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Tag: Plasmodium

Plasmodium knowlesi Cytoadhesion Involves SICA Variant Proteins

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

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

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

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

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).

Some conditions apply: Systems for studying Plasmodium falciparum protein function

Plasmodium falciparum life cycle
Fig 1. Conditional protein knockdown used throughout the Plasmodium falciparum life cycle.

Malaria, caused by infection with Plasmodium parasites, remains a significant global health concern. For decades, genetic intractability and limited tools hindered our ability to study essential proteins and pathways in Plasmodium falciparum, the parasite associated with the most severe malaria cases. However, recent years have seen major leaps forward in the ability to genetically manipulate P. falciparum parasites and conditionally control protein expression/function. The conditional knockdown systems used in P. falciparum target all 3 components of the central dogma, allowing researchers to conditionally control gene expression, translation, and protein function. Here, we review some of the common knockdown systems that have been adapted or developed for use in P. falciparum. Much of the work done using conditional knockdown approaches has been performed in asexual, blood-stage parasites, but we also highlight their uses in other parts of the life cycle and discuss new ways of applying these systems outside of the intraerythrocytic stages. With the use of these tools, the field’s understanding of parasite biology is ever increasing, and promising new pathways for antimalarial drug development are being discovered.

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.

A redox-active crosslinker reveals an essential and inhibitable oxidative folding network in the endoplasmic reticulum of malaria parasites

Oxidative folding in the P. falciparum ER

Malaria remains a major global health problem, creating a constant need for research to identify druggable weaknesses in P. falciparum biology. As important components of cellular redox biology, members of the Thioredoxin (Trx) superfamily of proteins have received interest as potential drug targets in Apicomplexans. However, the function and essentiality of endoplasmic reticulum (ER)-localized Trx-domain proteins within P. falciparum has not been investigated. We generated conditional mutants of the protein PfJ2-an ER chaperone and member of the Trx superfamily-and show that it is essential for asexual parasite survival. Using a crosslinker specific for redox-active cysteines, we identified PfJ2 substrates as PfPDI8 and PfPDI11, both members of the Trx superfamily as well, which suggests a redox-regulatory role for PfJ2. Knockdown of these PDIs in PfJ2 conditional mutants show that PfPDI11 may not be essential. However, PfPDI8 is required for asexual growth and our data suggest it may work in a complex with PfJ2 and other ER chaperones. Finally, we show that the redox interactions between these Trx-domain proteins in the parasite ER and their substrates are sensitive to small molecule inhibition. Together these data build a model for how Trx-domain proteins in the P. falciparum ER work together to assist protein folding and demonstrate the suitability of ER-localized Trx-domain proteins for antimalarial drug development.

David W. Cobb, Heather M. Kudyba, Alejandra Villegas, Michael R. Hoopmann, Rodrigo P. Baptista, Baylee Bruton, Michelle Krakowiak, Robert L. Moritz, Vasant Muralidharan. PLoS Pathog. 2021 Feb 3;17(2):e1009293. doi: 10.1371/journal.ppat.1009293.

Protozoan persister-like cells and drug treatment failure

Antimicrobial treatment failure threatens our ability to control infections. In addition to antimicrobial resistance, treatment failures are increasingly understood to derive from cells that survive drug treatment without selection of genetically heritable mutations. Parasitic protozoa, such as Plasmodium species that cause malaria, Toxoplasma gondii and kinetoplastid protozoa, including Trypanosoma cruzi and Leishmaniaspp., cause millions of deaths globally. These organisms can evolve drug resistance and they also exhibit phenotypic diversity, including the formation of quiescent or dormant forms that contribute to the establishment of long-term infections that are refractory to drug treatment, which we refer to as ‘persister-like cells’. In this Review, we discuss protozoan persister-like cells that have been linked to persistent infections and discuss their impact on therapeutic outcomes following drug treatment.

Michael P. Barrett, Dennis E. Kyle, L. David Sibley, Joshua B. Radke & Rick L. Tarleton. Nat Rev Microbiol. 2019 Aug 23. doi: 10.1038/s41579-019-0238-x.