Plasmodium Archives - Center for Tropical and Emerging Global Diseases

Generating Genetically Modified Plasmodium berghei Sporozoites

Donna Huber on May 5, 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.

Carson Bowers, Samarchith P Kurup. J Vis Exp. 2023 May 5;(195). doi: 10.3791/64992.

Cripowellins Pause Plasmodium falciparum Intraerythrocytic Development at the Ring Stage

Donna Huber on March 13, 2023

Cripowellins from Crinum erubescens are known pesticidal and have potent antiplasmodial activity. To gain mechanistic insights to this class of natural products, studies to determine the timing of action of cripowellins within the asexual intraerythrocytic cycle of Plasmodium falciparum were performed and led to the observation that this class of natural products induced reversible cytostasis in the ring stage within the first 24 h of treatment. The transcriptional program necessary for P. falciparum to progress through the asexual intraerythrocytic life cycle is well characterized. Whole transcriptome abundance analysis showed that cripowellin B “pauses” the transcriptional program necessary to progress through the intraerythrocytic life cycle coinciding with the lack of morphological progression of drug treated parasites. In addition, cripowellin B-treated parasites re-enter transcriptional progression after treatment was removed. This study highlights the use of cripowellins as chemical probes to reveal new aspects of cell cycle progression of the asexual ring stage of P. falciparum which could be leveraged for the generation of future antimalarial therapeutics.

Joshua H Butler, Heather J Painter, Emily K Bremers, Priscilla Krai, Manuel Llinás, Maria B Cassera. Molecules. 2023 Mar 13;28(6):2600. doi: 10.3390/molecules28062600.

AIM2 sensors mediate immunity to Plasmodium infection in hepatocytes

Donna Huber on January 3, 2023

Malaria, caused by Plasmodium parasites is a severe disease affecting millions of people around the world. Plasmodium undergoes obligatory development and replication in the hepatocytes, before initiating the life-threatening blood-stage of malaria. Although the natural immune responses impeding Plasmodium infection and development in the liver are key to controlling clinical malaria and transmission, those remain relatively unknown. Here we demonstrate that the DNA of Plasmodium parasites is sensed by cytosolic AIM2 (absent in melanoma 2) receptors in the infected hepatocytes, resulting in Caspase-1 activation. Remarkably, Caspase-1 was observed to undergo unconventional proteolytic processing in hepatocytes, resulting in the activation of the membrane pore-forming protein, Gasdermin D, but not inflammasome-associated proinflammatory cytokines. Nevertheless, this resulted in the elimination of Plasmodium-infected hepatocytes and the control of malaria infection in the liver. Our study uncovers a pathway of natural immunity critical for the control of malaria in the liver.

Camila Marques-da-Silva, Barun Poudel, Rodrigo P Baptista, Kristen Peissig, Lisa S Hancox, Justine C Shiau, Lecia L Pewe, Melanie J Shears, Thirumala-Devi Kanneganti, Photini Sinnis, Dennis E Kyle, Prajwal Gurung, John T Harty, Samarchith P Kurup. Proc Natl Acad Sci U S A. 2023 Jan 10;120(2):e2210181120. doi: 10.1073/pnas.2210181120.

Integrative genetic manipulation of Plasmodium cynomolgi reveals MultiDrug Resistance-1 Y976F associated with increased in vitro susceptibility to mefloquine

Donna Huber on December 7, 2022

The lack of a long-term in vitro culture method has severely restricted the study of Plasmodium vivax, in part because it limits genetic manipulation and reverse genetics. We used the recently optimized P. cynomolgi Berok in vitro culture model to investigate the putative P. vivax drug resistance marker MDR1 Y976F. Introduction of this mutation using CRISPR-Cas9 increased sensitivity to mefloquine, but had no significant effect on sensitivity to chloroquine, amodiaquine, piperaquine and artesunate. To our knowledge, this is the first reported use of CRISPR-Cas9 in P. cynomolgi, and the first reported integrative genetic manipulation of this species.

Kurt E Ward, Peter Christensen, Annie Racklyeft, Satish K Dhingra, Adeline C Y Chua, Caroline Remmert, Rossarin Suwanarusk, Jessica Matheson, Michael J Blackman, Osamu Kaneko, Dennis E Kyle, Marcus C S Lee, Robert W Moon, Georges Snounou, Laurent Rénia, David A Fidock, Bruce Russell, Pablo Bifani. J Infect Dis. 2022 Dec 7;jiac469. doi: 10.1093/infdis/jiac469. Online ahead of print.

MaHPIC malaria systems biology data from Plasmodium cynomolgi sporozoite longitudinal infections in macaques

Donna Huber on November 24, 2022

Plasmodium cynomolgi causes zoonotic malarial infections in Southeast Asia and this parasite species is important as a model for Plasmodium vivax and Plasmodium ovale. Each of these species produces hypnozoites in the liver, which can cause relapsing infections in the blood. Here we present methods and data generated from iterative longitudinal systems biology infection experiments designed and performed by the Malaria Host-Pathogen Interaction Center (MaHPIC) to delve deeper into the biology, pathogenesis, and immune responses of P. cynomolgi in the Macaca mulatta host. Infections were initiated by sporozoite inoculation. Blood and bone marrow samples were collected at defined timepoints for biological and computational experiments and integrative analyses revolving around primary illness, relapse illness, and subsequent disease and immune response patterns. Parasitological, clinical, haematological, immune response, and -omic datasets (transcriptomics, proteomics, metabolomics, and lipidomics) including metadata and computational results have been deposited in public repositories. The scope and depth of these datasets are unprecedented in studies of malaria, and they are projected to be a F.A.I.R., reliable data resource for decades.

Jeremy D DeBarry, Mustafa V Nural, Suman B Pakala, Vishal Nayak, Susanne Warrenfeltz, Jay Humphrey, Stacey A Lapp, Monica Cabrera-Mora, Cristiana F A Brito, Jianlin Jiang, Celia L Saney, Allison Hankus, Hannah M Stealey, Megan B DeBarry, Nicolas Lackman, Noah Legall, Kevin Lee, Yan Tang, Anuj Gupta, Elizabeth D Trippe, Robert R Bridger, Daniel Brent Weatherly, Mariko S Peterson, Xuntian Jiang, ViLinh Tran, Karan Uppal, Luis L Fonseca, Chester J Joyner, Ebru Karpuzoglu, Regina J Cordy, Esmeralda V S Meyer, Lance L Wells, Daniel S Ory, F Eun-Hyung Lee, Rabindra Tirouvanziam, Juan B Gutiérrez 1, Chris Ibegbu, Tracey J Lamb, Jan Pohl, Sarah T Pruett, Dean P Jones, Mark P Styczynski, Eberhard O Voit, Alberto Moreno, Mary R Galinski, Jessica C Kissinger. Sci Data. 2022 Nov 24;9(1):722. doi: 10.1038/s41597-022-01755-y.

Liver-stage fate determination in Plasmodium vivax parasites: Characterization of schizont growth and hypnozoite fating from patient isolates

Donna Huber on September 23, 2022

Plasmodium vivax, one species of parasite causing human malaria, forms a dormant liver stage, termed the hypnozoite, which activate weeks, months or years after the primary infection, causing relapse episodes. Relapses significantly contribute to the vivax malaria burden and are only killed with drugs of the 8-aminoquinoline class, which are contraindicated in many vulnerable populations. Development of new therapies targeting hypnozoites is hindered, in part, by the lack of robust methods to continuously culture and characterize this parasite. As a result, the determinants of relapse periodicity and the molecular processes that drive hypnozoite formation, persistence, and activation are largely unknown. While previous reports have described vastly different liver-stage growth metrics attributable to which hepatocyte donor lot is used to initiate culture, a comprehensive assessment of how different P. vivax patient isolates behave in the same lots at the same time is logistically challenging. Using our primary human hepatocyte-based P. vivax liver-stage culture platform, we aimed to simultaneously test the effects of how hepatocyte donor lot and P. vivax patient isolate influence the fate of sporozoites and growth of liver schizonts. We found that, while environmental factors such as hepatocyte donor lot can modulate hypnozoite formation rate, the P. vivax case is also an important determinant of the proportion of hypnozoites observed in culture. In addition, we found schizont growth to be mostly influenced by hepatocyte donor lot. These results suggest that, while host hepatocytes harbor characteristics making them more- or less-supportive of a quiescent versus growing intracellular parasite, sporozoite fating toward hypnozoites is isolate-specific. Future studies involving these host-parasite interactions, including characterization of individual P. vivax strains, should consider the impact of culture conditions on hypnozoite formation, in order to better understand this important part of the parasite’s lifecycle.

Amélie Vantaux, Julie Péneau, Caitlin A Cooper, Dennis E Kyle, Benoit Witkowski, Steven P Maher. Front Microbiol. 2022 Sep 23;13:976606. doi: 10.3389/fmicb.2022.976606.

UGA researcher uncovers humans’ natural weapon against malaria

Donna Huber on September 22, 2022

Samarchith “Sam” Kurup grew up in India, and he’s always been aware of the impact of malaria.

In 2020 there were an estimated 241 million cases of malaria worldwide and an estimated 627,000 deaths, according to a recently released World Health Organization Fact Sheet. Eighty percent of the malaria-related deaths in Africa are children under the age of 5. The relapsing nature of the disease leads to educational and employment loss that has long-term economic impacts for both the individual as well as society.

“Malaria is huge global problem,” said Kurup, a member of UGA’s Center for Tropical and Emerging Global Diseases. “Almost half of the world’s population is currently at risk of contracting malaria.”

Kurup began his training in veterinary medicine in India, where he became hooked on parasitology, then continued his studies at UGA. While pursuing his Ph.D. he worked in Rick Tarleton’s lab, studying a parasitic disease that affects both animals and humans—his first introduction to human immunology. He continued his training in immunology as a postdoctoral researcher in John Harty’s lab at the University of Iowa.

Combining parasitology with immunology prepared him to tackle malaria.

Malaria is one of the most studied parasitic diseases, yet the Plasmodium parasite that causes it keeps evading attempts to treat the infection in humans. This is largely due to its complex life cycle and the ability of the parasite to evolve drug resistance. In addition to life stages that occur in the mosquito, which transmits the Plasmodium parasite to humans, there are two life stages in humans—a short phase of initial development in the liver, followed by an infection of the blood cells that causes clinical disease.

“A lot of research has been focused on the blood stage in humans, as this is when a person is symptomatic,” said Kurup, assistant professor of cellular biology in the Franklin College of Arts and Sciences. “But we now recognize that if we want to stop malaria, we need to stop it in its tracks in the liver before accessing the blood, and for that we need to understand the liver stage.”

Kurup, a member of UGA’s Center for Tropical and Emerging Global Diseases, trained in parasitology and immunology. He hopes that uncovering how the human immune system naturally fights malaria in the liver stage will lead to an effective malaria vaccine. (photo credit: Lauren Corcino)

Kurup has been awarded a five-year National Institutes of Health grant to study the natural immune response to the Plasmodium parasite in liver cells.

“The liver stage is short and can be difficult to study in the laboratory,” he said. “There are also practical and ethical limitations to studying the liver stage of malaria in humans. We are hoping to tease apart the basic principles of immune responses during this stage using the mouse model.”

Kurup’s preliminary studies have shown that a group of signaling proteins called type 1 interferons play a role in the destruction of Plasmodium parasites in the liver. His newly funded project will fill a gap in the malaria knowledge base by using a combination of in vitro study and in vivo experiments to determine the molecular processes that eliminate Plasmodium parasites in liver cells. His group recently developed a transgenic parasite line that can be used to genetically alter its host cell.

“This strain is a game changer for our line of research because we can now determine how our liver cells would naturally eliminate the parasite, and maybe why it sometimes fails,” he said.

In a study recently published in Cell Reports, Kurup and colleagues used the genetically altered parasite to inhibit signaling by type 1 interferons and showed that this protein has a direct role in the control of malaria. Their study also revealed that other natural immune mechanisms may be active in controlling malaria in liver cells. The project funded by the new grant will delve further into these mechanisms.

“In addition to taking us a step closer to the control and possible eradication of malaria, this project will expand our knowledge so that we can better reduce the burdens of this illness in our society,” he said.

Kurup is hopeful that uncovering how the human immune system naturally fights malaria in the liver stage will lead to an effective malaria vaccine.

“I really believe that bringing together our knowledge in parasitology and approaches in immunology is key to uncovering new information on this elusive life stage in malaria,” he said. “There is no better place to do this, considering the intellectual and material resources we have at our disposal at UGA and the CTEGD.”

This story was first published at https://research.uga.edu/news/uga-researcher-uncovers-humans-natural-weapon-against-malaria/

Single-cell RNA profiling of Plasmodium vivax-infected hepatocytes reveals parasite- and host- specific transcriptomic signatures and therapeutic targets

Donna Huber on August 25, 2022

The resilience of Plasmodium vivax, the most widely-distributed malaria-causing parasite in humans, is attributed to its ability to produce dormant liver forms known as hypnozoites, which can activate weeks, months, or even years after an initial mosquito bite. The factors underlying hypnozoite formation and activation are poorly understood, as is the parasite’s influence on the host hepatocyte. Here, we shed light on transcriptome-wide signatures of both the parasite and the infected host cell by sequencing over 1,000 P. vivax-infected hepatocytes at single-cell resolution. We distinguish between replicating schizonts and hypnozoites at the transcriptional level, identifying key differences in transcripts encoding for RNA-binding proteins associated with cell fate. In infected hepatocytes, we show that genes associated with energy metabolism and antioxidant stress response are upregulated, and those involved in the host immune response downregulated, suggesting both schizonts and hypnozoites alter the host intracellular environment. The transcriptional markers in schizonts, hypnozoites, and infected hepatocytes revealed here pinpoint potential factors underlying dormancy and can inform therapeutic targets against P. vivax liver-stage infection.

Anthony A Ruberto, Steven P Maher, Amélie Vantaux, Chester J Joyner, Caitlin Bourke, Balu Balan, Aaron Jex, Ivo Mueller, Benoit Witkowski, Dennis E Kyle. Front Cell Infect Microbiol. 2022 Aug 25;12:986314. doi: 10.3389/fcimb.2022.986314. eCollection 2022.

Direct type I interferon signaling in hepatocytes controls malaria

Donna Huber on July 19, 2022

Malaria is a devastating disease impacting over half of the world’s population. Plasmodium parasites that cause malaria undergo obligatory development and replication in hepatocytes before infecting red blood cells and initiating clinical disease. While type I interferons (IFNs) are known to facilitate innate immune control to Plasmodium in the liver, how they do so has remained unresolved, precluding the manipulation of such responses to combat malaria. Utilizing transcriptomics, infection studies, and a transgenic Plasmodium strain that exports and traffics Cre recombinase, we show that direct type I IFN signaling in Plasmodium-infected hepatocytes is necessary to control malaria. We also show that the majority of infected hepatocytes naturally eliminate Plasmodium infection, revealing the potential existence of anti-malarial cell-autonomous immune responses in such hepatocytes. These discoveries challenge the existing paradigms in Plasmodium immunobiology and are expected to inspire anti-malarial drugs and vaccine strategies.

Camila Marques-da-Silva, Kristen Peissig, Michael P Walker, Justine Shiau, Carson Bowers, Dennis E Kyle, Rahul Vijay, Scott E Lindner, Samarchith P Kurup. Cell Rep. 2022 Jul 19;40(3):111098. doi: 10.1016/j.celrep.2022.111098.

Plasmodium knowlesi Cytoadhesion Involves SICA Variant Proteins

Donna Huber on June 23, 2022

Plasmodium knowlesi poses a health threat throughout Southeast Asian communities and currently causes most cases of malaria in Malaysia. This zoonotic parasite species has been studied in Macaca mulatta (rhesus monkeys) as a model for severe malarial infections, chronicity, and antigenic variation. The phenomenon of Plasmodium antigenic variation was first recognized during rhesus monkey infections. Plasmodium-encoded variant proteins were first discovered in this species and found to be expressed at the surface of infected erythrocytes, and then named the Schizont-Infected Cell Agglutination (SICA) antigens. SICA expression was shown to be spleen dependent, as SICA expression is lost after P. knowlesi is passaged in splenectomized rhesus. Here we present data from longitudinal P. knowlesi infections in rhesus with the most comprehensive analysis to date of clinical parameters and infected red blood cell sequestration in the vasculature of tissues from 22 organs. Based on the histopathological analysis of 22 tissue types from 11 rhesus monkeys, we show a comparative distribution of parasitized erythrocytes and the degree of margination of the infected erythrocytes with the endothelium. Interestingly, there was a significantly higher burden of parasites in the gastrointestinal tissues, and extensive margination of the parasites along the endothelium, which may help explain gastrointestinal symptoms frequently reported by patients with P. knowlesi malarial infections. Moreover, this margination was not observed in splenectomized rhesus that were infected with parasites not expressing the SICA proteins. This work provides data that directly supports the view that a subpopulation of P. knowlesi parasites cytoadheres and sequesters, likely via SICA variant antigens acting as ligands. This process is akin to the cytoadhesive function of the related variant antigen proteins, namely Erythrocyte Membrane Protein-1, expressed by Plasmodium falciparum.

Mariko S Peterson, Chester J Joyner, Stacey A Lapp, Jessica A Brady, Jennifer S Wood, Monica Cabrera-Mora, Celia L Saney, Luis L Fonseca, Wayne T Cheng, Jianlin Jiang, Stephanie R Soderberg, Mustafa V Nural, Allison Hankus, Deepa Machiah, Ebru Karpuzoglu, Jeremy D DeBarry, Rabindra Tirouvanziam, Jessica C Kissinger, Alberto Moreno, Sanjeev Gumber, Eberhard O Voit, Juan B Gutierrez, Regina Joice Cordy, Mary R Galinski. Front Cell Infect Microbiol. 2022 Jun 23;12:888496. doi: 10.3389/fcimb.2022.888496. eCollection 2022.

Tag: Plasmodium