodium infected red blood cell.”
Several species of Plasmodium parasites cause malaria in humans and results in nearly 450,000 deaths annually. The deadliest of these species is Plasmodium falciparum. Unfortunately, it is also drug resistance to many of the currently available treatments. Vasant Muralidharan, assistant professor in the department of cellular biology, and his research group at The Center for Tropical and Emerging Global Diseases at The University of Georgia reported on an essential protein in hopes of identifying new drug targets.
Plasmodium parasites contain an organelle known as the apicoplast that evolved via the endosymbiosis of a red alga. The apicoplast produces several essential metabolites required for parasite growth and survival. Therefore, drugs that target the apicoplast are clinically effective. However, there is still not a lot known about this organelle. Understanding the function, structure, and biogenesis of the apicoplast provides a gold mine of antimalarial drug targets.
The role of Clp proteins in Plasmodium apicoplast
Clp (Caseinolytic Proteases) are conserved prokaryotic proteins that serve a wide variety of biological functions in bacteria, the evolutionary ancestors of the apicoplast. Several Clp proteins have been reported to localize in the apicoplast of the parasite but their biological functions were unknown.
The research team used different genetic tools to conditionally inhibit the function of various apicoplast-Clp proteins. “It is similar to understanding the role of a single card in holding up a house of cards by removing it from the structure,” said Muralidharan.
Their data show that the Clp chaperone PfClpC is essential for parasite viability and that its inhibition resulted in morphological defects, and loss of the apicoplast. They also revealed that the chaperone activity is required to stabilize a Clp Protease, PfClpP, suggesting that, similar to bacteria and plants chloroplasts, these two proteins form a proteolytic complex. These data may be relevant to the function of bacterial and plant Clp complexes. “Our findings shed light on the biological roles of the apicoplast Clp Proteins and their involvement in apicoplast replication,” said Dr. Anat Florentin, lead author on the study.
Significance of the findings
The role that bacterial Clp proteins play in cell division, stress response and ability to cause disease have placed them at the center of several drug discovery programs. The new understanding of Clp proteins in Plasmodium provides an avenue for drug development in malaria in which highly active antibacterial compounds can be repurposed as effective anti-malarial agents.
An online version of this study is available: A. Florentin, D.W. Cobb, J.D. Fishburn, M. J. Cipriano, P.S. Kim, M.A. Fierro, B. Striepen, V. Muralidharan. 2017. PfClpC is an essential Clp chaperone required for plastid integrity and Clp protease stability in Plasmodium falciparum. Cell Reports 21, 1 – 11. http://dx.doi.org/10.1016/j.celrep.2017.10.081
Malaria is a mosquito-borne disease that has a disproportionate effect in poor and underdeveloped countries without access to western medicine. According to the WHO’s World Malaria Report, there were 212 million new cases of malaria worldwide in 2015 and an estimated 429,000 deaths.
Malaria is caused by the Plasmodium parasite. Plasmodium falciparum is the deadliest of the species infecting humans, causing 50% of all malaria cases. Unfortunately, it has become resistant to current drug treatment.
Belen Cassera and her laboratory group at the University of Georgia have been identifying possible compounds for new antimalarial medication from natural plant sources.
Cassera’s natural products team
History of anti-malarial drugs
Malaria has long been treated with plant-based medicine. Quinine, which comes from the bark of a cinchona tree, was first isolated as an antimalarial compound in the 1800s, though there is evidence that bark extracts have been used to treat malaria since the 1600s. The cinchona tree is native to Peru.
Quinine was the treatment of choice until the 1940s when other drugs, with fewer side effects, replaced it. One of those drugs was chloroquine, which was discovered in 1934. Following World War II, chloroquine became the preferred treatment for malaria and was prominent in mass drug administration programs of the 1950s. This wide-spread use, in part, led to chloroquine resistant strains of P. falciparum.
The rise of chloroquine-resistance led to the discovery of several potential synthetic alternatives. However, in 1972 Chinese scientists isolated artemisinin from Artemisia annua, commonly known as sweet wormwood. It is native to Asia but has been naturalized in several regions including North America. It has been the main treatment of malaria in south-east Asia. However, in recent years artemisinin resistance has also emerged. (Source: History of Antimalarials)
Cassera in collaboration with David Kingston at Virginia Tech and Michael Goetz and Jason Clement from the Natural Product Discovery Institute (NPDI) has a grant from the National Center for Complementary and Integrative Health (R01 AT008088) to study plants in the NPDI Repository to identify new antimalarial compounds.
Discovering new drugs from plants
In this project, they are concentrating on plants that have not been studied for their anti-malarial properties. Also, they are looking at plants that indigenous people have used to treat the various symptoms associated with malaria.
So far over 28,000 extracts have been screened and the team has identified over 100 compounds with anti-malarial activity.
In recently published findings, the group has reported the discovery of anti-malarial compounds in Malleastrum sp., Crinum firmifolium, and Magnolia grandiflora. The first two are plants found in Madagascar, but the last one is better known as the southern magnolia and can be found in backyards throughout the southeastern United States.
From the southern magnolia extracts, the Cassera and Kingston labs identified two new compounds with anti-malarial activity. It was also discovered that it contained six compounds that have been identified in other plants as possible malaria drug compounds. An online version of the study is available: https://doi.org/10.1002/cbdv.201700209
By Bury, Edward; Havell, Robert [Public domain], via Wikimedia CommonsExtracts from Crinum species, which are in the amaryllis family, have been used traditionally to treat ailments including fever, pain management, swelling, sores and wounds, cancer, and malaria. Following success in isolating new anti-malarial compounds from an extract of Crinum erubescens L. F. ex Aiton, they turned to C. firmifolium, which was already in their International Cooperative Biodiversity Group collection. Extracts yielded 4 known compounds and three new compounds with possible anti-malarial activity. It was observed that the potency of several of the compounds against the drug-resistant strain of P. falciparum was approximately the same as their potency against the drug-sensitive strain. An online version of the study is available: https://doi.org/10.1016/j.bmc.2017.06.017
An extract of the wood from a species of Malleastrum in the mahogany family was found to have moderate antimalarial activity against a drug-resistant strain of P. falciparum. The genus Malleastrum (Baill.) J.-F. Leroy is endemic to Madagascar and comprises 20 currently accepted species. However, there appear to be at least four previously unidentified species. The plant material in this study is almost certainly from one of the species that is still waiting to be named and described. An online version of the study is available: https://doi.org/10.1002/cbdv.201700331
Next steps in the drug discovery process
“We have identified some really promising compounds,” said Belen Cassera. “A few could be ready for pre-clinical studies in a few years.”
In addition to testing for anti-malarial activity, the Cassera lab is also looking at the mechanism of action – how the compound works. This is an important step in drug discovery; because once it is understood how the compound works a synthetic analog could be synthesized and manufactured at a cheaper cost and in a safer form.
Each compound they have identified has several molecules associated with it. In their current state, some of these compounds have too high of a toxicity to be considered for potential drug treatment. Therefore, it is important to strip the compound down to only those molecules that have potent antimalarial responses and hopefully they can remove the molecules associated with toxicity. Once this has been accomplished, then matching synthetic molecules can be created in the lab and scaled up for mass production.
Being able to create these synthetic molecules is a necessary step in the drug discovery process. Natural material can be costly to collect or not available in abundance. While the southern magnolia seems to be abundant in the yards of Georgia, its natural range only stretches from coastal North Carolina south to central Florida, and then west to eastern Texas and Oklahoma. In addition, due to environmental differences, many times compounds isolated from a plant from one part of the world cannot be found in the same plant grown in another which reinforces the need to focus on active compounds that can be resynthesized in the lab.
With the appearance of drug-resistant strains of the malaria parasite to all current medications, it is imperative new treatments be discovered. Since plant-based and traditional medicine have yielded a number of drugs historically it is likely that the next treatment option will again come from a plant source. There are countless numbers of plants that have yet to be studied for their anti-malarial uses. Belen Cassera and her team just might find the cure for malaria in your backyard.
Manuel’s project deals with understanding how calcium is regulated in the endoplasmic reticulum of Plasmodium falciparum, the parasite that causes malaria.
“My undergraduate training was in Dr. Silvia Moreno’s lab studying calcium signaling in Toxoplasma gondii and I wanted to answer the same type of questions in Plasmodium,” said Manuel.
Capstone Experience
Each T32 trainee is provided with the opportunity to complete a capstone experience at the end of their fellowship.
“My home country of Ecuador is approaching elimination of malaria,” said Manuel, “and I would like to work with some of the researchers in the field there who track populations of infected mosquitoes as well as monitor cases of infection in humans.”
T32 Fellowship helps trainee achieve goals
“I truly enjoy working in a lab, but it is not the same as experiencing what diseases are like in the real world,” said Manuel. “This fellowship will help me expand my understanding of malaria by giving me the opportunity to see it in a different setting.”
Manuel is currently considering a career in industry, but he is open to staying in academia.
Support trainees like Manuel Fierro by giving to the Center for Tropical & Emerging Global Diseases
Julie Moore and her colleagues at the University of Georgia have determined that oxidative stress plays a role in the poor pregnancy outcomes that occur during placental malaria. Furthermore, they have identified the antioxidant tempol (TPL) as a potential therapeutic treatment.
Being able to create these synthetic molecules is a necessary step in the drug discovery process. Natural material can be costly to collect or not available in abundance. While the southern magnolia seems to be abundant in the yards of Georgia, its natural range only stretches from coastal North Carolina south to central Florida, and then west to eastern Texas and Oklahoma. In addition, due to environmental differences, many times compounds isolated from a plant from one part of the world cannot be found in the same plant grown in another which reinforces the need to focus on active compounds that can be resynthesized in the lab.