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 parasite's structural integrity and highlights the alveolin domain as a key mediator of SPN architecture.

CDPK2A and CDPK1 form a signaling module upstream of Toxoplasma motility.

IMPORTANCE: This work uncovers interactions between various signaling pathways that govern Toxoplasma gondii egress. Specifically, we compare the function of three canonical calcium-dependent protein kinases (CDPKs) using chemical-genetic and conditional-depletion approaches. We describe the function of a previously uncharacterized CDPK, CDPK2A, in the Toxoplasma lytic cycle, demonstrating that it contributes to parasite fitness through regulation of microneme discharge, gliding motility, and egress from infected host cells. Comparison of analog-sensitive kinase alleles and conditionally depleted alleles uncovered epistasis between CDPK2A and CDPK1, implying a partial functional redundancy. Understanding the topology of signaling pathways underlying key events in the parasite life cycle can aid in efforts targeting kinases for anti-parasitic therapies.

MyosinA is a druggable target in the widespread protozoan parasite Toxoplasma gondii.

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 determine whether the parasite's motility and lytic cycle can be disrupted through pharmacological inhibition of TgMyoA, as an approach to altering disease progression in vivo. To this end, we first sought to identify inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant motor's 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, we present evidence that KNX-002 can slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. Taken together, these data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target in infections with T. gondii. 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.

A circular zone of attachment to the extracellular matrix provides directionality to the motility of Toxoplasma gondii in 3D.

Toxoplasma gondii is a protozoan parasite that infects 30-40% of the world's 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 toward 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 parasite's 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.

Blocking Palmitoylation of Toxoplasma gondii Myosin Light Chain 1 Disrupts Glideosome Composition but Has Little Impact on Parasite Motility.

Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in immunocompromised individuals and the developing fetus. Like other apicomplexans, T. gondii uses an unusual form of substrate-dependent gliding motility to invade cells of its hosts and to disseminate throughout the body during infection. It is well established that a myosin motor consisting of a class XIVa heavy chain (TgMyoA) and two light chains (TgMLC1 and TgELC1/2) plays an important role in parasite motility. The ability of the motor to generate force at the parasite periphery is thought to be reliant upon its anchoring and immobilization within a peripheral membrane-bound compartment, the inner membrane complex (IMC). The motor does not insert into the IMC directly; rather, this interaction is believed to be mediated by the binding of TgMLC1 to the IMC-anchored protein, TgGAP45. Therefore, the binding of TgMLC1 to TgGAP45 is considered a key element in the force transduction machinery of the parasite. TgMLC1 is palmitoylated, and we show here that palmitoylation occurs on two N-terminal cysteine residues, C8 and C11. Mutations that block TgMLC1 palmitoylation completely abrogate the binding of TgMLC1 to TgGAP45. Surprisingly, the loss of TgMLC1 binding to TgGAP45 in these mutant parasites has little effect on their ability to initiate or sustain movement. These results question a key tenet of the current model of apicomplexan motility and suggest that our understanding of gliding motility in this important group of human and animal pathogens is not yet complete.IMPORTANCE Gliding motility plays a central role in the life cycle of T. gondii and other apicomplexan parasites. The myosin motor thought to power motility is essential for virulence but distinctly different from the myosins found in humans. Consequently, an understanding of the mechanism(s) underlying parasite motility and the role played by this unusual myosin may reveal points of vulnerability that can be targeted for disease prevention or treatment. We show here that mutations that uncouple the motor from what is thought to be a key structural component of the motility machinery have little impact on parasite motility. This finding runs counter to predictions of the current, widely held "linear motor" model of motility, highlighting the need for further studies to fully understand how apicomplexan parasites generate the forces necessary to move into, out of, and between cells of the hosts they infect.

Differential requirements for cyclase-associated protein (CAP) in actin-dependent processes of Toxoplasma gondii.

Toxoplasma gondii contains a limited subset of actin binding proteins. Here we show that the putative actin regulator cyclase-associated protein (CAP) is present in two different isoforms and its deletion leads to significant defects in some but not all actin dependent processes. We observe defects in cell-cell communication, daughter cell orientation and the juxtanuclear accumulation of actin, but only modest defects in synchronicity of division and no defect in the replication of the apicoplast. 3D electron microscopy reveals that loss of CAP results in a defect in formation of a normal central residual body, but 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 F-actin regulation in Toxoplasma which appear to be achieved by partially overlapping functions of actin regulators.

Structural and mechanistic insights into the function of the unconventional class XIV myosin MyoA from Toxoplasma gondii.

Parasites of the phylum Apicomplexa are responsible for significant morbidity and mortality on a global scale. Central to the virulence of these pathogens are the phylum-specific, unconventional class XIV myosins that power the essential processes of parasite motility and host cell invasion. Notably, class XIV myosins differ from human myosins in key functional regions, yet they are capable of fast movement along actin filaments with kinetics rivaling previously studied myosins. Toward establishing a detailed molecular mechanism of class XIV motility, we determined the 2.6-Å resolution crystal structure of the Toxoplasma gondii MyoA (TgMyoA) motor domain. Structural analysis reveals intriguing strategies for force transduction and chemomechanical coupling that rely on a divergent SH1/SH2 region, the class-defining "HYAG"-site polymorphism, and the actin-binding surface. In vitro motility assays and hydrogen-deuterium exchange coupled with MS further reveal the mechanistic underpinnings of phosphorylation-dependent modulation of TgMyoA motility whereby localized regions of increased stability and order correlate with enhanced motility. Analysis of solvent-accessible pockets reveals striking differences between apicomplexan class XIV and human myosins. Extending these analyses to high-confidence homology models of Plasmodium and Cryptosporidium MyoA motor domains supports the intriguing potential of designing class-specific, yet broadly active, apicomplexan myosin inhibitors. The successful expression of the functional TgMyoA complex combined with our crystal structure of the motor domain provides a strong foundation in support of detailed structure-function studies and enables the development of small-molecule inhibitors targeting these devastating global pathogens.

Dissecting the molecular assembly of the Toxoplasma gondii MyoA motility complex.

Apicomplexan parasites such as Toxoplasma gondii rely on a unique form of locomotion known as gliding motility. Generating the mechanical forces to support motility are divergent class XIV myosins (MyoA) coordinated by accessory proteins known as light chains. Although the importance of the MyoA-light chain complex is well-established, the detailed mechanisms governing its assembly and regulation are relatively unknown. To establish a molecular blueprint of this dynamic complex, we first mapped the adjacent binding sites of light chains MLC1 and ELC1 on the MyoA neck (residues 775-818) using a combination of hydrogen-deuterium exchange mass spectrometry and isothermal titration calorimetry. We then determined the 1.85 Å resolution crystal structure of MLC1 in complex with its cognate MyoA peptide. Structural analysis revealed a bilobed architecture with MLC1 clamping tightly around the helical MyoA peptide, consistent with the stable 10 nm Kd measured by isothermal titration calorimetry. We next showed that coordination of calcium by an EF-hand in ELC1 and prebinding of MLC1 to the MyoA neck enhanced the affinity of ELC1 for the MyoA neck 7- and 8-fold, respectively. When combined, these factors enhanced ELC1 binding 49-fold (to a Kd of 12 nm). Using the full-length MyoA motor (residues 1-831), we then showed that, in addition to coordinating the neck region, ELC1 appears to engage the MyoA converter subdomain, which couples the motor domain to the neck. These data support an assembly model where staged binding events cooperate to yield high-affinity complexes that are able to maximize force transduction.

Parasites lacking the micronemal protein MIC2 are deficient in surface attachment and host cell egress, but remain virulent in vivo.

Background: Micronemal proteins of the thrombospondin-related anonymous protein (TRAP) family are believed to play essential roles during gliding motility and host cell invasion by apicomplexan parasites, and currently represent major vaccine candidates against Plasmodium falciparum, the causative agent of malaria. However, recent evidence suggests that they play multiple and different roles than previously assumed. Here, we analyse a null mutant for MIC2, the TRAP homolog in Toxoplasma gondii. Methods: We performed a careful analysis of parasite motility in a 3D-environment, attachment under shear stress conditions, host cell invasion and in vivo virulence. Results: We verified the role of MIC2 in efficient surface attachment, but were unable to identify any direct function of MIC2 in sustaining gliding motility or host cell invasion once initiated. Furthermore, we find that deletion of mic2 causes a slightly delayed infection in vivo, leading only to mild attenuation of virulence; like with wildtype parasites, inoculation with even low numbers of mic2 KO parasites causes lethal disease in mice. However, deletion of mic2 causes delayed host cell egress in vitro, possibly via disrupted signal transduction pathways. Conclusions: We confirm a critical role of MIC2 in parasite attachment to the surface, leading to reduced parasite motility and host cell invasion. However, MIC2 appears to not be critical for gliding motility or host cell invasion, since parasite speed during these processes is unaffected. Furthermore, deletion of MIC2 leads only to slight attenuation of the parasite.

Surface attachment, promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion.

BACKGROUND: Apicomplexan parasites employ a unique form of movement, termed gliding motility, in order to invade the host cell. This movement depends on the parasite's actomyosin system, which is thought to generate the force during gliding. However, recent evidence questions the exact molecular role of this system, since mutants for core components of the gliding machinery, such as parasite actin or subunits of the MyoA-motor complex (the glideosome), remain motile and invasive, albeit at significantly reduced efficiencies. While compensatory mechanisms and unusual polymerisation kinetics of parasite actin have been evoked to explain these findings, the actomyosin system could also play a role distinct from force production during parasite movement. RESULTS: In this study, we compared the phenotypes of different mutants for core components of the actomyosin system in Toxoplasma gondii to decipher their exact role during gliding motility and invasion. We found that, while some phenotypes (apicoplast segregation, host cell egress, dense granule motility) appeared early after induction of the act1 knockout and went to completion, a small percentage of the parasites remained capable of motility and invasion well past the point at which actin levels were undetectable. Those act1 conditional knockout (cKO) and mlc1 cKO that continue to move in 3D do so at speeds similar to wildtype parasites. However, these mutants are virtually unable to attach to a collagen-coated substrate under flow conditions, indicating an important role for the actomyosin system of T. gondii in the formation of attachment sites. CONCLUSION: We demonstrate that parasite actin is essential during the lytic cycle and cannot be compensated by other molecules. Our data suggest a conventional polymerisation mechanism in vivo that depends on a critical concentration of G-actin. Importantly, we demonstrate that the actomyosin system of the parasite functions in attachment to the surface substrate, and not necessarily as force generator.

Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail.

UNLABELLED: Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondii that plays a critical role in host cell invasion. The ligand to which T gondii AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the "moving junction," a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion. IMPORTANCE: Nearly one-third of the world's population is infected with the protozoan parasite Toxoplasma gondii, which causes life-threatening disease in neonates and immunocompromised individuals. T. gondii is a member of the phylum Apicomplexa, which includes many other parasites of veterinary and medical importance, such as those that cause coccidiosis, babesiosis, and malaria. Apicomplexan parasites grow within their hosts through repeated cycles of host cell invasion, parasite replication, and host cell lysis. Parasites that cannot invade host cells cannot survive or cause disease. AMA1 is a highly conserved protein on the surface of apicomplexan parasites that is known to be important for invasion, and the work presented here reveals new and unexpected insights into AMA1 function. A more complete understanding of the role of AMA1 in invasion may ultimately contribute to the development of new chemotherapeutics designed to disrupt AMA1 function and invasion-related signaling in this important group of human pathogens.

Global Analysis of Palmitoylated Proteins in Toxoplasma gondii.

Post-translational modifications (PTMs) such as palmitoylation are critical for the lytic cycle of the protozoan parasite Toxoplasma gondii. While palmitoylation is involved in invasion, motility, and cell morphology, the proteins that utilize this PTM remain largely unknown. Using a chemical proteomic approach, we report a comprehensive analysis of palmitoylated proteins in T. gondii, identifying a total of 282 proteins, including cytosolic, membrane-associated, and transmembrane proteins. From this large set of palmitoylated targets, we validate palmitoylation of proteins involved in motility (myosin light chain 1, myosin A), cell morphology (PhIL1), and host cell invasion (apical membrane antigen 1, AMA1). Further studies reveal that blocking AMA1 palmitoylation enhances the release of AMA1 and other invasion-related proteins from apical secretory organelles, suggesting a previously unrecognized role for AMA1. These findings suggest that palmitoylation is ubiquitous throughout the T. gondii proteome and reveal insights into the biology of this important human pathogen.

Yeast three-hybrid screen identifies TgBRADIN/GRA24 as a negative regulator of Toxoplasma gondii bradyzoite differentiation.

Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite's life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen.

A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.

Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 µm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 µm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.

Calcium-dependent phosphorylation alters class XIVa myosin function in the protozoan parasite Toxoplasma gondii.

Class XIVa myosins comprise a unique group of myosin motor proteins found in apicomplexan parasites, including those that cause malaria and toxoplasmosis. The founding member of the class XIVa family, Toxoplasma gondii myosin A (TgMyoA), is a monomeric unconventional myosin that functions at the parasite periphery to control gliding motility, host cell invasion, and host cell egress. How the motor activity of TgMyoA is regulated during these critical steps in the parasite's lytic cycle is unknown. We show here that a small-molecule enhancer of T. gondii motility and invasion (compound 130038) causes an increase in parasite intracellular calcium levels, leading to a calcium-dependent increase in TgMyoA phosphorylation. Mutation of the major sites of phosphorylation altered parasite motile behavior upon compound 130038 treatment, and parasites expressing a nonphosphorylatable mutant myosin egressed from host cells more slowly in response to treatment with calcium ionophore. These data demonstrate that TgMyoA undergoes calcium-dependent phosphorylation, which modulates myosin-driven processes in this important human pathogen.

Identification of TgCBAP, a novel cytoskeletal protein that localizes to three distinct subcompartments of the Toxoplasma gondii pellicle.

The cytoskeletons of Toxoplasma gondii and related apicomplexan parasites are highly polarized, with apical and basal regions comprised of distinct protein complexes. Components of these complexes are known to play important roles in parasite shape, cell division, and host cell invasion. During an effort to discover the biologically relevant target of a small-molecule inhibitor of T. gondii invasion (Conoidin A), we discovered a novel cytoskeletal protein that we named TgCBAP (Conserved Basal Apical Peripheral protein). Orthologs of TgCBAP are only found in the genomes of other apicomplexans; they contain no identifiable domains or motifs and their function(s) is unknown. As a first step toward elucidating the function of this highly conserved family of proteins, we disrupted the TgCBAP gene by double homologous recombination. Parasites lacking TgCBAP are as sensitive to the effects of Conoidin A as wild-type parasites, demonstrating that TgCBAP is not the biologically relevant target of Conoidin A. However, ΔTgCBAP parasites are significantly shorter than wild-type parasites and have a growth defect in culture. Furthermore, TgCBAP has an unusual subcellular localization, forming small rings at the apical and basal ends of the parasite and localizing to punctate, ring-like structures around the parasite periphery. These data identify a new marker of the apical and basal subcompartments of T. gondii, reveal a potentially novel compartment along the parasite periphery, and identify TgCBAP as a determinant of parasite size that is required for a maximally efficient lytic cycle.

Identification of T. gondii myosin light chain-1 as a direct target of TachypleginA-2, a small-molecule inhibitor of parasite motility and invasion.

Motility of the protozoan parasite Toxoplasma gondii plays an important role in the parasite's life cycle and virulence within animal and human hosts. Motility is driven by a myosin motor complex that is highly conserved across the Phylum Apicomplexa. Two key components of this complex are the class XIV unconventional myosin, TgMyoA, and its associated light chain, TgMLC1. We previously showed that treatment of parasites with a small-molecule inhibitor of T. gondii invasion and motility, tachypleginA, induces an electrophoretic mobility shift of TgMLC1 that is associated with decreased myosin motor activity. However, the direct target(s) of tachypleginA and the molecular basis of the compound-induced TgMLC1 modification were unknown. We show here by "click" chemistry labelling that TgMLC1 is a direct and covalent target of an alkyne-derivatized analogue of tachypleginA. We also show that this analogue can covalently bind to model thiol substrates. The electrophoretic mobility shift induced by another structural analogue, tachypleginA-2, was associated with the formation of a 225.118 Da adduct on S57 and/or C58, and treatment with deuterated tachypleginA-2 confirmed that the adduct was derived from the compound itself. Recombinant TgMLC1 containing a C58S mutation (but not S57A) was refractory to click labelling and no longer exhibited a mobility shift in response to compound treatment, identifying C58 as the site of compound binding on TgMLC1. Finally, a knock-in parasite line expressing the C58S mutation showed decreased sensitivity to compound treatment in a quantitative 3D motility assay. These data strongly support a model in which tachypleginA and its analogues inhibit the motility of T. gondii by binding directly and covalently to C58 of TgMLC1, thereby causing a decrease in the activity of the parasite's myosin motor.

Identification of Cryptosporidium parvum active chemical series by Repurposing the open access malaria box.

The apicomplexan parasites Cryptosporidium parvum and Cryptosporidium hominis are major etiologic agents of human cryptosporidiosis. The infection is typically self-limited in immunocompetent adults, but it can cause chronic fulminant diarrhea in immunocompromised patients and malnutrition and stunting in children. Nitazoxanide, the current standard of care for cryptosporidiosis, is only partially efficacious for children and is no more effective than a placebo for AIDS patients. Unfortunately, financial obstacles to drug discovery for diseases that disproportionately affect low-income countries and technical limitations associated with studies of Cryptosporidium biology impede the development of better drugs for treating cryptosporidiosis. Using a cell-based high-throughput screen, we queried the Medicines for Malaria Venture (MMV) Open Access Malaria Box for activity against C. parvum. We identified 3 novel chemical series derived from the quinolin-8-ol, allopurinol-based, and 2,4-diamino-quinazoline chemical scaffolds that exhibited submicromolar potency against C. parvum. Potency was conserved in a subset of compounds from each scaffold with varied physicochemical properties, and two of the scaffolds identified exhibit more rapid inhibition of C. parvum growth than nitazoxanide, making them excellent candidates for further development. The 2,4-diamino-quinazoline and allopurinol-based compounds were also potent growth inhibitors of the related apicomplexan parasite Toxoplasma gondii, and a good correlation was observed in the relative activities of the compounds in the allopurinol-based series against T. gondii and C. parvum. Taken together, these data illustrate the utility of the Open Access Malaria Box as a source of both potential leads for drug development and chemical probes to elucidate basic biological processes in C. parvum and other apicomplexan parasites.

Disruption of TgPHIL1 alters specific parameters of Toxoplasma gondii motility measured in a quantitative, three-dimensional live motility assay.

T. gondii uses substrate-dependent gliding motility to invade cells of its hosts, egress from these cells at the end of its lytic cycle and disseminate through the host organism during infection. The ability of the parasite to move is therefore critical for its virulence. T. gondii engages in three distinct types of gliding motility on coated two-dimensional surfaces: twirling, circular gliding and helical gliding. We show here that motility in a three-dimensional Matrigel-based environment is strikingly different, in that all parasites move in irregular corkscrew-like trajectories. Methods developed for quantitative analysis of motility parameters along the smoothed trajectories demonstrate a complex but periodic pattern of motility with mean and maximum velocities of 0.58 ± 0.07 µm/s and 2.01 ± 0.17 µm/s, respectively. To test how a change in the parasite's crescent shape might affect trajectory parameters, we compared the motility of Δphil1 parasites, which are shorter and wider than wild type, to the corresponding parental and complemented lines. Although comparable percentages of parasites were moving for all three lines, the Δphil1 mutant exhibited significantly decreased trajectory lengths and mean and maximum velocities compared to the parental parasite line. These effects were either partially or fully restored upon complementation of the Δphil1 mutant. These results show that alterations in morphology may have a significant impact on T. gondii motility in an extracellular matrix-like environment, provide a possible explanation for the decreased fitness of Δphil1 parasites in vivo, and demonstrate the utility of the quantitative three-dimensional assay for studying parasite motility.

A modular approach to triazole-containing chemical inducers of dimerisation for yeast three-hybrid screening.

The yeast three-hybrid (Y3H) approach shows considerable promise for the unbiased identification of novel small molecule-protein interactions. In recent years, it has been successfully used to link a number of bioactive molecules to novel protein binding partners. However despite its potential importance as a protein target identification method, the Y3H technique has not yet been widely adopted, in part due to the challenges associated with the synthesis of the complex chemical inducers of dimerisation (CIDs). The development of a modular approach using potentially "off the shelf" synthetic components was achieved and allowed the synthesis of a family of four triazole-containing CIDs, MTX-Cmpd2.2-2.5. These CIDs were then compared using the Y3H approach with three of them giving a strong positive interaction with a known target of compound 2, TgCDPK1. These results showed that the modular nature of our synthetic strategy may help to overcome the challenges currently encountered with CID synthesis and should contribute to the Y3H approach reaching its full potential as an unbiased target identification strategy.