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.
Filarial nematodes of the genus Brugia include parasites that are significant to both human and veterinary medicine. Accurate diagnosis is essential for managing infections by these parasites and supporting elimination programs. Traditional diagnostic methods, such as microscopy and serology, remain vital, especially in resource-limited settings. However, advancements in molecular diagnostics, including nucleic acid amplification tests, offer enhanced sensitivity and specificity. These techniques are becoming increasingly field-friendly, expanding their applications in diagnostics. By refining existing methods, developing novel biomarkers, and understanding the zoonotic potential of various Brugia species, it is possible to improve control measures and better support elimination efforts.
Fig 5 Immunofluorescence analysis of PIGJ-3×HA shows its localization both inside and outside the rER.
Glycosylphosphatidylinositols (GPIs) are highly conserved anchors for eukaryotic cell surface proteins. The apicomplexan parasite, Toxoplasma gondii, is a widespread intracellular parasite of warm-blooded animals whose plasma membrane is covered with GPI-anchored proteins, and free GPIs called GIPLs. While the glycan portion is conserved, species differ in sidechains added to the triple mannose core. The functional significance of the Glcα1,4GalNAcβ1- sidechain reported in Toxoplasma gondii has remained largely unknown without understanding its biosynthesis. Here we identify and disrupt two glycosyltransferase genes and confirm their respective roles by serology and mass spectrometry. Parasites lacking the sidechain on account of deletion of the first glycosyltransferase, PIGJ, exhibit increased virulence during primary and secondary infections, suggesting it is an important pathogenesis factor. Cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants are intact. By contrast, the scavenger receptor CD36 shows enhanced binding to PIGJ mutants, potentially explaining a subtle tropism for macrophages detected early in infection. Galectin-3, which binds GIPLs, exhibits an enhancement of binding to PIGJ mutants, and the protection of galectin-3 knockout mice from lethality suggests that Δpigj parasite virulence in this context is sidechain dependent. Parasite numbers are not affected by Δpigj early in the infection in wild-type mice, suggesting a breakdown of tolerance. However, increased tissue cysts in the brains of mice infected with Δpigj parasites indicate an advantage over wild-type strains. Thus, the GPI sidechain of T. gondii plays a crucial and diverse role in regulating disease outcomes in the infected host.IMPORTANCEThe functional significance of sidechain modifications to the glycosylphosphatidylinositol (GPI) anchor in parasites has yet to be determined because the glycosyltransferases responsible for these modifications have not been identified. Here we present identification and characterization of both Toxoplasmsa gondii GPI sidechain-modifying glycosyltransferases. Removal of the glycosyltransferase that adds the first GalNAc to the sidechain results in parasites without a sidechain on the GPI, and increased host susceptibility to infection. Loss of the second glycosyltransferase results in a sidechain with GalNAc alone, and no glucose added, and has negligible effect on disease outcomes. This indicates GPI sidechains are fundamental to host-parasite interactions.
Julia A Alvarez, Elisabet Gas-Pascual, Sahil Malhi, Juan C Sánchez-Arcila, Ferdinand Ngale Njume, Hanke van der Wel, Yanlin Zhao, Laura García-López, Gabriella Ceron, Jasmine Posada, Scott P Souza, George S Yap, Christopher M West, Kirk D C Jensen. mBio. 2024 Sep 20:e0052724. doi: 10.1128/mbio.00527-24
Figure 8. Immunolocalization of FBXO13-HA3 and FBXO14-HA3.
A dynamic proteome is required for cellular adaption to changing environments including levels of O2, and the SKP1/CULLIN-1/F-box protein/RBX1 (SCF) family of E3 ubiquitin ligases contributes importantly to proteasome-mediated degradation. We examine, in the apicomplexan parasite Toxoplasma gondii, the influence on the interactome of SKP1 by its novel glycan attached to a hydroxyproline generated by PHYa, the likely ortholog of the HIFα PHD2 oxygen-sensor of human host cells. Strikingly, the representation of several putative F-box proteins (FBPs) is substantially reduced in PHYaΔ parasites grown in fibroblasts. One, FBXO13, is a predicted lysyl hydroxylase related to the human JmjD6 oncogene except for its F-box domain. The abundance of FBXO13, epitope-tagged at its genetic locus, was reduced in PHYaΔ parasites thus explaining its diminished presence in the SKP1 interactome. A similar effect was observed for FBXO14, a cytoplasmic protein of unknown function that may have co-evolved with PHYa in apicomplexans. Similar findings in glycosylation-mutant cells, rescue by proteasomal inhibitors, and unchanged transcript levels, suggested the involvement of the SCF in their degradation. The effect was selective, because FBXO1 was not affected by loss of PHYa. These findings are physiologically significant because the effects were phenocopied in parasites reared at 0.5% O2. Modest impact on steady-state SKP1 modification levels suggests that effects are mediated during a lag phase in hydroxylation of nascent SKP1. The dependence of FBP abundance on O2-dependent SKP1 modification likely contributes to the reduced virulence of PHYaΔ parasites owing to impaired ability to sense O2 as an environmental signal.
Msano N Mandalasi, Elisabet Gas-Pascual, Carlos Gustavo Baptista, Bowen Deng, Hanke van der Wel, John A W Kruijtzer, Geert-Jan Boons, Ira J Blader, Christopher M West. J Biol Chem. 2024 Sep 20:107801. doi: 10.1016/j.jbc.2024.107801.
Fig 1 IFNγ stimulation following infection is countered by MYR1, preventing early tachyzoite egress and host cell death.
In both mice and humans, Type II interferon gamma (IFNγ) is crucial for the regulation of Toxoplasma gondii (T. gondii) infection, during acute or chronic phases. To thwart this defense, T. gondii secretes protein effectors hindering the host’s immune response. For example, T. gondii relies on the MYR translocon complex to deploy soluble dense granule effectors (GRAs) into the host cell cytosol or nucleus. Recent genome-wide loss-of-function screens in IFNγ-primed primary human fibroblasts identified MYR translocon components as crucial for parasite resistance against IFNγ-driven vacuole clearance. However, these screens did not pinpoint specific MYR-dependent GRA proteins responsible for IFNγ signaling blockade, suggesting potential functional redundancy. Our study reveals that T. gondii depends on the MYR translocon complex to prevent parasite premature egress and host cell death in human cells stimulated with IFNγ post-infection, a unique phenotype observed in various human cell lines but not in murine cells. Intriguingly, inhibiting parasite egress did not prevent host cell death, indicating this mechanism is distinct from those described previously. Genome-wide loss-of-function screens uncovered TgIST, GRA16, GRA24, and GRA28 as effectors necessary for a complete block of IFNγ response. GRA24 and GRA28 directly influenced IFNγ-driven transcription, GRA24’s action depended on its interaction with p38 MAPK, while GRA28 disrupted histone acetyltransferase activity of CBP/p300. Given the intricate nature of the immune response to T. gondii, it appears that the parasite has evolved equally elaborate mechanisms to subvert IFNγ signaling, extending beyond direct interference with the JAK/STAT1 pathway, to encompass other signaling pathways as well.
Figure 2. Presence of surface polyP in T. cruzi different stages.
Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that can lead to the development of cardiac fibrosis, which is characterized by the deposition of extracellular matrix (ECM) components in the interstitial region of the myocardium. The parasite itself can induce myofibroblast differentiation of cardiac fibroblast in vitro, leading to increased expression of ECM. Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that can also induce myofibroblast differentiation and deposition of ECM components and is highly abundant in T. cruzi. PolyP can modify proteins post-translationally by non-enzymatic polyphosphorylation of lysine residues of poly-acidic, serine-(S) and lysine (K)-rich (PASK) motifs. In this work, we used a bioinformatics screen and identified the presence of PASK domains in several surface proteins of T. cruzi. We also detected polyP in the external surface of its different life cycle stages and confirmed the stimulation of host cell fibrosis by trypomastigote infection. However, we were not able to detect significant secretion of the polymer or activation of transforming growth factor beta (TGF-β), an important factor for the generation of fibrosis by inorganic polyP- or trypomastigote-conditioned medium.
I’m Kaelynn Parker and I’m from Virginia where I received my BS in biology from the University of Mary Washington. I’m a cellular biology Ph.D. student in Deigo Huet‘s laboratory.
What made you want to study science?
I took a genetic course at Germanna Community College as an elective while pursuing an associate’s degree part-time and working as an assistant barn manager. We did an experiment where we transformed E. coli with GFP and I thought it was the coolest thing I had ever done. It was a turning point where I decided I wanted to be in science.
Why did you choose UGA?
I chose UGA because of my undergraduate research advisor, Dr. Swati Agrawal, a CTEGD alum. I fell in love with parasitology (something I never imagined would happen) working with her, which inspired me to continue in the field. She also organized a seminar series featuring graduate students from CTEGD labs. After hearing from the graduate students at CTEGD, UGA was the only place I wanted to go for graduate school.
What is your project and why did you choose this research focus?
My project focuses on understanding interorganellar communication in Toxoplasma gondii through discovery and characterization of membrane contact sites between the ER, mitochondrion, and apicoplast. I am also investigating mitochondrial dynamics and stress response in T. gondii. I came to UGA with the desire to work on T. gondii because my original undergraduate project was supposed to be characterizing proteins important for egress in T. gondii. However, the COVID-19 pandemic put a halt on that plan and I wanted to return to Toxoplasma for graduate school.
What are your career goals?
I would like to remain in academic parasitology.
What do you hope to do for your capstone experience? Is there a collaborator/field site you would like to visit?
For my capstone experience, my plan is to utilize the opportunity to go to another lab to learn techniques to apply to membrane contact site research.
What is your favorite thing about UGA and/or Athens?
I love to go bird watching at the botanical gardens and local parks.
Any advice for a student interested in this field?
Talk to people, take every opportunity to present your work and build connections.
Support trainees like Kaelynn by giving today to the Center for Tropical & Emerging Global Diseases.
Intracellular infection by a pathogen induces significant rewiring of host cell signaling and biological processes. Understanding how an intracellular pathogen such as Toxoplasma gondii modulates host cell metabolism with single-cell resolution has been challenged by the variability of infection within cultures and difficulties in separating host and parasite metabolic processes. A new study from Gallego-Lopez and colleagues (G. M. Gallego-López, E. C. Guzman, D. E. Desa, L. J. Knoll, M. C. Skala, mBio e00727-24, 2024, https://doi.org/10.1128/mbio.00727-24) applies a quantitative imaging approach to evaluate the host cell metabolism during intracellular infection with Toxoplasma. This study provides important insights into host metabolic responses to Toxoplasma infection and offers a valuable tool to dissect the mechanisms underlying parasite infection and pathophysiology.
CTEGD Director Dennis Kyle (Photo by Andrew Davis Tucker/UGA)
Dennis Kyle is the Director of CTEGD and the GRA Eminent Scholar in Antiparasitic Drug Discovery in the Departments of Cellular Biology and Infectious Diseases.
Brain-eating amoeba: Will the warming climate bring more cases? (MSN)
Michael Strand is a Regents Professor in the Department of Entomology and member of the Center for Tropical and Emerging Global Diseases. His mosquito research has recently been featured in a number of news stories.
What drives mosquitoes’ bloodlust? Their hormones (Nature)
The Science Behind What Makes Mosquitoes Bite You! Explained (News 9)
