Donna Huber, Author at Center for Tropical and Emerging Global Diseases

Shotgun Kinetic Target-Guided Synthesis Approach Enables the Discovery of Small-Molecule Inhibitors against Pathogenic Free-Living Amoeba Glucokinases

Donna Huber on October 11, 2023

Pathogenic free-living amoebae (pFLA) can cause life-threatening central nervous system (CNS) infections and warrant the investigation of new chemical agents to combat the rise of infection from these pathogens. Naegleria fowleri glucokinase (NfGlck), a key metabolic enzyme involved in generating glucose-6-phosphate, was previously identified as a potential target due to its limited sequence similarity with human Glck (HsGlck). Herein, we used our previously demonstrated multifragment kinetic target-guided synthesis (KTGS) screening strategy to identify inhibitors against pFLA glucokinases. Unlike the majority of previous KTGS reports, our current study implements a “shotgun” approach, where fragments were not biased by predetermined binding potentials. The study resulted in the identification of 12 inhibitors against 3 pFLA glucokinase enzymes─NfGlck, Balamuthia mandrillaris Glck (BmGlck), and Acanthamoeba castellanii Glck (AcGlck). This work demonstrates the utility of KTGS to identify small-molecule binders for biological targets where resolved X-ray crystal structures are not readily accessible.

Mintesinot Kassu, Prakash T Parvatkar, Jillian Milanes, Neil P Monaghan, Chungsik Kim, Matthew Dowgiallo, Yingzhao Zhao, Ami H Asakawa, Lili Huang, Alicia Wagner, Brandon Miller, Karissa Carter, Kayleigh F Barrett, Logan M Tillery, Lynn K Barrett, Isabelle Q Phan, Sandhya Subramanian, Peter J Myler, Wesley C Van Voorhis, James W Leahy, Christopher A Rice, Dennis E Kyle, James Morris, Roman Manetsch. ACS Infect Dis. 2023 Oct 11. doi: 10.1021/acsinfecdis.3c00284.

ATP synthase-associated coiled-coil-helix-coiled-coil-helix (CHCH) domain-containing proteins are critical for mitochondrial function in Toxoplasma gondii

Donna Huber on October 5, 2023

CHCH domain proteins associated with the T. gondii ATP synthase are essential for the lytic cycle. (A) Schematic representation of the CHCH domain size and location in ATPTG8 and ATPTG9. C represents cysteine residues and X represents any other amino acid residue.

Coiled-coil-helix-coiled-coil-helix (CHCH) domains consist of two pairs of cysteine residues that are oxidized to form disulfide bonds upon mitochondrial import. Proteins containing these domains play important roles in mitochondrial ultrastructure and in the biogenesis, function, and stability of electron transport chain complexes. Interestingly, recent investigations of the Toxoplasma gondii ATP synthase identified subunits containing CHCH domains. As CHCH domain proteins have never been found in any other ATP synthase, their role in T. gondii was unclear. Using conditional gene knockdown systems, we investigated two T. gondii ATP synthase subunits containing CHCH domains: ATPTG8 and ATPTG9. We show that these two subunits are essential for the lytic cycle as well as stability and function of the ATP synthase. Further, we illustrated that their knockdown disrupts multiple aspects of mitochondrial morphology, including ultrastructure and cristae density. Mutation of key cysteine residues in the CHCH domains also caused mis-localization of the proteins. Our work suggests that these proteins likely provide structural support to the exceptionally large T. gondii ATP synthase complex and that perturbations to the structural integrity of this complex result in deleterious downstream effects on the parasite mitochondrion. These investigations add to a growing body of work focused on the divergent aspects of the apicomplexan ATP synthase, which could ultimately uncover novel drug targets. IMPORTANCE Members of the coiled-coil-helix-coiled-coil-helix (CHCH) domain protein family are transported into the mitochondrial intermembrane space, where they play important roles in the biogenesis and function of the organelle. Unexpectedly, the ATP synthase of the apicomplexan Toxoplasma gondii harbors CHCH domain-containing subunits of unknown function. As no other ATP synthase studied to date contains this class of proteins, characterizing their function will be of broad interest to the fields of molecular parasitology and mitochondrial evolution. Here, we demonstrate that that two T. gondii ATP synthase subunits containing CHCH domains are required for parasite survival and for stability and function of the ATP synthase. We also show that knockdown disrupts multiple aspects of the mitochondrial morphology of T. gondii and that mutation of key residues in the CHCH domains caused mis-localization of the proteins. This work provides insight into the unique features of the apicomplexan ATP synthase, which could help to develop therapeutic interventions against this parasite and other apicomplexans, such as the malaria-causing parasite Plasmodium falciparum.

Madelaine M Usey, Diego Huet. mBio. 2023 Oct 5:e0176923. doi: 10.1128/mbio.01769-23.

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

Characterization of the extracellular vesicles, ultrastructural morphology, and intercellular interactions of multiple clinical isolates of the brain-eating amoeba, Naegleria fowleri

Donna Huber on September 27, 2023

SEM micrographs of each clinical isolate in axenic culture.

Introduction: As global temperatures rise to unprecedented historic levels, so too do the latitudes of habitable niches for the pathogenic free-living amoeba, Naegleria fowleri. This opportunistic parasite causes a rare, but >97% fatal, neurological infection called primary amoebic meningoencephalitis. Despite its lethality, this parasite remains one of the most neglected and understudied parasitic protozoans.

Methods: To better understand amoeboid intercellular communication, we elucidate the structure, proteome, and potential secretion mechanisms of amoeba-derived extracellular vesicles (EVs), which are membrane-bound communication apparatuses that relay messages and can be used as biomarkers for diagnostics in various diseases.

Results and discussion: Herein we propose that N. fowleri secretes EVs in clusters from the plasma membrane, from multivesicular bodies, and via beading of thin filaments extruding from the membrane. Uptake assays demonstrate that EVs are taken up by other amoebae and mammalian cells, and we observed a real-time increase in metabolic activity for mammalian cells exposed to EVs from amoebae. Proteomic analysis revealed >2,000 proteins within the N. fowleri-secreted EVs, providing targets for the development of diagnostics or therapeutics. Our work expands the knowledge of intercellular interactions among these amoebae and subsequently deepens the understanding of the mechanistic basis of PAM.

A Cassiopeia Russell, Peter Bush, Gabriela Grigorean, Dennis E Kyle. Front Microbiol. 2023 Sep 27:14:1264348. doi: 10.3389/fmicb.2023.1264348.

Sheptide A: an antimalarial cyclic pentapeptide from a fungal strain in the Herpotrichiellaceae

Donna Huber on September 20, 2023

Structure and amino acid sequence of the cyclic pentapeptide, sheptide A (1)

As part of ongoing efforts to isolate biologically active fungal metabolites, a cyclic pentapeptide, sheptide A (1), was discovered from strain MSX53339 (Herpotrichiellaceae). The structure and sequence of 1 were determined primarily by analysis of 2D NMR and HRMS/MS data, while the absolute configuration was assigned using a modified version of Marfey’s method. In an in vitro assay for antimalarial potency, 1 displayed a pEC₅₀ value of 5.75 ± 0.49 against malaria-causing Plasmodium falciparum. Compound 1 was also tested in a counter screen for general cytotoxicity against human hepatocellular carcinoma (HepG2), yielding a pCC₅₀ value of 5.01 ± 0.45 and indicating a selectivity factor of ~6. This makes 1 the third known cyclic pentapeptide biosynthesized by fungi with antimalarial activity.

Robert A Shepherd, Cody E Earp, Kristof B Cank, Huzefa A Raja, Joanna Burdette, Steven P Maher, Adriana A Marin, Anthony A Ruberto, Sarah Lee Mai, Blaise A Darveaux, Dennis E Kyle, Cedric J Pearce, Nicholas H Oberlies. J Antibiot (Tokyo). 2023 Sep 20. doi: 10.1038/s41429-023-00655-6.

Synergy between a cytoplasmic vWFA/VIT protein and a WD40-repeat F-box protein controls development in Dictyostelium

Donna Huber on September 14, 2023

Like most eukaryotes, the pre-metazoan social amoeba Dictyostelium depends on the SCF (Skp1/cullin-1/F-box protein) family of E3 ubiquitin ligases to regulate its proteome. In Dictyostelium, starvation induces a transition from unicellular feeding to a multicellular slug that responds to external signals to culminate into a fruiting body containing terminally differentiated stalk and spore cells. These transitions are subject to regulation by F-box proteins and O₂-dependent posttranslational modifications of Skp1. Here we examine in greater depth the essential role of FbxwD and Vwa1, an intracellular vault protein inter-alpha-trypsin (VIT) and von Willebrand factor-A (vWFA) domain containing protein that was found in the FbxwD interactome by co-immunoprecipitation. Reciprocal co-IPs using gene-tagged strains confirmed the interaction and similar changes in protein levels during multicellular development suggested co-functioning. FbxwD overexpression and proteasome inhibitors did not affect Vwa1 levels suggesting a non-substrate relationship. Forced FbxwD overexpression in slug tip cells where it is normally enriched interfered with terminal cell differentiation by a mechanism that depended on its F-box and RING domains, and on Vwa1 expression itself. Whereas vwa1-disruption alone did not affect development, overexpression of either of its three conserved domains arrested development but the effect depended on Vwa1 expression. Based on structure predictions, we propose that the Vwa1 domains exert their negative effect by artificially activating Vwa1 from an autoinhibited state, which in turn imbalances its synergistic function with FbxwD. Autoinhibition or homodimerization might be relevant to the poorly understood tumor suppressor role of the evolutionarily related VWA5A/BCSC-1 in humans.

Andrew W Boland, Elisabet Gas-Pascual, Hanke van der Wel, Hyun W Kim, Christopher M West. Front Cell Dev Biol. 2023 Sep 14;11:1259844. doi: 10.3389/fcell.2023.1259844. eCollection 2023.

Validation of a multiplex microsphere immunoassay for detection of antibodies to Trypanosoma cruzi in dogs

Donna Huber on September 5, 2023

Figure 2. Heatmap of multiplex microsphere immunoassay reactivity of 60 canine serum previously tested for Trypanosoma cruzi antibodies by an indirect fluorescent antibody (IFA) test (grouped by previous IFA result), and reactivity of Leishmania and canine parvovirus controls.

The vector-borne protozoan parasite Trypanosoma cruzi causes Chagas disease in humans, dogs, and many other mammalian hosts. Canine Chagas disease is increasingly diagnosed in dogs of the southern United States where triatomine insect vectors occur, and there are limited veterinary testing options; only the indirect fluorescent antibody (IFA) test is offered at a single accredited diagnostic laboratory. We evaluated a multiplex microsphere immunoassay (MIA) for the detection of antibodies against T. cruzi in dogs and compared it with existing serologic methods to establish cutoff values and relative sensitivity and specificity. We tested 135 canine sera that had been characterized using the IFA and off-label use of 2 commercial rapid assays with our multiplex MIA against 12 antigens: 9 T. cruzi antigens, a negative control recombinant protein (green fluorescent protein, GFP), a Leishmania antigen, and a canine parvovirus antigen (used as an antibody control given near-ubiquitous parvoviral vaccination). The median fluorescence intensity (MFI) ratio between each T. cruzi antigen and GFP was calculated for every sample. Samples with an antigen:GFP MFI ratio > 4 SDs above the mean of 25 known-negative sera were considered positive to that antigen. Samples testing positive to ≥ 2 antigens were considered positive for T. cruzi antibodies. Compared to the IFA, our multiplex MIA had a relative sensitivity of 100% and specificity of 97.0%. Given its precision, high-throughput format, potential for automation, and lack of subjective interpretation, our multiplex MIA should be considered a valid and improved assay for T. cruzi antibodies in dogs.

Carlos A Rodriguez, Rachel E Busselman, Huifeng Shen, Ashley B Saunders, Rick Tarleton, Sarah A Hamer. J Vet Diagn Invest. 2023 Sep 5;10406387231198525. doi: 10.1177/10406387231198525.

Chagas disease research in the news

Donna Huber on August 23, 2023

DDN Dialogues

Rick Tarleton was recently interviewed for the Drug Discovery News podcast. Listen as he talks about his research into new drug treatments for Chagas disease.

A written transcript is available on DDN’s website.

KFF Health News

Rick Tarleton, along with Drew Etheridge‘s lab, was featured in a KFF Health News story about Chagas disease that has been picked up by a number of media outlets.

KFF Health News (Spanish translation)

NBC News

California Healthline

News Medicine

CNN

Sun Herald

The San Diego Union-Tribune

cGAS-STING Pathway Activation during Trypanosoma cruzi Infection Leads to Tissue-Dependent Parasite Control

Donna Huber on August 21, 2023

FIGURE 1. T. cruzi activates the cGAS-STING pathway in primary cells to induce a modest IFN-I response.

Host cell invasion by Trypanosoma cruzi is a markedly silent process, with limited host transcriptional changes indicative of innate immune recognition, except for a modest type I IFN (IFN-I) response. In this study, we show that T. cruzi-induced IFN-β production was nearly abolished in primary murine cGAS-/- or stimulator of IFN genes (STING)-deficient (STINGGt) macrophages and fibroblasts. T. cruzi infection did not impact the ability of IFN-regulatory factor reporter macrophages to respond to classical cGAS-STING agonists, indicating that the limited IFN-β induction is not due to active parasite suppression. cGAS-/-, STINGGt, and IFN-α/β receptor-/- (IFNAR-/-) macrophages infected with T. cruzi yielded significantly higher numbers of amastigotes compared with wild-type macrophages; however, the impact of the STING pathway during infection in vivo is more complex. Despite an initial increase in parasite growth, STINGGt and IFNAR-/- mice ultimately had lower parasite burden in footpads as compared with wild-type mice, demonstrating a role for IFN-I expression in potentiating parasite growth at the infection site. STING pathway activation had little impact on parasite levels in the skeletal muscle; however, in the heart, cGAS-/- and STINGGt mice, but not IFNAR-/- mice, accumulated higher acute parasite loads, suggesting a protective role of STING sensing of T. cruzi in this organ that was independent of IFN-I. Together, these results demonstrate that host cGAS-STING senses T. cruzi infection, enhancing parasite growth at the site of entry, and contributes to acute-phase parasite restriction in the heart, a major site of tissue damage in chronic T. cruzi infection.

Natasha Perumal, Brooke White, Fernando Sanchez-Valdez, Rick L Tarleton. J Immunol. 2023 Aug 21;ji2300373. doi: 10.4049/jimmunol.2300373.

Interorganellar Communication Through Membrane Contact Sites in Toxoplasma gondii

Donna Huber on August 6, 2023

Figure 1. Reported and potential MCSs between organelles of Toxoplasma gondii. Schematic representation showing proteins recently reported to be involved in MCSs, along with putative MCS candidates (indicated with “?”). For clarity purposes, only the central part of the parasite is shown. Abbreviations: AP, apicoplast; ER, endoplasmic reticulum; PLVAC, plant-like vacuolar compartment; IMC, inner membrane complex; TgTPC, T. gondii two pore channel; VDAC, voltage-dependent anion channel; LMF1, lasso maintenance factor 1.; MCS, membrane contact site.

Apicomplexan parasites are a group of protists that cause disease in humans and include pathogens like Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, the etiological agent of toxoplasmosis and one of the most ubiquitous human parasites in the world. Membrane contact sites (MCSs) are widespread structures within eukaryotic cells but their characterization in apicomplexan parasites is only in its very beginnings. Basic biological features of the T. gondii parasitic cycle support numerous organellar interactions, including the transfer of Ca²⁺ and metabolites between different compartments. In T. gondii, Ca²⁺ signals precede a series of interrelated molecular processes occurring in a coordinated manner that culminate in the stimulation of key steps of the parasite life cycle. Calcium transfer from the endoplasmic reticulum to other organelles via MCSs would explain the precision, speed, and efficiency that is needed during the lytic cycle of T. gondii. In this short review, we discuss the implications of these structures in cellular signaling, with an emphasis on their potential role in Ca²⁺ signaling.

Diego Huet, Silvia N J Moreno. Contact (Thousand Oaks). 2023 Aug 6;6:25152564231189064. doi: 10.1177/25152564231189064. eCollection 2023 Jan-Dec.

Author: Donna Huber