RESUMEN
T. gondii is a eukaryotic parasite that has evolved a stage called tachyzoite which multiplies in host cells by producing two daughter cells internally. These nascent tachyzoites bud off their mother and repeat the division process until the expanding progenies escape to settle and multiply in other host cells. Over these intra- and extra-cellular phases, the tachyzoite maintains an essential apicobasal polarity that emerges through a unique bidirectional budding process of the elongating cells. This process requires the assembly of several molecular complexes that, at the nascent pole, encompass structural and myosin motor elements. To characterize a recently identified basal pole marker named BCC7 with respect to the posterior myosin J and myosin C motors, we used conventional biochemistry as well as advanced proteomic and in silico analysis in conjunction with live and super resolution microscopy of transgenic fluorescent tachyzoites. We document that BCC7 forms a ribbed ring below which myosin C motor entities distribute regularly. In addition, we identified-among 13 BCC7 putative partners-two novel and five known members of the inner membrane complex (IMC) family which ends at the apical side of the ring. Therefore, BCC7 could assist the stabilization of the IMC plaques and contribute to the parasite biomechanical properties.
Asunto(s)
Toxoplasma , División Celular , Miosinas/metabolismo , Proteómica , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismoRESUMEN
N-myristoylation is a ubiquitous class of protein lipidation across eukaryotes and N-myristoyl transferase (NMT) has been proposed as an attractive drug target in several pathogens. Myristoylation often primes for subsequent palmitoylation and stable membrane attachment, however, growing evidence suggests additional regulatory roles for myristoylation on proteins. Here we describe the myristoylated proteome of Toxoplasma gondii using chemoproteomic methods and show that a small-molecule NMT inhibitor developed against related Plasmodium spp. is also functional in Toxoplasma. We identify myristoylation on a transmembrane protein, the microneme protein 7 (MIC7), which enters the secretory pathway in an unconventional fashion with the myristoylated N-terminus facing the lumen of the micronemes. MIC7 and its myristoylation play a crucial role in the initial steps of invasion, likely during the interaction with and penetration of the host cell. Myristoylation of secreted eukaryotic proteins represents a substantial expansion of the functional repertoire of this co-translational modification.
A microscopic parasite known as Toxoplasma gondii infects around 30% of the human population. Most infections remain asymptomatic, but in people with a compromised immune system, developing fetuses and people infected with particular virulent strains of the parasite, infection can be fatal. T. gondii is closely related to other parasites that also infect humans, including the one that causes malaria. These parasites have complex lifecycles that involve successive rounds of invading the cells of their hosts, growing and then exiting these cells. Signaling proteins found at specific locations within parasite cells regulate the ability of the parasites to interact with and invade host cells. Sometimes these signaling proteins are attached to membranes using lipid anchors, for example through a molecule called myristic acid. An enzyme called NMT can attach myristic acid to one end of its target proteins. The myristic acid tag can influence the ability of target proteins to bind to other proteins, or to membranes. Previous studies have found that drugs that inhibit the NMT enzyme prevent the malaria parasite from successfully invading and growing inside host cells. The NMT enzyme from T. gondii is very similar to that of the malaria parasite. Broncel et al. have shown that the drug developed against P. falciparum also inhibits the ability of T. gondii to grow. These findings suggest that drugs against the NMT enzyme may be useful to treat diseases caused by T. gondii and other closely-related parasites. Broncel et al. also identified 65 proteins in T. gondii that contain a myristic acid tag using an approach called proteomics. One of the unexpected 'myristoylated' proteins identified in the experiments is known as MIC7. This protein was found to be transported onto the surface of T. gondii parasites and is required in its myristoylated form for the parasite to successfully invade host cells. This was surprising as myristoylated proteins are generally thought to not enter the pathway that brings proteins to the outside of cell. These findings suggest that myristic acid on proteins that are secreted can facilitate interactions between cells, maybe by inserting the myristic acid into the cell membrane.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Fibroblastos/parasitología , Proteínas de la Membrana/metabolismo , Ácidos Mirísticos/química , Proteínas Protozoarias/metabolismo , Toxoplasma/genética , Toxoplasma/fisiología , Aciltransferasas/fisiología , Animales , Animales Modificados Genéticamente , Proteínas de Unión al Calcio/genética , Línea Celular , Línea Celular Tumoral , Membrana Celular/fisiología , Humanos , Proteínas de la Membrana/genética , Microscopía por Video , Dominios Proteicos , Proteómica , Proteínas Protozoarias/genéticaRESUMEN
Among the eukaryotic cells that navigate through fully developed metazoan tissues, protozoans from the Apicomplexa phylum have evolved motile developmental stages that move much faster than the fastest crawling cells owing to a peculiar substrate-dependent type of motility, known as gliding. Best-studied models are the Plasmodium sporozoite and the Toxoplasma tachyzoite polarized cells for which motility is vital to achieve their developmental programs in the metazoan hosts. The gliding machinery is shared between the two parasites and is largely characterized. Localized beneath the cell surface, it includes actin filaments, unconventional myosin motors housed within a multimember glideosome unit, and apically secreted transmembrane adhesins. In contrast, less is known about the force mechanisms powering cell movement. Pioneered biophysical studies on the sporozoite and phenotypic analysis of tachyzoite actin-related mutants have added complexity to the general view that force production for parasite forward movement directly results from the myosin-driven rearward motion of the actin-coupled adhesion sites. Here, we have interrogated how forces and substrate adhesion-de-adhesion cycles operate and coordinate to allow the typical left-handed helical gliding mode of the tachyzoite. By combining quantitative traction force and reflection interference microscopy with micropatterning and expansion microscopy, we unveil at the millisecond and nanometer scales the integration of a critical apical anchoring adhesion with specific traction and spring-like forces. We propose that the acto-myoA motor directs the traction force which allows transient energy storage by the microtubule cytoskeleton and therefore sets the thrust force required for T. gondii tachyzoite vital helical gliding capacity.
Asunto(s)
Parásitos , Toxoplasma , Actinas , Animales , Proteínas Protozoarias , Torque , TracciónRESUMEN
Apicomplexan parasites are unicellular eukaryotic pathogens that must obtain and combine lipids from both host cell scavenging and de novo synthesis to maintain parasite propagation and survival within their human host. Major questions on the role and regulation of each lipid source upon fluctuating host nutritional conditions remain unanswered. Characterization of an apicoplast acyltransferase, TgATS2, shows that the apicoplast provides (lyso)phosphatidic acid, required for the recruitment of a critical dynamin (TgDrpC) during parasite cytokinesis. Disruption of TgATS2 also leads parasites to shift metabolic lipid acquisition from de novo synthesis toward host scavenging. We show that both lipid scavenging and de novo synthesis pathways in wild-type parasites exhibit major metabolic and cellular plasticity upon sensing host lipid-deprived environments through concomitant (1) upregulation of de novo fatty acid synthesis capacities in the apicoplast and (2) parasite-driven host remodeling to generate multi-membrane-bound structures from host organelles that are imported toward the parasite.
Asunto(s)
Adaptación Fisiológica , Apicoplastos/metabolismo , División Celular , Interacciones Huésped-Parásitos , Metabolismo de los Lípidos , Parásitos/metabolismo , Toxoplasma/metabolismo , Toxoplasma/fisiología , Aciltransferasas/metabolismo , Animales , Membrana Celular/metabolismo , Citocinesis , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/biosíntesis , Eliminación de Gen , Humanos , Espacio Intracelular/parasitología , Estadios del Ciclo de Vida , Lipidómica , Masculino , Modelos Biológicos , Cuerpos Multivesiculares/metabolismo , Cuerpos Multivesiculares/ultraestructura , Mutación/genética , Nutrientes , Parásitos/crecimiento & desarrollo , Parásitos/fisiología , Parásitos/ultraestructura , Proteínas Protozoarias/metabolismo , Toxoplasma/crecimiento & desarrollo , Toxoplasma/ultraestructuraRESUMEN
Biofilms are currently considered as a predominant lifestyle of many bacteria in nature. While they promote survival of microbes, biofilms also potentially increase the threats to animal and public health in case of pathogenic species. They not only facilitate bacteria transmission and persistence, but also promote spreading of antibiotic resistance leading to chronic infections. In the case of Francisella tularensis, the causative agent of tularemia, biofilms have remained largely enigmatic. Here, applying live and static confocal microscopy, we report growth and ultrastructural organization of the biofilms formed in vitro by these microorganisms over the early transition from coccobacillary into coccoid shape during biofilm assembly. Using selective dispersing agents, we provided evidence for extracellular DNA (eDNA) being a major and conserved structural component of mature biofilms formed by both F. subsp. novicida and a human clinical isolate of F. philomiragia. We also observed a higher physical robustness of F. novicida biofilm as compared to F. philomiragia one, a feature likely promoted by specific polysaccharides. Further, F. novicida biofilms resisted significantly better to ciprofloxacin than their planktonic counterparts. Importantly, when grown in biofilms, both Francisella species survived longer in cold water as compared to free-living bacteria, a trait possibly associated with a gain in fitness in the natural aquatic environment. Overall, this study provides information on survival of Francisella when embedded with biofilms that should improve both the future management of biofilm-related infections and the design of effective strategies to tackle down the problematic issue of bacteria persistence in aquatic ecosystems.
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Biopelículas , Farmacorresistencia Bacteriana , Francisella/fisiología , Agua Dulce/microbiología , Adaptación Fisiológica , Antibacterianos/farmacología , Ciprofloxacina/farmacología , Secuencia Conservada , ADN Bacteriano/química , Francisella/efectos de los fármacos , Francisella/genética , Francisella/patogenicidad , Infecciones por Bacterias Gramnegativas/microbiología , HumanosRESUMEN
We previously synthesized an hydroxamate derivative (N-hydroxy-4-[2-(3- methoxyphenyl)acetamido]benzamide) named 363 with potent anti-Toxoplasma gondii activity and histone deacetylase inhibitor (HDACi) effects. Here we show that 1-N-hydroxy-4-N- [(2-methoxyphenyl)methyl]benzene-1,4-dicarboxamide, a 363 isomer, does not have antiparasitic potency and has a 13-fold decrease in HDACi activity. The in silico modeling of T. gondii HDACs of the type II strain discloses identity varying from 25% to 62% on more than 250 residues for S8EP32_TOXG and A0A125YPH4_TOXGM. We observed a high conservation degree with the human HDAC2 (53% and 64% identity, respectively) and a moderate one with the human HDAC8 (30-40%). Two other TgHDACs, S8F6L4_TOXGM and S8GEI3_TOXGM, were identified as displaying a higher similarity with some bacterial orthologs (~35%) than with the human enzymes (~25%). The docking in parallel of the two compounds on the models generated allowed us to gain insights on the docking of these hydroxamate derivatives that guide their specificity and potency against T. gondii histone deacetylase. This information would constitute the rationale from which more specific derivatives can be synthetized.
Asunto(s)
Antiparasitarios/química , Antiparasitarios/farmacología , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Sitios de Unión , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Histona Desacetilasas/química , Humanos , Ligandos , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Unión Proteica , Relación Estructura-Actividad , Toxoplasma/efectos de los fármacosRESUMEN
The protozoan parasite Toxoplasma gondii has co-evolved with its homeothermic hosts (humans included) strategies that drive its quasi-asymptomatic persistence in hosts, hence optimizing the chance of transmission to new hosts. Persistence, which starts with a small subset of parasites that escape host immune killing and colonize the so-called immune privileged tissues where they differentiate into a low replicating stage, is driven by the interleukin 12 (IL-12)-interferon-γ (IFN-γ) axis. Recent characterization of a family of Toxoplasma effectors that are delivered into the host cell, in which they rewire the host cell gene expression, has allowed the identification of regulators of the IL-12-IFN-γ axis, including repressors. We now report on the dense granule-resident effector, called TEEGR (Toxoplasma E2F4-associated EZH2-inducing gene regulator) that counteracts the nuclear factor-κB (NF-κB) signalling pathway. Once exported into the host cell, TEEGR ends up in the nucleus where it not only complexes with the E2F3 and E2F4 host transcription factors to induce gene expression, but also promotes shaping of a non-permissive chromatin through its capacity to switch on EZH2. Remarkably, EZH2 fosters the epigenetic silencing of a subset of NF-κB-regulated cytokines, thereby strongly contributing to the host immune equilibrium that influences the host immune response and promotes parasite persistence in mice.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/metabolismo , FN-kappa B/metabolismo , Proteínas Protozoarias/metabolismo , Transducción de Señal/genética , Toxoplasma/fisiología , Animales , Línea Celular , Núcleo Celular/metabolismo , Citocinas/metabolismo , Factores de Transcripción E2F/genética , Factores de Transcripción E2F/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Expresión Génica , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Mutación , Carga de Parásitos , Regiones Promotoras Genéticas , Multimerización de Proteína , Proteínas Protozoarias/genética , Toxoplasma/genética , Toxoplasma/inmunología , Toxoplasmosis/metabolismo , Toxoplasmosis/parasitologíaRESUMEN
Dinitroanilines are chemical compounds with high selectivity for plant cell α-tubulin in which they promote microtubule depolymerization. They target α-tubulin regions that have diverged over evolution and show no effect on non-photosynthetic eukaryotes. Hence, they have been used as herbicides over decades. Interestingly, dinitroanilines proved active on microtubules of eukaryotes deriving from photosynthetic ancestors such as Toxoplasma gondii and Plasmodium falciparum, which are responsible for toxoplasmosis and malaria, respectively. By combining differential in silico screening of virtual chemical libraries on Arabidopsis thaliana and mammal tubulin structural models together with cell-based screening of chemical libraries, we have identified dinitroaniline related and non-related compounds. They inhibit plant, but not mammalian tubulin assembly in vitro, and accordingly arrest A. thaliana development. In addition, these compounds exhibit a moderate cytotoxic activity towards T. gondii and P. falciparum. These results highlight the potential of novel herbicidal scaffolds in the design of urgently needed anti-parasitic drugs.
Asunto(s)
Apicomplexa/fisiología , Plantas/metabolismo , Plantas/parasitología , Tubulina (Proteína)/metabolismo , Animales , Células HeLa , Humanos , Microtúbulos/metabolismo , Modelos Moleculares , Fotosíntesis , Células Vegetales/metabolismo , Plasmodium falciparum , Conformación Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/genéticaRESUMEN
The intracellular parasite Toxoplasma gondii, hijacks evolutionarily conserved host processes by delivering effector proteins into the host cell that shift gene expression in a timely fashion. We identified a parasite dense granule protein as GRA18 that once released in the host cell cytoplasm forms versatile complexes with regulatory elements of the ß-catenin destruction complex. By interacting with GSK3/PP2A-B56, GRA18 drives ß-catenin up-regulation and the downstream effects on host cell gene expression. In the context of macrophages infection, GRA18 induces the expression of a specific set of genes commonly associated with an anti-inflammatory response that includes those encoding chemokines CCL17 and CCL22. Overall, this study adds another original strategy by which T. gondii tachyzoites reshuffle the host cell interactome through a GSK3/ß-catenin axis to selectively reprogram immune gene expression.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Inflamación/metabolismo , Inflamación/patología , Transducción de Señal , Toxoplasma/metabolismo , beta Catenina/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Quimiocinas/metabolismo , Citoplasma/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Células RAW 264.7 , Transcripción Genética , Transcriptoma/genéticaRESUMEN
Toxoplasma gondii is an intracellular protozoan parasite widely distributed in animals and humans. Infection of host cells and parasite proliferation are essential steps in Toxoplasma pathology. The objective of this study was to develop and validate a novel automatic High Content Imaging (HCI) assay to study T. gondii infection and proliferation. We tested various fluorescent markers and strategies of image analysis to obtain an automated method providing results comparable to those from gold standard infection and proliferation assays. No significant difference was observed between the results obtained from the HCI assay and the standard assays (manual fluorescence microscopy and incorporation of [3H]-uracil). We developed here a robust and time-saving assay. This automated technology was then used to screen a library of compounds belonging to four classes of either natural compounds or synthetic derivatives. Inhibition of parasite proliferation and host cell toxicity were measured in the same assay and led to the identification of one hit, a thiosemicarbazone that allows important inhibition of Toxoplasma proliferation while being relatively safe for the host cells.
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Colorantes Fluorescentes/metabolismo , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis/diagnóstico por imagen , Uracilo/metabolismo , Animales , Células Cultivadas , Fibroblastos/citología , Fibroblastos/parasitología , Prepucio/citología , Prepucio/parasitología , Humanos , Masculino , Microscopía Fluorescente , Programas Informáticos , Tiosemicarbazonas/farmacología , Toxoplasma/efectos de los fármacos , Toxoplasmosis/metabolismoRESUMEN
To invade cells, the parasite Toxoplasma gondii injects a multi-unit nanodevice into the target cell plasma membrane (PM). The core nanodevice, which is composed of the RhOptry Neck (RON) protein complex, connects Toxoplasma and host cell through a circular tight junction (TJ). We now report that this RON nanodevice mechanically promotes membrane scission at the TJ-PM interface, directing a physical rotation driven by the parasite twisting motion that enables the budding parasitophorous vacuole (PV) to seal and separate from the host cell PM as a bona fide subcellular Toxoplasma-loaded PV. Mechanically impairing the process induces swelling of the budding PV and death of the parasite but not host cell. Moreover, this study reveals that the parasite nanodevice functions as a molecular trigger to promote PV membrane remodeling and rapid onset of T. gondii to intracellular lifestyle.
Asunto(s)
Membrana Celular/metabolismo , Fibroblastos/parasitología , Proteínas Protozoarias/metabolismo , Toxoplasma/patogenicidad , Vacuolas/parasitología , Animales , Línea Celular , Femenino , Fibroblastos/ultraestructura , Interacciones Huésped-Parásitos , Humanos , Ratones , Ratones Transgénicos , Imagen Óptica , Técnicas de Placa-Clamp , Proteínas Protozoarias/genética , Rotación , Uniones Estrechas/metabolismo , Toxoplasma/genéticaRESUMEN
Developing anti-parasitic lead compounds that act on key vulnerabilities are necessary for new anti-infectives. Malaria, leishmaniasis, toxoplasmosis, cryptosporidiosis and coccidiosis together kill >500,000 humans annually. Their causative parasites Plasmodium, Leishmania, Toxoplasma, Cryptosporidium and Eimeria display high conservation in many housekeeping genes, suggesting that these parasites can be attacked by targeting invariant essential proteins. Here, we describe selective and potent inhibition of prolyl-tRNA synthetases (PRSs) from the above parasites using a series of quinazolinone-scaffold compounds. Our PRS-drug co-crystal structures reveal remarkable active site plasticity that accommodates diversely substituted compounds, an enzymatic feature that can be leveraged for refining drug-like properties of quinazolinones on a per parasite basis. A compound we termed In-5 exhibited a unique double conformation, enhanced drug-like properties, and cleared malaria in mice. It thus represents a new lead for optimization. Collectively, our data offer insights into the structure-guided optimization of quinazolinone-based compounds for drug development against multiple human eukaryotic pathogens.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Inhibidores Enzimáticos/administración & dosificación , Infecciones por Protozoos/tratamiento farmacológico , Quinazolinonas/administración & dosificación , Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Animales , Dominio Catalítico/efectos de los fármacos , Coccidiosis/tratamiento farmacológico , Criptosporidiosis/tratamiento farmacológico , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Leishmaniasis/tratamiento farmacológico , Malaria/tratamiento farmacológico , Ratones , Modelos Moleculares , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Quinazolinonas/química , Quinazolinonas/farmacología , Relación Estructura-Actividad , Toxoplasmosis/tratamiento farmacológicoRESUMEN
Toxoplasma gondii is an important food and waterborne pathogen causing toxoplasmosis, a potentially severe disease in immunocompromised or congenitally infected humans. Available therapeutic agents are limited by suboptimal efficacy and frequent side effects that can lead to treatment discontinuation. Here we report that the benzoxaborole AN3661 had potent in vitro activity against T. gondii Parasites selected to be resistant to AN3661 had mutations in TgCPSF3, which encodes a homologue of cleavage and polyadenylation specificity factor subunit 3 (CPSF-73 or CPSF3), an endonuclease involved in mRNA processing in eukaryotes. Point mutations in TgCPSF3 introduced into wild-type parasites using the CRISPR/Cas9 system recapitulated the resistance phenotype. Importantly, mice infected with T. gondii and treated orally with AN3661 did not develop any apparent illness, while untreated controls had lethal infections. Therefore, TgCPSF3 is a promising novel target of T. gondii that provides an opportunity for the development of anti-parasitic drugs.
Asunto(s)
Antiprotozoarios/farmacología , Compuestos de Boro/farmacología , Factor de Especificidad de Desdoblamiento y Poliadenilación/antagonistas & inhibidores , Toxoplasma/efectos de los fármacos , Toxoplasma/enzimología , Toxoplasmosis/tratamiento farmacológico , Administración Oral , Animales , Antiprotozoarios/administración & dosificación , Compuestos de Boro/administración & dosificación , Modelos Animales de Enfermedad , Resistencia a Medicamentos , Ratones , Pruebas de Sensibilidad Parasitaria , Mutación Puntual , Análisis de SupervivenciaRESUMEN
BACKGROUND: The several-micrometer-sized Toxoplasma gondii protozoan parasite invades virtually any type of nucleated cell from a warm-blooded animal within seconds. Toxoplasma initiates the formation of a tight ring-like junction bridging its apical pole with the host cell membrane. The parasite then actively moves through the junction into a host cell plasma membrane invagination that delineates a nascent vacuole. Recent high resolution imaging and kinematics analysis showed that the host cell cortical actin dynamics occurs at the site of entry while gene silencing approaches allowed motor-deficient parasites to be generated, and suggested that the host cell could contribute energetically to invasion. In this study we further investigate this possibility by analyzing the behavior of parasites genetically impaired in different motor components, and discuss how the uncovered mechanisms illuminate our current understanding of the invasion process by motor-competent parasites. RESULTS: By simultaneously tracking host cell membrane and cortex dynamics at the site of interaction with myosin A-deficient Toxoplasma, the junction assembly step could be decoupled from the engagement of the Toxoplasma invasive force. Kinematics combined with functional analysis revealed that myosin A-deficient Toxoplasma had a distinct host cell-dependent mode of entry when compared to wild-type or myosin B/C-deficient Toxoplasma. Following the junction assembly step, the host cell formed actin-driven membrane protrusions that surrounded the myosin A-deficient mutant and drove it through the junction into a typical vacuole. However, this parasite-entry mode appeared suboptimal, with about 40 % abortive events for which the host cell membrane expansions failed to cover the parasite body and instead could apply deleterious compressive forces on the apical pole of the zoite. CONCLUSIONS: This study not only clarifies the key contribution of T. gondii tachyzoite myosin A to the invasive force, but it also highlights a new mode of entry for intracellular microbes that shares early features of macropinocytosis. Given the harmful potential of the host cell compressive forces, we propose to consider host cell invasion by zoites as a balanced combination between host cell membrane dynamics and the Toxoplasma motor function. In this light, evolutionary shaping of myosin A with fast motor activity could have contributed to optimize the invasive potential of Toxoplasma tachyzoites and thereby their fitness.
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Miosinas/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Toxoplasma/patogenicidad , Técnica del Anticuerpo Fluorescente , Células HeLa , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/fisiología , Humanos , Microscopía Confocal , Microscopía Electrónica de Rastreo , Microscopía por Video , Miosinas/genética , Proteínas Protozoarias/genética , Toxoplasma/genéticaRESUMEN
An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of T. gondii-targeting activities in immune and nonimmune cells but can also contribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ-stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1(+) inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-γ-mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism.
Asunto(s)
Cromatina/fisiología , Interferón gamma/farmacología , Proteínas Protozoarias/fisiología , Factor de Transcripción STAT1/fisiología , Toxoplasma/fisiología , Animales , Regulación de la Expresión Génica , Factor 1 Regulador del Interferón/análisis , Macrófagos/fisiología , Ratones , Ratones Endogámicos BALB C , Monocitos/fisiología , Fosforilación , Regiones Promotoras Genéticas , Factor de Transcripción STAT1/antagonistas & inhibidoresRESUMEN
Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection.
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Técnicas de Inactivación de Genes , Fenotipo , Proteínas Protozoarias/genética , Sitios de Carácter Cuantitativo , Toxoplasma/fisiología , Animales , Eliminación de Gen , Orden Génico , Marcación de Gen , Interacciones Huésped-Parásitos , Ratones , Plásmidos/genética , Toxoplasma/patogenicidad , Toxoplasma/ultraestructura , Toxoplasmosis/parasitología , Virulencia/genéticaRESUMEN
The apicomplexan parasites Cryptosporidium and Toxoplasma are serious threats to human health. Cryptosporidiosis is a severe diarrheal disease in malnourished children and immunocompromised individuals, with the only FDA-approved drug treatment currently being nitazoxanide. The existing therapies for toxoplasmosis, an important pathology in immunocompromised individuals and pregnant women, also have serious limitations. With the aim of developing alternative therapeutic options to address these health problems, we tested a number of benzoxaboroles, boron-containing compounds shown to be active against various infectious agents, for inhibition of the growth of Cryptosporidium parasites in mammalian cells. A 3-aminomethyl benzoxaborole, AN6426, with activity in the micromolar range and with activity comparable to that of nitazoxanide, was identified and further characterized using biophysical measurements of affinity and crystal structures of complexes with the editing domain of Cryptosporidium leucyl-tRNA synthetase (LeuRS). The same compound was shown to be active against Toxoplasma parasites, with the activity being enhanced in the presence of norvaline, an amino acid that can be mischarged by LeuRS. Our observations are consistent with AN6426 inhibiting protein synthesis in both Cryptosporidium and Toxoplasma by forming a covalent adduct with tRNA(Leu) in the LeuRS editing active site and suggest that further exploitation of the benzoxaborole scaffold is a valid strategy to develop novel, much needed antiparasitic agents.
Asunto(s)
Antiprotozoarios/farmacología , Compuestos de Boro/farmacología , Cryptosporidium parvum/efectos de los fármacos , Leucina-ARNt Ligasa/antagonistas & inhibidores , Leucina-ARNt Ligasa/química , Toxoplasma/efectos de los fármacos , Animales , Antiprotozoarios/química , Antiprotozoarios/metabolismo , Compuestos de Boro/química , Cristalografía por Rayos X , Perros , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/parasitología , Humanos , Leucina-ARNt Ligasa/metabolismo , Células de Riñón Canino Madin Darby/parasitología , Simulación del Acoplamiento Molecular , Conformación ProteicaRESUMEN
The Chinese herb Dichroa febrifuga has traditionally treated malaria-associated fever. Its active component febrifugine (FF) and derivatives such as halofuginone (HF) are potent anti-malarials. Here, we show that FF-based derivatives arrest parasite growth by direct interaction with and inhibition of the protein translation enzyme prolyl-tRNA synthetase (PRS). Dual administration of inhibitors that target different tRNA synthetases suggests high utility of these drug targets. We reveal the ternary complex structure of PRS-HF and adenosine 5'-(ß,γ-imido)triphosphate where the latter facilitates HF integration into the PRS active site. Structural analyses also highlight spaces within the PRS architecture for HF derivatization of its quinazolinone, but not piperidine, moiety. We also show a remarkable ability of HF to kill the related human parasite Toxoplasma gondii, suggesting wider HF efficacy against parasitic PRSs. Hence, our cell-, enzyme-, and structure-based data on FF-based inhibitors strengthen the case for their inclusion in anti-malarial and anti-toxoplasmosis drug development efforts.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Antimaláricos/farmacología , Piperidinas/farmacología , Plasmodium falciparum/efectos de los fármacos , Quinazolinonas/farmacología , Aminoacil-ARNt Sintetasas/metabolismo , Antimaláricos/química , Dominio Catalítico/efectos de los fármacos , Cristalografía , Humanos , Modelos Moleculares , Complejos Multiproteicos/química , Piperidinas/química , Plasmodium falciparum/química , Plasmodium falciparum/enzimología , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Quinazolinonas/química , Relación Estructura-Actividad , Toxoplasma/química , Toxoplasma/efectos de los fármacos , Toxoplasma/enzimologíaRESUMEN
Natural immunity or resistance to pathogens most often relies on the genetic make-up of the host. In a LEW rat model of refractoriness to toxoplasmosis, we previously identified on chromosome 10 the Toxo1 locus that directs toxoplasmosis outcome and controls parasite spreading by a macrophage-dependent mechanism. Now, we narrowed down Toxo1 to a 891 kb interval containing 29 genes syntenic to human 17p13 region. Strikingly, Toxo1 is included in a haplotype block strictly conserved among all refractory rat strains. The sequencing of Toxo1 in nine rat strains (5 refractory and 4 susceptible) revealed resistant-restricted conserved polymorphisms displaying a distribution gradient that peaks at the bottom border of Toxo1, and highlighting the NOD-like receptor, Nlrp1a, as a major candidate. The Nlrp1 inflammasome is known to trigger, upon pathogen intracellular sensing, pyroptosis programmed-cell death involving caspase-1 activation and cleavage of IL-1ß. Functional studies demonstrated that the Toxo1-dependent refractoriness in vivo correlated with both the ability of macrophages to restrict T. gondii growth and a T. gondii-induced death of intracellular parasites and its host macrophages. The parasite-induced cell death of infected macrophages bearing the LEW-Toxo1 alleles was found to exhibit pyroptosis-like features with ROS production, the activation of caspase-1 and IL1-ß secretion. The pharmacological inactivation of caspase-1 using YVAD and Z-VAD inhibitors prevented the death of both intravacuolar parasites and host non-permissive macrophages but failed to restore parasite proliferation. These findings demonstrated that the Toxo1-dependent response of rat macrophages to T. gondii infection may trigger two pathways leading to the control of parasite proliferation and the death of parasites and host macrophages. The NOD-like receptor NLRP1a/Caspase-1 pathway is the best candidate to mediate the parasite-induced cell death. These data represent new insights towards the identification of a major pathway of innate resistance to toxoplasmosis and the prediction of individual resistance.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 1/metabolismo , Sitios Genéticos , Haplotipos , Macrófagos Peritoneales/metabolismo , Toxoplasma/metabolismo , Toxoplasmosis/metabolismo , Animales , Caspasa 1/genética , Inhibidores de Caspasas/farmacología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Humanos , Inflamasomas/genética , Inflamasomas/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos Peritoneales/parasitología , Macrófagos Peritoneales/patología , Ratones , Oligopéptidos/farmacología , Ratas , Toxoplasmosis/genética , Toxoplasmosis/patologíaRESUMEN
BACKGROUND: New chemotherapy drugs should be investigated to improve survival of patients with advanced bladder cancer. Here, we report the synthesis and evaluation of AG11, a new flavanone derivative obtained through cyclization of its chalcone precursor CB11. MATERIALS AND METHODS: The effect of AG11 on cell viability was evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay and apoptotic cell death was analyzed by flow cytometry. Finally, the effect of AG11 on tubulin polymerization in vitro and microtubule distribution across the cells was investigated. RESULTS: AG11 was found to have an IC50 (half-maximal inhibitory concentration) of 4.6 µM and its inhibitory effect on RT4 cells proliferation is associated with a cell-cycle arrest in G2+M phases followed by apoptosis after a 48 h treatment. AG11 prevented polymerization of purified tubulin in a concentration-dependent manner in vitro and disrupted mitotic spindle formation in cells. CONCLUSION: AG11 appears to be an attractive scaffold for further development of a structurally simpler new anti-microtubule agents.