Plasmodium Archives - Center for Tropical and Emerging Global Diseases

Plasmodium RON11 triggers biogenesis of the merozoite rhoptry pair and is essential for erythrocyte invasion

Donna Huber on September 30, 2024

Fig 1. RON11 is essential for intraerythrocytic growth.

Malaria is a global and deadly human disease caused by the apicomplexan parasites of the genus Plasmodium. Parasite proliferation within human red blood cells (RBCs) is associated with the clinical manifestations of the disease. This asexual expansion within human RBCs begins with the invasion of RBCs by P. falciparum, which is mediated by the secretion of effectors from 2 specialized club-shaped secretory organelles in merozoite-stage parasites known as rhoptries. We investigated the function of the Rhoptry Neck Protein 11 (RON11), which contains 7 transmembrane domains and calcium-binding EF-hand domains. We generated conditional mutants of the P. falciparum RON11. Knockdown of RON11 inhibits parasite growth by preventing merozoite invasion. The loss of RON11 did not lead to any defects in processing of rhoptry proteins but instead led to a decrease in the amount of rhoptry proteins. We utilized ultrastructure expansion microscopy (U-ExM) to determine the effect of RON11 knockdown on rhoptry biogenesis. Surprisingly, in the absence of RON11, fully developed merozoites had only 1 rhoptry each. The single rhoptry in RON11-deficient merozoites were morphologically typical with a bulb and a neck oriented into the apical polar ring. Moreover, rhoptry proteins are trafficked accurately to the single rhoptry in RON11-deficient parasites. These data show that in the absence of RON11, the first rhoptry is generated during schizogony but upon the start of cytokinesis, the second rhoptry never forms. Interestingly, these single-rhoptry merozoites were able to attach to host RBCs but are unable to invade RBCs. Instead, RON11-deficient merozoites continue to engage with RBC for prolonged periods eventually resulting in echinocytosis, a result of secreting the contents from the single rhoptry into the RBC. Together, our data show that RON11 triggers the de novo biogenesis of the second rhoptry and functions in RBC invasion.

David Anaguano, Opeoluwa Adewale-Fasoro, Grace W Vick, Sean Yanik, James Blauwkamp, Manuel A Fierro, Sabrina Absalon, Prakash Srinivasan, Vasant Muralidharan. PLoS Biol. 2024 Sep 18;22(9):e3002801. doi: 10.1371/journal.pbio.3002801. eCollection 2024 Sep.

A kalihinol analog disrupts apicoplast function and vesicular trafficking in P. falciparum malaria

Donna Huber on September 27, 2024

A systems biology approach for antimalarial drug discovery.

We report the discovery of MED6-189, an analog of the kalihinol family of isocyanoterpene natural products that is effective against drug-sensitive and drug-resistant Plasmodium falciparum strains, blocking both asexual replication and sexual differentiation. In vivo studies using a humanized mouse model of malaria confirm strong efficacy of the compound in animals with no apparent hemolytic activity or toxicity. Complementary chemical, molecular, and genomics analyses revealed that MED6-189 targets the parasite apicoplast and acts by inhibiting lipid biogenesis and cellular trafficking. Genetic analyses revealed that a mutation in PfSec13, which encodes a component of the parasite secretory machinery, reduced susceptibility to the drug. Its high potency, excellent therapeutic profile, and distinctive mode of action make MED6-189 an excellent addition to the antimalarial drug pipeline.

Z Chahine, S Abel, T Hollin, G L Barnes, J H Chung, M E Daub, I Renard, J Y Choi, P Vydyam, A Pal, M Alba-Argomaniz, C A S Banks, J Kirkwood, A Saraf, I Camino, P Castaneda, M C Cuevas, J De Mercado-Arnanz, E Fernandez-Alvaro, A Garcia-Perez, N Ibarz, S Viera-Morilla, J Prudhomme, C J Joyner, A K Bei, L Florens, C Ben Mamoun, C D Vanderwal, K G Le Roch. Science. 2024 Sep 27;385(6716):eadm7966. doi: 10.1126/science.adm7966.

A Drug Repurposing Approach Reveals Targetable Epigenetic Pathways in Plasmodium vivax Hypnozoites

Donna Huber on May 29, 2024

Hypnozonticidal hit detection and confirmation.

Radical cure of Plasmodium vivax malaria must include elimination of quiescent ‘hypnozoite’ forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase (DNMT) inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax. The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.

S. P. Maher, M. A. Bakowski, A. Vantaux, E. L. Flannery, C. Andolina, M. Gupta, Y. Antonova-Koch, M. Argomaniz, M. Cabrera-Mora, B. Campo, A. T. Chao, A. K. Chatterjee, W. T. Cheng, E. Chuenchob, C. A. Cooper, K. Cottier, M. R. Galinski, A. Harupa-Chung, H. Ji, S. B. Joseph, T. Lenz, S. Lonardi, J. Matheson, S. A. Mikolajczak, T. Moeller, A. Orban, V. Padín-Irizarry, K. Pan, J. Péneau, J. Prudhomme, C. Roesch, A. A. Ruberto, S. S. Sabnis, C. L. Saney, J. Sattabongkot, S. Sereshki, S. Suriyakan, R. Ubalee, Y. Wang, P. Wasisakun, J. Yin, J. Popovici, C. W. McNamara, C. J. Joyner, F. Nosten, B. Witkowski, K. G. Le Roch, D. E. Kyle. 2024. eLife13:RP98221, https://doi.org/10.7554/eLife.98221.1

Trainee Spotlight: Grace Vick

Donna Huber on May 16, 2024

My name is Grace Vick and I am a 4th year infectious diseases PhD candidate in Vasant Muralidharan’s lab. I’m originally from North Carolina and received my Bachelor’s of Science in Biology from Western Carolina University. After graduating undergraduate, I completed an internship at the Defense Forensic Science Center doing forensic biology research. After that, I spent 2 years as an ORISE Fellow at the Centers for Disease Control and Prevention, studying and identifying genetic markers of multi-drug resistant strains of Neisseria gonorrhoeae. I came straight to UGA through the ILS program after my fellowship at CDC.

What made you want to study science?

Ever since I was little, I’ve always spent a lot of time being outside in nature and enjoyed figuring out the intricacies of how things work. During my undergraduate, I was able to explore the different areas of science and found the molecular biology of genetics to be an interesting field that is highly translatable and still vastly unknown. After I spent a few years gaining lab experience and an appreciation for the public health concerns of infectious diseases at the CDC, I knew I wanted to pursue a PhD in that field which brought me to UGA.

Why did you choose UGA?

My experience at the CDC offered the opportunity to learn about diseases and public health issues across all sectors and countries, which led me to learn more about parasitic diseases. Previously, I knew nothing about these diseases but as I learned more about their complex and fascinating life cycles and how these diseases of poverty impact people around the world, I was captivated by this research. Because I was really interested in spending my PhD studying infectious and parasitic diseases, I found out about the CTEGD at UGA and that is what brought me here. The CTEGD is a really wonderful environment for trainees to be exposed to exciting and diverse parasitology research, and I’ve really enjoyed my experience here.

What is your research focus and why did you choose it?

Our lab works on the deadliest form of malaria, Plasmodium falciparum. P. falciparum kills over half a million people each year, with the majority of those deaths being children under the age of 5. Our lab is interested in understanding the molecular mechanisms that are essential to asexual blood stage of this parasite. My work specifically focuses on determining the role of previously unknown proteins that we have discovered are essential for asexual stage invasion of merozoites into host red blood cells. Using a combination of genetic engineering, molecular, and cellular biology techniques, I aim to determine the molecular function of these proteins in the human asexual stage invasion of red blood cells.

Have you received any awards or honors?

In addition to receiving the NIH T32 Predoctoral Fellowship, I have been invited to present at multiple national and international conferences such as Molecular Parasitology Meeting in Massachusetts and Molecular Approaches in Malaria in Lorne, Australia where I won a poster award.

What are your career goals?

When I graduate with my doctoral degree, I hope to either join governmental research or the industry sector. If I decided to head into governmental work, I would choose a career at the CDC where I could continue working in the parasitology research field and apply current public health policies to the international parasitology field. If I decide to join the biomedical industry sector, I would want to work in Research and Design at a company that designs therapeutics and diagnostics for disease prevention and treatment.

What do you hope to do for your capstone experience?

I would really love to experience fieldwork in a malaria-endemic region. I think having the experience of meeting people and learning firsthand how this disease affects millions of people every day would be very eye-opening for me since I have only seen the lab side of malaria. The ability to experience fieldwork would give me a broader experience with how malaria is researched and treated outside of the lab environment and in rural lab environments. I would love to visit Africa or South East Asia to conduct fieldwork in a malaria-endemic environment.

What is your favorite thing about Athens?

Obviously, I love the food in Athens! I love going downtown to grab food and drinks on the weekend. Otherwise, I enjoy getting out and exploring the green spaces and parks that Athens has to offer such as Sandy Creek and the North Oconee Greenway with my husband and dog.

Any advice for a student interested in this field?

I would say the best advice is to read and soak up as much as you can about parasitology both before you get into the field and after. A lot of research has overlap between different parasites and it’s helpful to know about other parasitic diseases that might not be your main focus. Plus, parasites are fun! 🙂 My other advice in general for starting graduate school is to always reach out to students in labs you’re interested in joining. Students are pretty much always willing to help give clear insight into lab dynamics, mentorship of the PI, and generally how life working in that lab is. That information is all really helpful to know when choosing which lab to join!

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Hepatocytes and the art of killing Plasmodium softly

Donna Huber on May 6, 2024

Figure 1. The gap in our understanding of how hepatocytes eliminate Plasmodium.

The Plasmodium parasites that cause malaria undergo asymptomatic development in the parenchymal cells of the liver, the hepatocytes, prior to infecting erythrocytes and causing clinical disease. Traditionally, hepatocytes have been perceived as passive bystanders that allow hepatotropic pathogens such as Plasmodium to develop relatively unchallenged. However, now there is emerging evidence suggesting that hepatocytes can mount robust cell-autonomous immune responses that target Plasmodium, limiting its progression to the blood and reducing the incidence and severity of clinical malaria. Here we discuss our current understanding of hepatocyte cell-intrinsic immune responses that target Plasmodium and how these pathways impact malaria.

Camila Marques-da-Silva, Clyde Schmidt-Silva, Samarchith P Kurup. Trends Parasitol. 2024 May 6:S1471-4922(24)00086-2. doi: 10.1016/j.pt.2024.04.004.

In Vitro Antimalarial Activity of Trichothecenes against Liver and Blood Stages of Plasmodium Species

Donna Huber on January 23, 2024

Trichothecenes (TCNs) are a large group of tricyclic sesquiterpenoid mycotoxins that have intriguing structural features and remarkable biological activities. Herein, we focused on three TCNs (anguidine, verrucarin A, and verrucarol) and their ability to target both the blood and liver stages of Plasmodium species, the parasite responsible for malaria. Anguidine and verrucarin A were found to be highly effective against the blood and liver stages of malaria, while verrucarol had no effect at the highest concentration tested. However, these compounds were also found to be cytotoxic and, thus, not selective, making them unsuitable for drug development. Nonetheless, they could be useful as chemical probes for protein synthesis inhibitors due to their direct impact on parasite synthesis processes.

Prakash T Parvatkar, Steven P Maher, Yingzhao Zhao, Caitlin A Cooper, Sagan T de Castro, Julie Péneau, Amélie Vantaux, Benoît Witkowski, Dennis E Kyle, Roman Manetsch. J Nat Prod. 2024 Jan 23. doi: 10.1021/acs.jnatprod.3c01019.

Inherently Reduced Expression of ASC Restricts Caspase-1 Processing in Hepatocytes and Promotes Plasmodium Infection

Donna Huber on December 27, 2023

Fig. 1 Inherently reduced expression of pro–caspase-1 and ASC in hepatocytes. — Inherently reduced expression of pro–caspase-1 and ASC in hepatocytes.

Inflammasome-mediated caspase-1 activation facilitates innate immune control of Plasmodium in the liver, thereby limiting the incidence and severity of clinical malaria. However, caspase-1 processing occurs incompletely in both mouse and human hepatocytes and precludes the generation of mature IL-1β or IL-18, unlike in other cells. Why this is so or how it impacts Plasmodium control in the liver has remained unknown. We show that an inherently reduced expression of the inflammasome adaptor molecule apoptosis-associated specklike protein containing CARD (ASC) is responsible for the incomplete proteolytic processing of caspase-1 in murine hepatocytes. Transgenically enhancing ASC expression in hepatocytes enabled complete caspase-1 processing, enhanced pyroptotic cell death, maturation of the proinflammatory cytokines IL-1β and IL-18 that was otherwise absent, and better overall control of Plasmodium infection in the liver of mice. This, however, impeded the protection offered by live attenuated antimalarial vaccination. Tempering ASC expression in mouse macrophages, on the other hand, resulted in incomplete processing of caspase-1. Our work shows how caspase-1 activation and function in host cells are fundamentally defined by ASC expression and offers a potential new pathway to create better disease and vaccination outcomes by modifying the latter.

Camila Marques-da-Silva, Clyde Schmidt-Silva, Rodrigo P Baptista, Samarchith P Kurup. J Immunol. 2023 Dec 27:ji2300440. doi: 10.4049/jimmunol.2300440. Online ahead of print.

Atlas of Plasmodium falciparum intraerythrocytic development using expansion microscopy

Donna Huber on December 18, 2023

Ultrastructural expansion microscopy (U-ExM) workflow and summary of parasite structures imaged in this study.

Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample by ~4.5×. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have cataloged 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.

Benjamin Liffner, Ana Karla Cepeda Diaz, James Blauwkamp, David Anaguano, Sonja Frolich, Vasant Muralidharan, Danny W Wilson, Jeffrey D Dvorin, Sabrina Absalon. Elife. 2023 Dec 18:12:RP88088. doi: 10.7554/eLife.88088.

Blood meals from ‘dead-end’ vertebrate hosts enhance transmission potential of malaria-infected mosquitoes

Donna Huber on November 30, 2023

Ingestion of an additional blood meal(s) by a hematophagic insect can accelerate development of several vector-borne parasites and pathogens. Most studies, however, offer blood from the same vertebrate host species as the original challenge (for e.g., human for primary and additional blood meals). Here, we show a second blood meal from bovine and canine hosts can also enhance sporozoite migration in Anopheles stephensi mosquitoes infected with the human- and rodent-restricted Plasmodium falciparum and P. berghei, respectively. The extrinsic incubation period (time to sporozoite appearance in salivary glands) showed more consistent reductions with blood from human and bovine donors than canine blood, although the latter’s effect may be confounded by the toxicity, albeit non-specific, associated with the anticoagulant used to collect whole blood from donors. The complex patterns of enhancement highlight the limitations of a laboratory system but are nonetheless reminiscent of parasite host-specificity and mosquito adaptations, and the genetic predisposition of An. stephensi for bovine blood. We suggest that in natural settings, a blood meal from any vertebrate host could accentuate the risk of human infections by P. falciparum: targeting vectors that also feed on animals, via endectocides for instance, may reduce the number of malaria-infected mosquitoes and thus directly lower residual transmission. Since endectocides also benefit animal health, our results underscore the utility of the One Health framework, which postulates that human health and well-being is interconnected with that of animals. We posit this framework will be further validated if our observations also apply to other vector-borne diseases which together are responsible for some of the highest rates of morbidity and mortality in socio-economically disadvantaged populations.

Ashutosh K Pathak, Justine C Shiau, Rafael C S Freitas, Dennis E Kyle. One Health. 2023 Jun 9:17:100582. doi: 10.1016/j.onehlt.2023.100582. eCollection 2023 Dec.

Time-resolved proximity biotinylation implicates a porin protein in export of transmembrane malaria parasite effectors

Donna Huber on September 29, 2023

Figure 1 Generation of SBP1TbID mutants. — Generation of SBP1TbID mutants.

The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including transmembrane proteins, across multiple membranes to the RBC. However, the process by which these exported membrane proteins are extracted from the parasite plasma membrane for export remains unknown. To address this question, we fused the exported membrane protein, skeleton binding protein 1 (SBP1), with TurboID, a rapid, efficient, and promiscuous biotin ligase (SBP1TbID). Using time-resolved, proximity biotinylation, and label-free quantitative proteomics, we identified two groups of SBP1TbID interactors: early interactors (pre-export) and late interactors (post-export). Notably, two promising membrane-associated proteins were identified as pre-export interactors, one of which possesses a predicted translocon domain, that could facilitate the export of membrane proteins. Further investigation using conditional mutants of these candidate proteins showed that these proteins were essential for asexual growth and localize to the host-parasite interface during early stages of the intraerythrocytic cycle. These data suggest that they may play a role in ushering membrane proteins from the PPM for export to the host RBC.

David Anaguano, Watcharatip Dedkhad, Carrie F Brooks, David W Cobb, Vasant Muralidharan. J Cell Sci. 2023 Sep 29;jcs.260506. doi: 10.1242/jcs.260506

Tag: Plasmodium