RÉSUMÉ
Plasmodium sporozoites are the infective forms of the malaria parasite in the mosquito and vertebrate host. Gliding motility allows sporozoites to migrate and invade mosquito salivary glands and mammalian hosts. Motility and invasion are powered by an actin-myosin motor complex linked to the glideosome, which contains glideosome-associated proteins (GAPs), MyoA and the myosin A tail-interacting protein (MTIP). However, the role of several proteins involved in gliding motility remains unknown. We identified that the S14 gene is upregulated in sporozoite from transcriptome data of Plasmodium yoelii and further confirmed its transcription in P. berghei sporozoites using real-time PCR. C-terminal 3×HA-mCherry tagging revealed that S14 is expressed and localized on the inner membrane complex of the sporozoites. We disrupted S14 in P. berghei and demonstrated that it is essential for sporozoite gliding motility, and salivary gland and hepatocyte invasion. The gliding and invasion-deficient S14 knockout sporozoites showed normal expression and organization of inner membrane complex and surface proteins. Taken together, our data show that S14 plays a role in the function of the glideosome and is essential for malaria transmission.
Sujet(s)
Paludisme , Plasmodium berghei , Protéines de protozoaire , Sporozoïtes , Sporozoïtes/métabolisme , Plasmodium berghei/métabolisme , Plasmodium berghei/génétique , Protéines de protozoaire/métabolisme , Protéines de protozoaire/génétique , Animaux , Souris , Paludisme/parasitologie , Glandes salivaires/parasitologie , Glandes salivaires/métabolisme , Anopheles/parasitologieRÉSUMÉ
Cyclic invasion of red blood cells (RBCs) by Plasmodium merozoites is associated with the symptoms and pathology of malaria. Merozoite invasion is powered actively and rapidly by a parasite actomyosin motor called the glideosome. The ability of the glideosome to generate force to support merozoite entry into the host RBCs is thought to rely on its stable anchoring within the inner membrane complex (IMC) through membrane-resident proteins, such as GAP50 and GAP40. Using a conditional knockdown (KD) approach, we determined that PfGAP40 was required for asexual blood-stage replication. PfGAP40 is not needed for merozoite egress from host RBCs or for the attachment of merozoites to new RBCs. PfGAP40 coprecipitates with PfGAP45 and PfGAP50. During merozoite invasion, PfGAP40 is associated strongly with stabilizing the expression levels of PfGAP45 and PfGAP50 in the schizont stage. Although PfGAP40 KD did not influence IMC integrity, it impaired the maturation of gametocytes. In addition, PfGAP40 is phosphorylated, and mutations that block phosphorylation of PfGAP40 at the C-terminal serine residues S370, S372, S376, S405, S409, S420, and S445 reduced merozoite invasion efficiency. Overall, our findings implicate PfGAP40 as an important regulator for the gliding activity of merozoites and suggest that phosphorylation is required for PfGAP40 function. IMPORTANCE Red blood cell invasion is central to the pathogenesis of the malaria parasite, and the parasite proteins involved in this process are potential therapeutic targets. Gliding motility powers merozoite invasion and is driven by a unique molecular motor termed the glideosome. The glideosome is stably anchored to the parasite inner membrane complex (IMC) through membrane-resident proteins. In the present study, we demonstrate the importance of an IMC-resident glideosome component, PfGAP40, that plays a critical role in stabilizing the expression levels of glideosome components in the schizont stage. We determined that phosphorylation of PfGAP40 at C-terminal residues is required for efficient merozoite invasion.
Sujet(s)
Paludisme , Plasmodium falciparum , Animaux , Plasmodium falciparum/génétique , Plasmodium falciparum/métabolisme , Mérozoïtes/métabolisme , Protéines de protozoaire/métabolisme , Protéines membranaires/métabolisme , Paludisme/parasitologieRÉSUMÉ
The phylum of Apicomplexa groups intracellular parasites that employ substrate-dependent gliding motility to invade host cells, egress from the infected cells, and cross biological barriers. The glideosome-associated connector (GAC) is a conserved protein essential to this process. GAC facilitates the association of actin filaments with surface transmembrane adhesins and the efficient transmission of the force generated by myosin translocation of actin to the cell surface substrate. Here, we present the crystal structure of Toxoplasma gondii GAC and reveal a unique, supercoiled armadillo repeat region that adopts a closed ring conformation. Characterisation of the solution properties together with membrane and F-actin binding interfaces suggests that GAC adopts several conformations from closed to open and extended. A multi-conformational model for assembly and regulation of GAC within the glideosome is proposed.
Sujet(s)
Toxoplasma , Toxoplasma/métabolisme , Actines/métabolisme , Cytosquelette d'actine/métabolisme , Membrane cellulaire/métabolisme , Myosines/métabolisme , Protéines de protozoaire/métabolismeRÉSUMÉ
The inner membrane complex (IMC) is a defining feature of apicomplexans comprising of lipid and protein components involved in gliding motility and host cell invasion. Motility of Plasmodium parasites is accomplished by an actin and myosin based glideosome machinery situated between the parasite plasma membrane (PPM) and IMC. Here, we have studied in vivo expression and localization of a Plasmodium falciparum (Pf) IMC protein 'PfIMC1l' and characterized it functionally by using biochemical assays. We have identified cytoskeletal protein 'actin' and motor protein 'myosin' as novel binding partners of PfIMC1l, alongside its interaction with the lipids 'cholesterol' and 'phosphatidyl-inositol 4, 5 bisphosphate' (PIP2). While actin and myosin compete for interaction with PfIMC1l, actin and either of the lipids (cholesterol or PIP2) simultaneously bind PfIMC1l. Interestingly, PfIMC1l showed enhanced binding with actin in the presence of calcium ions, and displayed direct binding with calcium. Based on our in silico analysis and experimental data showing PfIMC1l-actin/myosin and PfIMC1l-lipid interactions, we propose that this protein may anchor the IMC membrane with the parasite gliding apparatus. Considering its binding with key proteins involved in motility viz. myosin and actin (with calcium dependence), we suggest that PfIMC1l may have a role in the locomotion of Plasmodium.
Sujet(s)
Actines/métabolisme , Cytosquelette/métabolisme , Lipides membranaires/métabolisme , Myosines/métabolisme , Plasmodium falciparum/métabolisme , Protéines de protozoaire/métabolisme , Fixation compétitive , Calcium/métabolisme , Cholestérol/métabolisme , Sérums immuns/métabolisme , Ions , Protéines membranaires/composition chimique , Protéines membranaires/métabolisme , Modèles biologiques , Phosphatidylinositol diphosphate-4,5/métabolisme , Liaison aux protéines , Domaines protéiques , Protéines de protozoaire/composition chimique , Protéines recombinantes/composition chimique , Protéines recombinantes/métabolisme , SolutionsRÉSUMÉ
Toxoplasma gondii is a highly prevalent protozoon that can infect all warm-blooded animals, including humans. It is frequently used as an Apicomplexan parasite model in research. In this review, the invasion mechanism of T. gondii is described as a representative Apicomplexan parasite. The invasion machinery of T. gondii consists of the moving junction and the glideosome, which is a specific motor system for Apicomplexan parasites. I provide details about the moving junction, parasite-secreted proteins and host adhesion receptors, the glideosome, and calcium signaling, which generates the power for the gliding mobility of T. gondii. A detailed understanding of parasite invasion can be useful for the development of new effective drugs to inhibit this event and disrupt the Apicomplexan life cycle.
Sujet(s)
Toxoplasma/physiologie , Animaux , Régulation de l'expression des gènes , Humains , Protéines membranaires , Protéines de protozoaire/génétique , Protéines de protozoaire/métabolismeRÉSUMÉ
Apicomplexa are obligate intracellular parasites that actively invade, replicate within, and egress from host cells. The parasite actinomyosin-based molecular motor complex (often referred to as the glideosome) is considered an important mediator of parasite motility and virulence. Mature intracellular parasites often become motile just prior to egress from their host cells, and in some genera, this motility is important for successful egress as well as for subsequent invasion of new host cells. To determine whether actinomyosin-based motility is important in the red blood cell egress and invasion activities of the malaria parasite, we have used a conditional genetic approach to delete GAP45, a primary component of the glideosome, in asexual blood stages of Plasmodium falciparum Our results confirm the essential nature of GAP45 for invasion but show that P. falciparum does not require a functional motor complex to undergo egress from the red blood cell. Malarial egress therefore differs fundamentally from induced egress in the related apicomplexan Toxoplasma gondiiIMPORTANCE Clinical malaria results from cycles of replication of single-celled parasites of the genus Plasmodium in red blood cells. Intracellular parasite replication is followed by a highly regulated, protease-dependent process called egress, in which rupture of the bounding membranes allows explosive release of daughter merozoites which rapidly invade fresh red cells. A parasite actinomyosin-based molecular motor (the glideosome) has been proposed to provide the mechanical force to drive invasion. Studies of the related parasite Toxoplasma gondii have shown that induced egress requires parasite motility, mediated by a functional glideosome. However, whether the glideosome has a similar essential role in egress of malaria merozoites from red blood cells is unknown. Here, we show that although a functional glideosome is required for red blood cell invasion by Plasmodium falciparum merozoites, it is not required for egress. These findings place further emphasis on the key role of the protease cascade in malarial egress.
Sujet(s)
Endocytose , Érythrocytes/parasitologie , Protéines membranaires/métabolisme , Plasmodium falciparum/physiologie , Délétion de gène , Protéines membranaires/génétique , Plasmodium falciparum/génétiqueRÉSUMÉ
Motility of the apicomplexan malaria parasite Plasmodium falciparum is enabled by a multiprotein glideosome complex, whose core is the class XIV myosin motor, PfMyoA, and a divergent Plasmodium actin (PfAct1). Parasite motility is necessary for host-cell invasion and virulence, but studying its molecular basis has been hampered by unavailability of sufficient amounts of PfMyoA. Here, we expressed milligram quantities of functional full-length PfMyoA with the baculovirus/Sf9 cell expression system, which required a UCS (UNC-45/CRO1/She4p) family myosin chaperone from Plasmodium spp. In addition to the known light chain myosin tail interacting protein (MTIP), we identified an essential light chain (PfELC) that co-purified with PfMyoA isolated from parasite lysates. The speed at which PfMyoA moved actin was fastest with both light chains bound, consistent with the light chain-binding domain acting as a lever arm to amplify nucleotide-dependent motions in the motor domain. Surprisingly, PfELC binding to the heavy chain required that MTIP also be bound to the heavy chain, unlike MTIP that bound the heavy chain independently of PfELC. Neither the presence of calcium nor deletion of the MTIP N-terminal extension changed the speed of actin movement. Of note, PfMyoA moved filaments formed from Sf9 cell-expressed PfAct1 at the same speed as skeletal muscle actin. Duty ratio estimates suggested that as few as nine motors can power actin movement at maximal speed, a feature that may be necessitated by the dynamic nature of Plasmodium actin filaments in the parasite. In summary, we have reconstituted the essential core of the glideosome, enabling drug targeting of both of its core components to inhibit parasite invasion.
Sujet(s)
Actines/métabolisme , Complexes multiprotéiques/métabolisme , Muscles squelettiques/métabolisme , Myosine non-musculaire de type IIA/métabolisme , Plasmodium falciparum/métabolisme , Protéines de protozoaire/métabolisme , Séquence d'acides aminés , Animaux , Mouvement cellulaire , Modèles moléculaires , Chaperons moléculaires , Conformation des protéines , Similitude de séquencesRÉSUMÉ
Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain-interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion.
Sujet(s)
Étapes du cycle de vie/physiologie , Protéines membranaires , Myosines , Plasmodium berghei , Plasmodium falciparum , Protéines de protozoaire , Animaux , Humains , Protéines membranaires/génétique , Protéines membranaires/métabolisme , Souris , Myosines/génétique , Myosines/métabolisme , Plasmodium berghei/génétique , Plasmodium berghei/métabolisme , Plasmodium falciparum/génétique , Plasmodium falciparum/métabolisme , Protéines de protozoaire/génétique , Protéines de protozoaire/métabolismeRÉSUMÉ
Apicomplexa exhibit a unique form of substrate-dependent gliding motility central for host cell invasion and parasite dissemination. Gliding is powered by rearward translocation of apically secreted transmembrane adhesins via their interaction with the parasite actomyosin system. We report a conserved armadillo and pleckstrin homology (PH) domain-containing protein, termed glideosome-associated connector (GAC), that mediates apicomplexan gliding motility, invasion, and egress by connecting the micronemal adhesins with the actomyosin system. TgGAC binds to and stabilizes filamentous actin and specifically associates with the transmembrane adhesin TgMIC2. GAC localizes to the apical pole in invasive stages of Toxoplasma gondii and Plasmodium berghei, and apical positioning of TgGAC depends on an apical lysine methyltransferase, TgAKMT. GAC PH domain also binds to phosphatidic acid, a lipid mediator associated with microneme exocytosis. Collectively, these findings indicate a central role for GAC in spatially and temporally coordinating gliding motility and invasion.
Sujet(s)
Apicomplexa/cytologie , Apicomplexa/physiologie , Lipides , Protéines des microfilaments/physiologie , Moteurs moléculaires/physiologie , Protéines de protozoaire/physiologie , Cytosquelette d'actine/physiologie , Actines/physiologie , Animaux , Apicomplexa/métabolisme , Molécules d'adhérence cellulaire/physiologie , Mouvement cellulaire , Exocytose/physiologie , Protéines membranaires/métabolisme , Protéines membranaires/physiologie , Methyltransferases/métabolisme , Protéines des microfilaments/métabolisme , Modèles moléculaires , Organites , Acides phosphatidiques/métabolisme , Plasmodium berghei/métabolisme , Plasmodium berghei/physiologie , Conformation des protéines , Protozooses/parasitologie , Protéines de protozoaire/métabolisme , Lapins , Toxoplasma/cytologie , Toxoplasma/métabolisme , Toxoplasma/physiologie , Toxoplasmose/parasitologieRÉSUMÉ
The apicomplexan family of pathogens, which includes Plasmodium spp. and Toxoplasma gondii, are primarily obligate intracellular parasites and invade multiple cell types. These parasites express extracellular membrane protein receptors, adhesins, to form specific pathogen-host cell interaction complexes. Various adhesins are used to invade a variety of cell types. The receptors are linked to an actomyosin motor, which is part of a complex comprised of many proteins known as the invasion machinery or glideosome. To date, reviews on invasion have focused primarily on the molecular pathways and signals of invasion, with little or no structural information presented. Over 75 structures of parasite receptors and glideosome proteins have been deposited with the Protein Data Bank. These structures include adhesins, motor proteins, bridging proteins, inner membrane complex and cytoskeletal proteins, as well as co-crystal structures with peptides and antibodies. These structures provide information regarding key interactions necessary for target receptor engagement, machinery complex formation, how force is transmitted, and the basis of inhibitory antibodies. Additionally, these structures can provide starting points for the development of antibodies and inhibitory molecules targeting protein-protein interactions, with the aim to inhibit invasion. This review provides an overview of the parasite adhesin protein families, the glideosome components, glideosome architecture, and discuss recent work regarding alternative models.
Sujet(s)
Actomyosine/métabolisme , Apicomplexa/génétique , Apicomplexa/métabolisme , Interactions hôte-pathogène/physiologie , Protéines membranaires/métabolisme , Modèles moléculaires , Complexes multiprotéiques/métabolisme , Bases de données de protéines , Interactions hôte-pathogène/génétique , Protéines membranaires/génétique , Liaison aux protéines , Conformation des protéines , Cartographie d'interactions entre protéines , Structure tertiaire des protéinesRÉSUMÉ
The apicomplexan parasite Toxoplasma gondii must invade host cells to continue its lifecycle. It invades different cell types using an actomyosin motor that is connected to extracellular adhesins via the bridging protein fructose-1,6-bisphosphate aldolase. During invasion, aldolase serves in the role of a structural bridging protein, as opposed to its normal enzymatic role in the glycolysis pathway. Crystal structures of the homologous Plasmodium falciparum fructose-1,6-bisphosphate aldolase have been described previously. Here, T. gondii fructose-1,6-bisphosphate aldolase has been crystallized in space group P22121, with the biologically relevant tetramer in the asymmetric unit, and the structure has been determined via molecular replacement to a resolution of 2.0â Å. An analysis of the quality of the model and of the differences between the four chains in the asymmetric unit and a comparison between the T. gondii and P. falciparum aldolase structures is presented.
Sujet(s)
Fructose bisphosphate aldolase/composition chimique , Fructose diphosphate/composition chimique , Toxoplasma/enzymologie , Séquence d'acides aminés , Animaux , Cristallographie aux rayons X , Données de séquences moléculaires , Conformation des protéinesRÉSUMÉ
The myosin motor of the malaria parasite's invasion machinery moves over actin fibers while it is making critical contacts with the myosin-tail interacting protein (MTIP). Previously, in a "compact" Plasmodium falciparum MTIPâ¢MyoA complex, MTIP domains 2 (D2) and 3 (D3) make contacts with the MyoA helix, and the central helix is kinked, but in an "extended" Plasmodium knowlesi MTIPâ¢MyoA complex only D3 interacts with the MyoA helix, and the central helix is fully extended. Here we report the crystal structure of the compact P. knowlesi MTIPâ¢MyoA complex. It appears that, depending on the pH, P. knowlesi MTIP can adopt either the compact or the extended conformation to interact with MyoA. Only at pH values above ~7.0, can key hydrogen bonds can be formed by the imidazole group of MyoA His810 with an aspartate carboxylate from the hinge of MTIP and a lysine amino group of MyoA simultaneously.
Sujet(s)
Protéines du cytosquelette/composition chimique , Plasmodium knowlesi/composition chimique , Cristallographie aux rayons X , Protéines du cytosquelette/métabolisme , Concentration en ions d'hydrogène , Modèles moléculaires , Plasmodium knowlesi/métabolisme , Liaison aux protéines , Conformation des protéines/effets des médicaments et des substances chimiquesRÉSUMÉ
Apicomplexan parasites enter host cells by many sophisticated steps including use of an ATP-powered invasion machinery. The machinery consists of multiple proteins, including a special myosin (MyoA) which moves along an actin fiber and which is connected to the myosin tail interaction protein (MTIP). Here we report a crystal structure of the major MyoA-binding domain (D3) of Plasmodium falciparum MTIP in complex with an anti-MTIP nanobody. In this complex, the MyoA-binding groove in MTIP-D3 is considerably less accessible than when occupied by the MyoA helix, due to a shift of two helices. The nanobody binds to an area slightly overlapping with the MyoA binding groove, covering a hydrophobic region next to the groove entrance. This provides a new avenue for arriving at compounds interfering with the invasion machinery since small molecules binding simultaneously to the nanobody binding site and the adjacent MyoA binding groove would prevent MyoA binding by MTIP.