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While recent advances in cryo-EM, coupled with single particle analysis, have the potential to allow structure determination in a near-native state from vanishingly few individual particles, this vision has yet to be realised in practise. Requirements for particle numbers that currently far exceed the theoretical lower limits, challenges with the practicalities of achieving high concentrations for difficult-to-produce samples, and inadequate sample-dependent imaging conditions, all result in significant bottlenecks preventing routine structure determination using cryo-EM. Therefore, considerable efforts are being made to circumvent these bottlenecks by developing affinity purification of samples on-grid; at once obviating the need to produce large amounts of protein, as well as more directly controlling the variable, and sample-dependent, process of grid preparation. In this proof-of-concept study, we demonstrate a further practical step towards this paradigm, developing a 3D-printable flow-cell device to allow on-grid affinity purification from raw inputs such as whole cell lysates, using graphene oxide-based affinity grids. Our flow-cell device can be interfaced directly with routinely-used laboratory equipment such as liquid chromatographs, or peristaltic pumps, fitted with standard chromatographic (1/16") connectors, and can be used to allow binding of samples to affinity grids in a controlled environment prior to the extensive washing required to remove impurities. Furthermore, by designing a device which can be 3D printed and coupled to routinely used laboratory equipment, we hope to increase the accessibility of the techniques presented herein to researchers working towards single-particle macromolecular structures.
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Impresión Tridimensional , Proteínas , Microscopía por Crioelectrón/métodos , Microscopía ElectrónicaRESUMEN
The mammalian gut and its microbiome form a temporally dynamic and spatially heterogeneous environment. The inaccessibility of the gut and the spatially restricted nature of many gut diseases translate into difficulties in diagnosis and therapy for which novel tools are needed. Engineered bacterial whole-cell biosensors and therapeutics have shown early promise at addressing these challenges. Natural and engineered sensing systems can be repurposed in synthetic genetic circuits to detect spatially specific biomarkers during health and disease. Heat, light, and magnetic signals can also activate gene circuit function with externally directed spatial precision. The resulting engineered bacteria can report on conditions in situ within the complex gut environment or produce biotherapeutics that specifically target host or microbiome activity. Here, we review the current approaches to engineering spatial precision for in vivo bacterial diagnostics and therapeutics using synthetic circuits, and the challenges and opportunities this technology presents.
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Infection with Plasmodium falciparum parasites results in approximately 627,000 deaths from malaria annually. Key to the parasite's success is their ability to invade and subsequently grow within human erythrocytes. Parasite proteins involved in parasite invasion and proliferation are therefore intrinsically of great interest, as targeting these proteins could provide novel means of therapeutic intervention. One such protein is P113 which has been reported to be both an invasion protein and an intracellular protein located within the parasitophorous vacuole (PV). The PV is delimited by a membrane (PVM) across which a plethora of parasite-specific proteins are exported via the Plasmodium Translocon of Exported proteins (PTEX) into the erythrocyte to enact various immune evasion functions. To better understand the role of P113 we isolated its binding partners from in vitro cultures of P. falciparum. We detected interactions with the protein export machinery (PTEX and exported protein-interacting complex) and a variety of proteins that either transit through the PV or reside on the parasite plasma membrane. Genetic knockdown or partial deletion of P113 did not significantly reduce parasite growth or protein export but did disrupt the morphology of the PVM, suggesting that P113 may play a role in maintaining normal PVM architecture.
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Malaria Falciparum , Parásitos , Animales , Eritrocitos/parasitología , Humanos , Malaria Falciparum/parasitología , Parásitos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Transporte de Proteínas/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Vacuolas/metabolismoRESUMEN
Synthetic gene oscillators have the potential to control timed functions and periodic gene expression in engineered cells. Such oscillators have been refined in bacteria in vitro, however, these systems have lacked the robustness and precision necessary for applications in complex in vivo environments, such as the mammalian gut. Here, we demonstrate the implementation of a synthetic oscillator capable of keeping robust time in the mouse gut over periods of days. The oscillations provide a marker of bacterial growth at a single-cell level enabling quantification of bacterial dynamics in response to inflammation and underlying variations in the gut microbiota. Our work directly detects increased bacterial growth heterogeneity during disease and differences between spatial niches in the gut, demonstrating the deployment of a precise engineered genetic oscillator in real-life settings.
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Relojes Biológicos/genética , Microbioma Gastrointestinal , Biología Sintética/métodos , Animales , División Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Ratones , Microorganismos Modificados Genéticamente/metabolismo , Microorganismos Modificados Genéticamente/fisiología , Imagen ÓpticaRESUMEN
In nature, microbes interact antagonistically, neutrally, or beneficially. To shed light on the effects of positive interactions in microbial consortia, we introduced metabolic dependencies and metabolite overproduction into four bacterial species. While antagonistic interactions govern the wild-type consortium behavior, the genetic modifications alleviated antagonistic interactions and resulted in beneficial interactions. Engineered cross-feeding increased population evenness, a component of ecological diversity, in different environments, including in a more complex gnotobiotic mouse gut environment. Our findings suggest that metabolite cross-feeding could be used as a tool for intentionally shaping microbial consortia in complex environments.IMPORTANCE Microbial communities are ubiquitous in nature. Bacterial consortia live in and on our body and in our environment, and more recently, biotechnology is applying microbial consortia for bioproduction. As part of our body, bacterial consortia influence us in health and disease. Microbial consortium function is determined by its composition, which in turn is driven by the interactions between species. Further understanding of microbial interactions will help us in deciphering how consortia function in complex environments and may enable us to modify microbial consortia for health and environmental benefits.
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Engineering synthetic circuits into intestinal bacteria to sense, record, and respond to in vivo signals is a promising new approach for the diagnosis, treatment, and prevention of disease. However, because the design of disease-responsive circuits is limited by a relatively small pool of known biosensors, there is a need for expanding the capacity of engineered bacteria to sense and respond to the host environment. Here, we apply a robust genetic memory circuit in Escherichia coli to identify new bacterial biosensor triggers responding in the healthy and diseased mammalian gut, which may be used to construct diagnostic or therapeutic circuits. We developed a pipeline for rapid systems-level library construction and screening, using next-generation sequencing and computational analysis, which demonstrates remarkably reliable identification of responsive biosensor triggers from pooled libraries. By testing libraries of potential triggers-each consisting of a promoter and ribosome binding site (RBS)-and using RBS variation to augment the range of trigger sensitivity, we identify and validate triggers that selectively activate our synthetic memory circuit during transit through the gut. We further identify biosensor triggers with increased response in the inflamed gut through comparative screening of one of our libraries in healthy mice and those with intestinal inflammation. Our results demonstrate the power of systems-level screening for the identification of novel biosensor triggers in the gut and provide a platform for disease-specific screening that is capable of contributing to both the understanding and clinical management of intestinal illness.IMPORTANCE The gut is a largely obscure and inaccessible environment. The use of live, engineered probiotics to detect and respond to disease signals in vivo represents a new frontier in the management of gut diseases. Engineered probiotics have also shown promise as a novel mechanism for drug delivery. However, the design and construction of effective strains that respond to the in vivo environment is hindered by our limited understanding of bacterial behavior in the gut. Our work expands the pool of environmentally responsive synthetic circuits for the healthy and diseased gut, providing insight into host-microbe interactions and enabling future development of increasingly complex biosensors. This method also provides a framework for rapid prototyping of engineered systems and for application across bacterial strains and disease models, representing a practical step toward the construction of clinically useful synthetic tools.
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Our ability to generate bacterial strains with unique and increasingly complex functions has rapidly expanded in recent times. The capacity for DNA synthesis is increasing and costing less; new tools are being developed for fast, large-scale genetic manipulation; and more tested genetic parts are available for use, as is the knowledge of how to use them effectively. These advances promise to unlock an exciting array of 'smart' bacteria for clinical use but will also challenge scientists to better optimize preclinical testing regimes for early identification and validation of promising strains and strategies. Here, we review recent advances in the development and testing of engineered bacterial diagnostics and therapeutics. We highlight new technologies that will assist the development of more complex, robust and reliable engineered bacteria for future clinical applications, and we discuss approaches to more efficiently evaluate engineered strains throughout their preclinical development.
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Bacterias/genética , Ingeniería Genética , Animales , Diseño de Fármacos , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , HumanosRESUMEN
Bacteria can be engineered to function as diagnostics or therapeutics in the mammalian gut but commercial translation of technologies to accomplish this has been hindered by the susceptibility of synthetic genetic circuits to mutation and unpredictable function during extended gut colonization. Here, we report stable, engineered bacterial strains that maintain their function for 6 months in the mouse gut. We engineered a commensal murine Escherichia coli strain to detect tetrathionate, which is produced during inflammation. Using our engineered diagnostic strain, which retains memory of exposure in the gut for analysis by fecal testing, we detected tetrathionate in both infection-induced and genetic mouse models of inflammation over 6 months. The synthetic genetic circuits in the engineered strain were genetically stable and functioned as intended over time. The durable performance of these strains confirms the potential of engineered bacteria as living diagnostics.
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Escherichia coli/genética , Escherichia coli/metabolismo , Gastroenteritis/diagnóstico , Gastroenteritis/microbiología , Microbioma Gastrointestinal , Ácido Tetratiónico/metabolismo , Animales , Supervivencia Celular , Escherichia coli/aislamiento & purificación , Femenino , Ingeniería Genética/métodos , Intestinos , Ratones , Ratones Endogámicos C57BLRESUMEN
It is only in the last decade that sub-cellular resolution of red cell invasion by the malaria parasite Plasmodium falciparum has been possible. Here we look back on the development of methodologies that led to this possibility and the subsequent advancements made in understanding this key event in malaria disease.
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Endocitosis , Eritrocitos/parasitología , Imagen Óptica/métodos , Plasmodium falciparum/fisiología , Eritrocitos/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Parasitología/métodos , Plasmodium falciparum/ultraestructuraRESUMEN
Microscopy-based localisation of proteins during malaria parasite (Plasmodium) invasion of the erythrocyte is widely used for tentative assignment of protein function. To date, however, imaging has been limited by the rarity of invasion events and the poor resolution available, given the micron size of the parasite, which leads to a lack of quantitative measures for definitive localisation. Here, using computational image analysis we have attempted to assign relative protein localisation during invasion using wide-field deconvolution microscopy. By incorporating three-dimensional information we present a detailed assessment of known parasite effectors predicted to function during entry but as yet untested or for which data are equivocal. Our method, termed longitudinal intensity profiling, resolves confusion surrounding the localisation of apical membrane antigen 1 (AMA1) at the merozoite-erythrocyte junction and predicts that the merozoite thrombospondin-related anonymous protein (MTRAP) is unlikely to play a direct role in the mechanics of entry, an observation supported with additional biochemical evidence. This approach sets a benchmark for imaging of complex micron-scale events and cautions against simplistic interpretations of small numbers of representative images for the assignment of protein function or prioritisation of candidates as therapeutic targets.
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Antígenos de Protozoos/metabolismo , Eritrocitos/parasitología , Imagenología Tridimensional , Plasmodium falciparum/inmunología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/metabolismo , Actinas/metabolismo , Anticuerpos Antiprotozoarios/metabolismo , Especificidad de Anticuerpos , Epítopos/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Humanos , Merozoítos/metabolismo , Modelos Biológicos , Estructura Terciaria de Proteína , Transporte de Proteínas , Uniones Estrechas/metabolismoRESUMEN
BACKGROUND: Gliding motility in Plasmodium parasites, the aetiological agents of malaria disease, is mediated by an actomyosin motor anchored in the outer pellicle of the motile cell. Effective motility is dependent on a parasite myosin motor and turnover of dynamic parasite actin filaments. To date, however, the basis for directional motility is not known. Whilst myosin is very likely orientated as a result of its anchorage within the parasite, how actin filaments are orientated to facilitate directional force generation remains unexplained. In addition, recent evidence has questioned the linkage between actin filaments and secreted surface antigens leaving the way by which motor force is transmitted to the extracellular milieu unknown. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which few are predicted to interact with filamentous (F)-actin directly. One of these, PF3D7_1251200, shows strong homology to the coronin family of actin-filament binding proteins, herein referred to as PfCoronin. METHODS: Here the N terminal beta propeller domain of PfCoronin (PfCor-N) was expressed to assess its ability to bind and bundle pre-formed actin filaments by sedimentation assay, total internal reflection fluorescence (TIRF) microscopy and confocal imaging as well as to explore its ability to bind phospholipids. In parallel a tagged PfCoronin line in Plasmodium falciparum was generated to determine the cellular localization of the protein during asexual parasite development and blood-stage merozoite invasion. RESULTS: A combination of biochemical approaches demonstrated that the N-terminal beta-propeller domain of PfCoronin is capable of binding F-actin and facilitating formation of parallel filament bundles. In parasites, PfCoronin is expressed late in the asexual lifecycle and localizes to the pellicle region of invasive merozoites before and during erythrocyte entry. PfCoronin also associates strongly with membranes within the cell, likely mediated by interactions with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane. CONCLUSIONS: These data suggest PfCoronin may fulfil a key role as the critical determinant of actin filament organization in the Plasmodium cell. This raises the possibility that macro-molecular organization of actin mediates directional motility in gliding parasites.
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Citoesqueleto de Actina/química , Proteínas de Microfilamentos/química , Plasmodium falciparum/química , Plasmodium falciparum/fisiología , Proteínas Protozoarias/química , Citoesqueleto de Actina/metabolismo , Animales , Eritrocitos/parasitología , Humanos , Malaria Falciparum/parasitología , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/metabolismo , ConejosRESUMEN
The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells.
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Membrana Celular/parasitología , Eritrocitos/parasitología , Interacciones Huésped-Parásitos , Plasmodium falciparum/patogenicidad , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Humanos , Merozoítos/fisiologíaRESUMEN
BACKGROUND: A highly effective vaccine against Plasmodium falciparum malaria should induce potent, strain transcending immunity that broadly protects against the diverse population of parasites circulating globally. We aimed to identify vaccine candidates that fulfill the criteria. METHODS: We have measured growth inhibitory activity of antibodies raised to a range of antigens to identify those that can efficiently block merozoite invasion for geographically diverse strains of P. falciparum. RESULTS: This has shown that the conserved Region III-V, of the P. falciparum erythrocyte-binding antigen (EBA)-175 was able to induce antibodies that potently inhibit merozoite invasion across diverse parasite strains, including those reliant on invasion pathways independent of EBA-175 function. Additionally, the conserved RIII-V domain of EBA-140 also induced antibodies with strong in vitro parasite growth inhibitory activity. CONCLUSION: We identify an alternative, highly conserved region (RIV-V) of EBA-175, present in all EBA proteins, that is the target of potent, strain transcending neutralizing antibodies, that represents a strong candidate for development as a component in a malaria vaccine.
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Anticuerpos Neutralizantes/sangre , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Vacunación , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antiprotozoarios/farmacología , Antígenos de Protozoos/química , Secuencia Conservada , Mapeo Epitopo , Interacciones Huésped-Parásitos/inmunología , Humanos , Inmunoglobulina G/sangre , Concentración 50 Inhibidora , Vacunas contra la Malaria/inmunología , Malaria Falciparum/sangre , Malaria Falciparum/inmunología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/química , Conejos , Especificidad de la EspecieRESUMEN
Erythrocyte invasion by merozoites forms of the malaria parasite is a key step in the establishment of human malaria disease. To date, efforts to understand cellular events underpinning entry have been limited to insights from non-human parasites, with no studies at sub-micrometer resolution undertaken using the most virulent human malaria parasite, Plasmodium falciparum. This leaves our understanding of the dynamics of merozoite sub-cellular compartments during infectionincomplete, in particular that of the secretory organelles. Using advances in P. falciparum merozoite isolation and new imaging techniques we present a three-dimensional study of invasion using electron microscopy, cryo-electron tomography and cryo-X-ray tomography. We describe the core architectural features of invasion and identify fusion between rhoptries at the commencement of invasion as a hitherto overlooked event that likely provides a critical step that initiates entry. Given the centrality of merozoite organelle proteins to vaccine development, these insights provide a mechanistic framework to understand therapeutic strategies targeted towards the cellular events of invasion.
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Tomografía con Microscopio Electrónico , Endocitosis , Eritrocitos/parasitología , Eritrocitos/ultraestructura , Merozoítos/ultraestructura , Plasmodium falciparum/fisiología , Plasmodium falciparum/ultraestructura , Interacciones Huésped-Patógeno , Humanos , Imagenología TridimensionalRESUMEN
Export of proteins into the infected erythrocyte is critical for malaria parasite survival. The majority of effector proteins are thought to export via a proteinaceous translocon, resident in the parasitophorous vacuole membrane surrounding the parasite. Identification of the Plasmodium translocon of exported proteins and its biochemical association with exported proteins suggests it performs this role. Direct evidence for this, however, is lacking. Here using viable purified Plasmodium falciparum merozoites and three-dimensional structured illumination microscopy, we investigate remodelling events immediately following parasite invasion. We show that multiple complexes of the Plasmodium translocon of exported proteins localize together in foci that dynamically change in clustering behaviour. Furthermore, we provide conclusive evidence of spatial association between exported proteins and exported protein 2, a core component of the Plasmodium translocon of exported proteins, during native conditions and upon generation of translocation intermediates. These data provide the most direct cellular evidence to date that protein export occurs at regions of the parasitophorous vacuole membrane housing the Plasmodium translocon of exported proteins complex.
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Eritrocitos/parasitología , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Análisis por Conglomerados , Eritrocitos/metabolismo , Eritrocitos/patología , Eritrocitos/ultraestructura , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Merozoítos/citología , Merozoítos/metabolismo , Merozoítos/ultraestructura , Modelos Biológicos , Parásitos/citología , Parásitos/metabolismo , Parásitos/ultraestructura , Plasmodium falciparum/citología , Plasmodium falciparum/ultraestructura , Estructura Terciaria de Proteína , Transporte de Proteínas , Desplegamiento Proteico , Proteínas Protozoarias/química , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
BACKGROUND: The apicoplast is a plastid organelle derived from a secondary endosymbiosis, containing biosynthetic pathways essential for the survival of apicomplexan parasites. The Toxoplasma apicoplast clearly possesses four membranes but in related Plasmodium spp. the apicoplast has variably been reported to have either three or four membranes. METHODS: Cryo-electron tomography was employed to image merozoites of Plasmodium falciparum and Plasmodium berghei frozen in their near-native state. Three-dimensional reconstructions revealed the number of apicoplast membranes and the association of the apicoplast with other organelles. Routine transmission electron microscopy of parasites preserved by high-pressure freezing followed by freeze substitution techniques was also used to analyse apicoplast morphology. RESULTS: Cryo-preserved parasites showed clearly four membranes surrounding the apicoplast. A wider gap between the second and third apicoplast membranes was frequently observed. The apicoplast was found in close proximity to the nucleus and to the rhoptries. The apicoplast matrix showed ribosome-sized particles and membranous whorls. CONCLUSIONS: The Plasmodium apicoplast possesses four membranes, as do the apicoplasts of other apicomplexan parasites. This is consistent with a four-membraned secondary endosymbiotic plastid ancestor.
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Membranas Intracelulares/ultraestructura , Plasmodium berghei/ultraestructura , Plasmodium falciparum/ultraestructura , Plastidios/ultraestructura , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Imagenología Tridimensional , Merozoítos/ultraestructura , Microscopía Electrónica de TransmisiónRESUMEN
Cellular imaging has reemerged in recent years as a powerful approach to provide researchers with a direct measure of essential molecular events in a cell's life, ranging in scale from broad morphological observations of whole cells to intricate single molecule imaging. When combined with quantitative image analysis, the available imaging techniques can act as a critical means to confirm hypotheses, drive the formation of new theories or provide accurate determination of protein localization at subcellular and nanometer scales. Here, we describe two methodological approaches for imaging the transient step of malaria parasite invasion of the human erythrocyte. When applied to image the most virulent human malaria parasite, Plasmodium falciparum, the first approach, using live time-lapse wide-field microscopy, allows the capture of transient events during invasion and postinvasion intra-erythrocytic development, while the second, using immunofluorescence assay (IFA) of fixed samples, allows high-definition exploration of parasite architecture on multiple platforms.
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Eritrocitos/parasitología , Microscopía/métodos , Plasmodium falciparum/crecimiento & desarrollo , Animales , Eritrocitos/patología , Humanos , Estadios del Ciclo de Vida , Malaria Falciparum/diagnóstico , Malaria Falciparum/parasitología , Merozoítos/citología , Merozoítos/crecimiento & desarrollo , Plasmodium falciparum/citología , Plasmodium falciparum/aislamiento & purificaciónRESUMEN
Host cell infection by apicomplexan parasites plays an essential role in lifecycle progression for these obligate intracellular pathogens. For most species, including the etiological agents of malaria and toxoplasmosis, infection requires active host-cell invasion dependent on formation of a tight junction - the organising interface between parasite and host cell during entry. Formation of this structure is not, however, shared across all Apicomplexa or indeed all parasite lifecycle stages. Here, using an in silico integrative genomic search and endogenous gene-tagging strategy, we sought to characterise proteins that function specifically during junction-dependent invasion, a class of proteins we term invasins to distinguish them from adhesins that function in species specific host-cell recognition. High-definition imaging of tagged Plasmodium falciparum invasins localised proteins to multiple cellular compartments of the blood stage merozoite. This includes several that localise to distinct subcompartments within the rhoptries. While originating from the same organelle, however, each has very different dynamics during invasion. Apical Sushi Protein and Rhoptry Neck protein 2 release early, following the junction, whilst a novel rhoptry protein PFF0645c releases only after invasion is complete. This supports the idea that organisation of proteins within a secretory organelle determines the order and destination of protein secretion and provides a localisation-based classification strategy for predicting invasin function during apicomplexan parasite invasion.
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Eritrocitos/parasitología , Malaria/parasitología , Orgánulos/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/metabolismo , Western Blotting , Técnica del Anticuerpo Fluorescente , Interacciones Huésped-Parásitos , Humanos , Microscopía Inmunoelectrónica , Orgánulos/ultraestructuraRESUMEN
Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu.