Plasmodium Archives - Page 2 of 3 - Center for Tropical and Emerging Global Diseases

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.

Improving in vitro continuous cultivation of Plasmodium cynomolgi, a model for P. vivax

Donna Huber on April 22, 2022

The absence of a routine continuous in vitro cultivation method for Plasmodium vivax, an important globally distributed parasite species causing malaria in humans, has restricted investigations to field and clinical sampling. Such a method has recently been developed for the Berok strain of P. cynomolgi, a parasite of macaques that has long been used as a model for P. vivax, as these two parasites are nearly indistinguishable biologically and are genetically closely related. The availability of the P. cynomolgi Berok in routine continuous culture provides for the first time an opportunity to conduct a plethora of functional studies. However, the initial cultivation protocol proved unsuited for investigations requiring extended cultivation times, such as reverse genetics and drug resistance. Here we have addressed some of the critical obstacles to this, and we propose a set of modifications that help overcome them.

Peter Christensen, Annie Racklyeft, Kurt E Ward, Jessica Matheson, Rossarin Suwanarusk, Adeline C Y Chua, Osamu Kaneko, Htin Lin Aung, Laurent Rénia, Nadia Amanzougaghene, Victor Magneron, Julien Lemaitre, Roger Le Grand, Dennis Kyle, Pablo Bifani, Gregory M Cook, Georges Snounou, Bruce Russell. Parasitol Int. 2022 Apr 22;89:102589. doi: 10.1016/j.parint.2022.102589. Online ahead of print.

A Phenotypic Screen for the Liver Stages of Plasmodium vivax

Donna Huber on December 5, 2021

Control of malaria caused by Plasmodium vivax can be improved by the discovery and development of novel drugs against the parasite’s liver stage, which includes relapse-causing hypnozoites. Several recent reports describe breakthroughs in the culture of the P. vivax liver stage in 384-well microtiter plates, with the goal of enabling a hypnozoite-focused drug screen. Herein we describe assay details, protocol developments, and different assay formats to interrogate the chemical sensitivity of the P. vivax liver stage in one such medium-throughput platform. The general assay protocol includes seeding of primary human hepatocytes which are infected with P. vivax sporozoites generated from the feeding of Anopheles dirus mosquitoes on patient isolate bloodmeals. This protocol is unique in that, after source drug plates are supplied, all culture-work steps have been optimized to preclude the need for automated liquid handling, thereby allowing the assay to be performed within resource-limited laboratories in malaria-endemic countries. Throughput is enhanced as complex culture methods, such as extracellular matrix overlays, multiple cell types in co-culture, or hepatic spheroids, are excluded as the workflow consists entirely of routine culture methods for adherent cells. Furthermore, installation of a high-content imager at the study site enables assay data to be read and transmitted with minimal logistical delays. Herein we detail distinct assay improvements which increase data quality, provide a means to limit the confounding effect of hepatic metabolism on assay data, and detect activity of compounds with a slow-clearance phenotype.

Steven P. Maher, Amélie Vantaux, Caitlin A. Cooper, Nathan M. Chasen, Wayne T. Cheng, Chester J. Joyner, Roman Manetsch, Benoît Witkowski, Dennis Kyle. 2021. Bio-Protocol. 11(23): DOI: 10.21769/BioProtoc.4253

Diagnostic Characteristics of Lactate Dehydrogenase on a Multiplex Assay for Malaria Detection Including the Zoonotic Parasite Plasmodium knowlesi

Donna Huber on November 15, 2021

Plasmodium lactate dehydrogenase (pLDH) is a common target in malaria rapid diagnostic tests (RDTs). These commercial antibody capture assays target either Plasmodium falciparum-specific pLDH (PfLDH), P. vivax-specific pLDH (PvLDH), or a conserved epitope in all human malaria pLDH (PanLDH). However, there are no assays specifically targeting P. ovale, P. malariae or zoonotic parasites such as P. knowlesi and P. cynomolgi. A malaria multiplex array, carrying the specific antibody spots for PfLDH, PvLDH, and PanLDH has been previously developed. This study aimed to assess potential cross-reactivity between pLDH from various Plasmodium species and this array. We tested recombinant pLDH proteins, clinical samples for P. vivax, P. falciparum, P. ovale curtisi, and P. malariae; and in vitro cultured P. knowlesi and P. cynomolgi. P. ovale-specific pLDH (PoLDH) and P. malariae-specific pLDH (PmLDH) cross-reacted with the PfLDH and PanLDH spots. Plasmodium knowlesi-specific pLDH (PkLDH) and P. cynomolgi-specific pLDH (PcLDH) cross-reacted with the PvLDH spot, but only PkLDH was recognized by the PanLDH spot. Plasmodium ovale and P. malariae can be differentiated from P. falciparum by the concentration ratios of PanLDH/PfLDH, which had mean (range) values of 4.56 (4.07-5.16) and 4.56 (3.43-6.54), respectively, whereas P. falciparum had a lower ratio of 1.12 (0.56-2.61). Plasmodium knowlesi had a similar PanLDH/PvLDH ratio value, with P. vivax having a mean value of 2.24 (1.37-2.79). The cross-reactivity pattern of pLDH can be a useful predictor to differentiate certain Plasmodium species. Cross-reactivity of the pLDH bands in RDTs requires further investigation.

Becky Barney, Miguel Velasco, Caitlin Cooper, Andrew Rashid, Dennis Kyle, Robert Moon, Gonzalo Domingo, Ihn Kyung Jang. Am J Trop Med Hyg. 2021 Nov 15;tpmd210532. doi: 10.4269/ajtmh.21-0532

Probing the distinct chemosensitivity of Plasmodium vivax liver stage parasites and demonstration of 8-aminoquinoline radical cure activity in vitro

Donna Huber on October 7, 2021

Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade.

Steven P. Maher, Amélie Vantaux, Victor Chaumeau, Adeline C. Y. Chua, Caitlin A. Cooper, Chiara Andolina, Julie Péneau, Mélanie Rouillier, Zaira Rizopoulos, Sivchheng Phal, Eakpor Piv, Chantrea Vong, Sreyvouch Phen, Chansophea Chhin, Baura Tat, Sivkeng Ouk, Bros Doeurk, Saorin Kim, Sangrawee Suriyakan, Praphan Kittiphanakun, Nana Akua Awuku, Amy J. Conway, Rays H. Y. Jiang, Bruce Russell, Pablo Bifani, Brice Campo, François Nosten, Benoît Witkowski & Dennis E. Kyle. Sci Rep 11, 19905 (2021). https://doi.org/10.1038/s41598-021-99152-9

Some conditions apply: Systems for studying Plasmodium falciparum protein function

Donna Huber on April 22, 2021

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.

Heather M Kudyba, David W Cobb, Joel Vega-Rodríguez, Vasant Muralidharan. PLoS Pathog. 2021 Apr 22;17(4):e1009442. doi: 10.1371/journal.ppat.1009442. eCollection 2021 Apr.

Tag: Plasmodium