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Tag: Plasmodium

PfFBXO1 is essential for inner membrane complex formation in Plasmodium falciparum during both asexual and transmission stages

PfFBXO1 localization by immunofluorescence stained with anti-V5 (PfFBXO1)

Plasmodium species replicate via schizogony, which involves asynchronous nuclear divisions followed by semi-synchronous segmentation and cytokinesis. Successful segmentation requires a double-membranous structure known as the inner membrane complex (IMC). Here we demonstrate that PfFBXO1 (PF3D7_0619700) is critical for both asexual segmentation and gametocyte maturation. In Toxoplasma gondii, the FBXO1 homolog, TgFBXO1, is essential for the development of the daughter cell scaffold and a component of the daughter cell IMC. We demonstrate PfFBXO1 forming a similar IMC initiation scaffold near the apical region of developing merozoites and unilaterally positioned in gametocytes of P. falciparum. While PfFBXO1 initially localizes to the apical region of dividing parasites, it displays an IMC-like localization as segmentation progresses. Similarly, PfFBXO1 localizes to the IMC region in gametocytes. Following inducible knockout of PfFBXO1, parasites undergo abnormal segmentation and karyokinesis, generating inviable daughters. PfFBXO1-deficient gametocytes are abnormally shaped and fail to fully mature. Proteomic analysis identified PfSKP1 as one of PfBXO1’s stable interacting partners, while other major proteins included multiple IMC pellicle and membrane proteins. We hypothesize that PfFBXO1 is necessary for IMC biogenesis, chromosomal maintenance, vesicular transport, and ubiquitin-mediated translational regulation of proteins in both sexual and asexual stages of P. falciparum.

Sreelakshmi K Sreenivasamurthy, Carlos Gustavo Baptista, Christopher M West, Ira J Blader, Jeffrey D Dvorin. Commun Biol. 2025 Feb 7;8(1):190. doi: 10.1038/s42003-025-07619-6.

Screening the Global Health Priority Box against Plasmodium berghei liver stage parasites using an inexpensive luciferase detection protocol

Optimization of conditions for a luciferase endpoint with in-house reagents (FLAR).

Background: Malaria, a disease caused by parasites of the genus Plasmodium, continues to impact many regions globally. The rise in resistance to artemisinin-based anti-malarial drugs highlights the need for new treatments. Ideally, new anti-malarials will kill the asymptomatic liver stages as well as the symptomatic blood stages. While blood stage screening assays are routine and efficient, liver stage screening assays are more complex and costly. To decrease the cost of liver stage screening, a previously reported luciferase detection protocol requiring only common laboratory reagents was adapted for testing against luciferase-expressing Plasmodium berghei liver stage parasites.

Methods: After optimizing cell lysis conditions, the concentration of reagents, and the density of host hepatocytes (HepG2), the protocol was validated with 28 legacy anti-malarials to show this simple protocol produces a stable signal useful for obtaining quality small molecule potency data similar to that obtained from a high content imaging endpoint. The protocol was then used to screen the Global Health Priority Box (GHPB) and confirm the potency of hits in dose-response assays. Selectivity was determined using a galactose-based, 72 h HepG2 assay to avoid missing mitochondrial-toxic compounds due to the Crabtree effect. Receiver-operator characteristic plots were used to retroactively characterize the screens’ predictive value.

Results: Optimal luciferase signal was achieved using a lower HepG2 seed density (5 × 103 cells/well of a 384-well microtitre plate) compared to many previously reported luciferase-based screens. While producing lower signal compared to a commercial alternative, this luciferase detection method was found much more stable, with a > 3 h half-life, and robust enough for producing dose-response plots with as few as 500 sporozoites/well. A screen of the GHPB resulted in 9 hits with selective activity against P. berghei liver schizonts, including MMV674132 which exhibited 30.2 nM potency. Retrospective analyses show excellent predictive value for both anti-malarial activity and cytotoxicity.

Conclusions: This method is suitable for high-throughput screening at a cost nearly 20-fold less than using commercial luciferase detection kits, thereby enabling larger liver stage anti-malarial screens and hit optimization make-test cycles. Further optimization of the hits detected using this protocol is ongoing.

Gia-Bao Nguyen, Caitlin A Cooper, Olivia McWhorter, Ritu Sharma, Anne Elliot, Anthony Ruberto, Rafael Freitas, Ashutosh K Pathak, Dennis E Kyle, Steven P Maher. Malar J. 2024 Nov 23;23(1):357. doi: 10.1186/s12936-024-05155-y.

β-Carboline-3-carboxamide Antimalarials: Structure-Activity Relationship, ADME-Tox Studies, and Resistance Profiling

Graphical abstract

The development of parasite resistance to both artemisinin derivatives and their partner drugs jeopardizes the effectiveness of the artemisinin combination therapy. Thus, the discovery of new antimalarial drugs, with new mechanisms of action, is urgently needed. We recently disclosed that β-carboline 1a was orally efficacious in Plasmodium berghei-infected mice and that it showed low cross-resistance between susceptible Plasmodium falciparum and four different drug-resistant strains. In this report, we describe the synthesis and in vitro antimalarial evaluation of 91 new derivatives of 1a. The asexual blood stage growth inhibition data show a clear preference for a 3,4-dihalogenated, 3,5-dihalogenated, 3,4,5-trichloro-, or 4-trifluoromethyphenyl ring at the C1-position. The most potent compound, 3,4,5-trichlorophenyl-substituted 42a, is twice as potent as 1a. Six potent analogues were assessed for their drug-like properties, and four of these were subjected to in vitro barcoded cross-resistance profiling. Compounds 1a, 1m, 42a, and 42m showed no cross-resistance to 32 resistance mutations on the Dd2 genetic background and 10 resistance mutations on the 3D7 genetic background. These data suggest that compounds in this scaffold possess a novel mechanism of antimalarial action.

Jopaul Mathew, Bo Zhou, Reagan S Haney, Kevin A Kunz, Leticia S Do Amaral, Rudraneel Roy Chowdhury, Joshua H Butler, Haibo Li, Amarraj J Chakraborty, Anika Tabassum, Emily K Bremers, Emilio F Merino, Rachael Coyle, Marcus C S Lee, Delphine Baud, Stephen Brand, Maxim Totrov, Maria Belen Cassera, Paul R Carlier. ACS Infect Dis. 2024 Oct 28. doi: 10.1021/acsinfecdis.4c00653.

Researchers discover malaria gene needed to make pair of invasion organelles

by Donna Huber

Vasant Muralidharan and his research group at the University of Georgia’s Center for Tropical and Emerging Global Diseases have uncovered the role of an essential protein in Plasmodium falciparum, the parasite that causes the deadliest form of malaria. The discovery offers new insights for vaccine and drug development.

The parasite that causes malaria was discovered more than 125 years ago, but much is still unknown about this complex, single-celled organism. Researchers in the University of Georgia’s Center for Tropical and Emerging Global Diseases, however, have uncovered the role of one of the parasite’s essential proteins, offering new insights for vaccine and drug development.

Plasmodium falciparum causes the deadliest form of malaria, a disease the World Health Organization estimates killed more than 600,000 people worldwide died in 2022. A large majority of those deaths were children under the age of 5.

Historically, the parasite has been difficult to study due to its complex lifecycle, which includes three stages. One occurs in the mosquito, while the liver and blood stages take place in humans. The blood stage is when the infected person exhibits symptoms of malaria.

In the blood stage, the parasite invades red blood cells (RBCs) where they replicate and can be transmitted to the mosquito. The receptor-ligand complexes that enable RBC invasion have been well-studied and it is one of the targets of anti-malarial vaccines currently in clinical trials. But questions still remain.

“How does the parasite know it has encountered a red blood cell?” asked Vasant Muralidharan, associate professor in Franklin College’s Department of Cellular Biology and leader of the Muralidharan Research Group, where the study took place.

Interested, the team took a closer look at a protein called RON11, which is sent to a pair of unique club-shaped secretory organelles known as the rhoptry (Greek for club) that houses proteins needed to invade the RBC.

Click play to listen to an excerpt of Vasant Muralidharan discussing the cellular mechanics of malaria infection.

“When we knocked out this protein, we found that the parasite could do everything it usually does – create a putative pore in the membrane of the RBC, send proteins needed for parasite invasion through this putative opening into the RBC – but the parasite itself cannot enter the red blood cell,” Muralidharan explained. “If a parasite cannot enter the red blood cell, the life cycle is interrupted and the parasite dies.”

And then things got really interesting.

“We found that the parasites lacking RON11 were only producing half the rhoptry proteins, which are used in invasion,” Muralidharan said.

While it is known that Plasmodium parasites have two rhoptry organelles, they are so teeny-tiny they have been relatively understudied due to a lack of proper tools. However, new tools and techniques are emerging. David Anaguano, a cellular biology graduate student who led the study, traveled on a Daniel G. Colley Training in Parasitology fellowship to the Absalon Laboratory at Indiana University School of Medicine to learn a new tool known as Ultrastructure Expansion Microscopy.

Vasant Muralidharan is an associate professor in Franklin College’s Department of Cellular Biology. (Photo by Lauren Corcino)

“Electron microscopy is labor intensive, and since it uses thin slices of the parasite you are never sure if what you’re looking for really isn’t there or just not in the slice of the sample you have,” Muralidharan said. “Expansion microscopy is like using light microscopy but with a special gel to expand the cell proportionately in all directions. Thus, you don’t get the distortion you would with just an enlarged cell and you can image the entire infected cell in all dimensions. It has been a real game changer.”

As reported in the PLoS Biology paper, the Muralidharan group generated for the first time a Plasmodium cell with only one rhoptry organelle when they removed RON11 from malaria parasites.

“It’s not unusual for an organism to have a backup copy, but we can see that the parasite can create the first rhoptry just fine – without defect – but the second one that should form during the end of the replication cycle never forms,” Muralidharan said. “Why is that?”

As it appears that this second rhoptry is needed for RBC invasion, understanding the mechanisms that control its development could open up new targets for vaccine and drug treatment discovery as well as answering crucial questions like whether the two rhoptries are identical.

“This has been a long unanswered question,” Muralidharan said. “Now with this RON11 knockout parasite that doesn’t form a second rhoptry, we have the tools to answer it.”

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

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

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

Hypnozonticidal hit detection and confirmation.
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

Ph.D. student Grace Woods

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

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