Biology subjects

Ward, G. E.

Publications and source records attributed to Ward, G. E..

9 recordsLinked to original sources

bioRxiv2026

Identification of UCB-9721 as a potent inhibitor of MyoA, the essential class XIV myosin motor of apicomplexan parasites

The virulence of Toxoplasma gondii and other apicomplexan parasites relies on a unique form of cellular motility driven by MyoA, an unconventional class XIV myosin motor protein. To identify new chemical probes for investigating the molecular mechanisms of parasite motility, we screened over 50,000 small molecules for inhibitors of T. gondii MyoA (TgMyoA). The top hit from the screen, UCB-9721, is almost 40-fold more potent as an inhibitor of TgMyoA actin-activated ATPase activity than the previously described TgMyoA inhibitor, KNX-002, and 45-fold more potent at inhibiting parasite motility, with no detectable toxicity towards mammalian cells. UCB-9721 also inhibited the motility and/or growth of the related apicomplexan parasites Plasmodium falciparum, Cryptosporidium parvum, and Babesia duncani, suggesting that this compound will be a useful new chemical probe for studying motility and MyoA function in apicomplexan parasites more broadly. While UCB-9721 and KNX-002 were identified independently, they share a similar chemical scaffold. To determine why UCB-9721 is so much more potent than KNX-002 and to inform future development of this inhibitor class, we undertook comparative molecular docking analyses, targeted TgMyoA mutagenesis, and a directed structure-activity relationship analysis. The results identified the sulfonamide group of UCB-9721 and its hydrogen bond interactions with R249, E275 and a stabilized water network within the TgMyoA binding pocket as key to the compounds increased potency. Further development of UCB-9721, informed by the results presented here, may transform this promising new chemical class into actionable drug development leads against this important group of human and animal pathogens.

microbiology↗

bioRxiv2025

Single-cell analysis of brain-derived Toxoplasma bradyzoites reveals a novel cell cycle regulated by AP2XI-6.

Toxoplasma gondii prevalence is due, in part, to its ability to persist in hosts while retaining the capacity to transmit and recrudesce. A process that is poorly understood. Through single-cell RNA profiling of in vivo-derived bradyzoites, we discovered that they are heterogeneous and not G1-arrested, as expected from in vitro studies. Instead, they progress through two cell cycles that branch from a single G1. While in G1b, in vivo-derived bradyzoites express cyst wall proteins predicted to replenish the wall that shields cysts. One cell cycle is common amongst tachyzoites and in vitro- and in vivo-derived bradyzoites; the other is unique to in vivo-derived bradyzoites (BCC). We demonstrated that AP2XI-6, expressed in G1 in vivo, is dispensable for tachyzoite growth and cyst formation in vitro but necessary for encystation in vivo. We propose that AP2XI-6 is a driver of replication through the BCC that is required for cyst maturation and persistence.

microbiology↗

bioRxiv2024

Perforation of the host cell plasma membrane during Toxoplasma gondii invasion requires rhoptry exocytosis

Toxoplasma gondii is an obligate intracellular parasite, and the delivery of effector proteins from the parasite into the host cell during invasion is critical for invasion itself and for parasite virulence. The effector proteins are released from specialized apical secretory organelles known as rhoptries. While much has been learned recently about the structure and composition of the rhoptry exocytic machinery and the function of individual rhoptry effector proteins that are exocytosed, virtually nothing is known about how the released proteins are translocated across the host cell plasma membrane. Previous electrophysiology experiments reported an unanticipated observation that invasion by T. gondii is preceded by a transient increase in host cell plasma membrane conductance. Here, we confirm this electrophysiological observation and propose that the conductance transient represents a parasite-induced perforation in the host cell plasma membrane through which rhoptry proteins are delivered. As a first step towards testing this hypothesis, and to provide higher throughput than patch clamp electrophysiology, we developed an alternative assay to detect the perforation. This assay utilizes high-speed, multi-wavelength fluorescence imaging to enable simultaneous visualization of host cell perforation and parasite invasion. Using this assay, we interrogated a panel of mutant parasites conditionally depleted of key invasion-related proteins. Parasites lacking signaling proteins involved in triggering rhoptry secretion (e.g., CLAMP) or components of the rhoptry exocytic machinery (e.g., Nd9, RASP2) are defective in their ability to induce the perforation. These data are consistent with a model in which the perforating agents that disrupt host cell membrane integrity during invasion - and may thereby provide the conduit for delivery of rhoptry effector proteins - are stored within the rhoptries themselves and released upon contact with the host cell.

microbiology↗

bioRxiv2024

Motility-dependent processes in Toxoplasma gondii tachyzoites and bradyzoites: same same but different

The tachyzoite stage of the apicomplexan parasite Toxoplasma gondii utilizes motility for multiple purposes during its lytic cycle, including host cell invasion, egress from infected cells, and migration to new uninfected host cells to repeat the process. Bradyzoite stage parasites, which establish a new infection in a naive host, must also use motility to escape from the cysts that are ingested by the new host and then migrate to the gut wall, where they either invade cells of the intestinal epithelium or squeeze between these cells to infect the underlying connective tissue. We know very little about the motility of bradyzoites, which we analyze in detail here and compare to the well-characterized motility and motility-dependent processes of tachyzoites. Unexpectedly, bradyzoites were found to be as motile as tachyzoites in a 3D model extracellular matrix, and they showed increased invasion into and transmigration across certain cell types, consistent with their need to establish the infection in the gut. The motility of the two stages was inhibited to the same extent by cytochalasin D and KNX-002, compounds known to target the parasites actomyosin-based motor. In contrast, other compounds that impact tachyzoite motility (tachyplegin and enhancer 5) have less of an effect on bradyzoites, and rapid bradyzoite egress from infected cells is not triggered by treatment with calcium ionophores, as it is with tachyzoites. The similarities and differences between these two life cycle stages highlight the need to characterize both tachyzoites and bradyzoites for a more complete understanding of the role of motility in the parasite life cycle and the effect that potential therapeutics targeting parasite motility will have on disease establishment and progression.

microbiology↗

bioRxiv2023

The Toxoplasma subpellicular network is highly interconnected and defines parasite shape for efficient motility and replication

Apicomplexan parasites possess several specialized structures to invade their host cells and replicate successfully. One of these is the inner membrane complex (IMC), a peripheral membrane-cytoskeletal system underneath the plasma membrane. It is composed of a series of flattened, membrane-bound vesicles and a cytoskeletal subpellicular network (SPN) comprised of intermediate filament-like proteins called alveolins. While the alveolin proteins are conserved throughout the Apicomplexa and the broader Alveolata, their precise functions and interactions remain poorly understood. Here, we describe the function of one of these alveolin proteins, TgIMC6. Disruption of IMC6 resulted in striking morphological defects that led to aberrant motility, invasion, and replication. Deletion analyses revealed that the alveolin domain alone is largely sufficient to restore localization and partially sufficient for function. As this highlights the importance of the IMC6 alveolin domain, we implemented unnatural amino acid photoreactive crosslinking to the alveolin domain and identified multiple binding interfaces between IMC6 and two other cytoskeletal proteins - IMC3 and ILP1. To our knowledge, this provides the first direct evidence of protein-protein interactions in the alveolin domain and supports the long-held hypothesis that the alveolin domain is responsible for filament formation. Collectively, our study features the conserved alveolin proteins as critical components that maintain the parasites structural integrity and highlights the alveolin domain as a key mediator of SPN architecture.

microbiology↗

bioRxiv2022

Identification of KNX-002, a novel inhibitor of Toxoplasma motility targeting the Class XIV myosin, TgMyoA

Toxoplasma gondii is a widespread apicomplexan parasite that can cause severe disease in its human hosts. The ability of T. gondii and other apicomplexan parasites to invade into, egress from, and move between cells of the hosts they infect is critical to parasite virulence and disease progression. An unusual and highly conserved parasite myosin motor (TgMyoA) plays a central role in T. gondii motility. The goal of this work was to test whether pharmacological inhibition of TgMyoA can alter disease progression in an animal model of infection. To this end, we sought to identify small molecule inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant motors actin-activated ATPase activity. The top hit to emerge from the screen, KNX-002, inhibited TgMyoA with little to no effect on any of the vertebrate myosins tested. KNX-002 was also active against parasites, inhibiting parasite motility and growth in culture in a dose-dependent manner. We used chemical mutagenesis, selection in KNX-002, and targeted sequencing to identify a mutation in TgMyoA (T130A) that renders the recombinant motor less sensitive to compound. Compared to wild-type parasites, parasites expressing the T130A mutation showed reduced sensitivity to KNX-002 in motility and growth assays, confirming TgMyoA as a biologically relevant target of KNX-002. Finally, KNX-002 was shown to slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. These data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target for toxoplasmosis. Since TgMyoA is essential for virulence, conserved in apicomplexan parasites, and distinctly different from the myosins found in humans, pharmacological inhibition of MyoA offers a promising new approach to treating the devastating diseases caused by T. gondii and other apicomplexan parasites.

microbiology↗

bioRxiv2022

A circular zone of attachment to the extracellular matrix acts as a guidance system for Toxoplasma gondii 3D motility

Toxoplasma gondii is a protozoan parasite that infects 30-40% of the worlds population. Infections are typically subclinical but can be severe and, in some cases, life threatening. Central to the virulence of T. gondii is an unusual form of substrate-dependent motility that enables the parasite to invade cells of its host and to disseminate throughout the body. A hetero-oligomeric complex of proteins that functions in motility has been characterized, but how these proteins work together to drive forward motion of the parasite remains controversial. A key piece of information needed to understand the underlying mechanism(s) is the directionality of the forces that a moving parasite exerts on the external environment. The linear motor model of motility, which has dominated the field for the past two decades, predicts continuous anterior-to-posterior force generation along the length of the parasite. We show here using three-dimensional traction force mapping that the predominant forces exerted by a moving parasite are instead periodic and directed in towards the parasite at a fixed circular location within the extracellular matrix. These highly localized forces, which are generated by the parasite pulling on the matrix, create a visible constriction in the parasites plasma membrane. We propose that the ring of inward-directed force corresponds to a circumferential attachment zone between the parasite and the matrix, through which the parasite propels itself to move forward. The combined data suggest a closer connection between the mechanisms underlying parasite motility and host cell invasion than previously recognized. In parasites lacking the major surface adhesin, TgMIC2, neither the inward-directed forces nor the constriction of the parasite membrane are observed. The trajectories of the TgMIC2-deficient parasites are less straight than those of wild-type parasites, suggesting that the annular zone of TgMIC2-mediated attachment to the extracellular matrix normally constrains the directional options available to the parasite as it migrates through its surrounding environment.

microbiology↗

bioRxiv2022

The potent Toxoplasma gondii growth inhibitor QQ-437 does not bind to its predicted target, parasite adaptin3β, in yeast three-hybrid assays

New drugs are needed to treat infections with Toxoplasma gondii, a ubiquitous protozoan parasite that can cause miscarriage, blindness, and life-threatening encephalitis in its human hosts. A novel N-benzoyl-2-hydroxybenzamide named QQ-437 was recently shown to be a potent inhibitor of T. gondii growth in vitro (EC50 = 16nM), and to reduce parasite load in a mouse model of infection. Intriguingly, mutations in the parasite protein TgAP3{beta}, which is thought to play an important role in intracellular protein trafficking, confer resistance to QQ-437. In this report, we use yeast three-hybrid analysis to test the hypothesis that QQ-437 inhibits parasite growth via a direct effect on TgAP3{beta}. We see no evidence for the ability of QQ-437 to bind directly to TgAP3{beta}, suggesting that the mutation in TgAP3{beta} that confers resistance does so through an indirect mechanism. Further studies are needed to identify the direct molecular target(s) of this promising class of compounds.

microbiology↗

bioRxiv2019

Differential requirements of cyclase associated protein (CAP) for actin turnover during the lytic cycle of Toxoplasma gondii

Toxoplasma gondii contains a limited subset of actin binding proteins. Here we show that ablation of the putative actin regulator cyclase-associated protein (TgCAP) leads to significant defects in some but not all actin dependent processes, including a defect in cell-cell communication, but surprisingly not synchronicity of division. Two CAP isoforms originate from alternative translational start sites and are beneficial for parasite fitness while a single isoform is sufficient for virulence in mice. Examination of the mutant parasites by 3D electron microscopy reveals that loss of CAP results in a defect to form a normal residual body, but all parasites remain connected within the vacuole. This dissociates synchronicity of division and parasite rosetting and reveals that establishment and maintenance of the residual body may be more complex than previously thought. These results highlight the different spatial requirements for actin turnover in Toxoplasma, controlled by a reduced subset of actin binding proteins.

microbiology↗