RESUMO
Cytokinetic abscission marks the final stage of cell division, during which the daughter cells physically separate through the generation of new barriers, such as the plasma membrane or cell wall. While the contractile ring plays a central role during cytokinesis in bacteria, fungi and animal cells, the process diverges in Apicomplexa. In Toxoplasma gondii, two daughter cells are formed within the mother cell by endodyogeny. The mechanism by which the progeny cells acquire their plasma membrane during the disassembly of the mother cell, allowing daughter cells to emerge, remains unknown. Here we identify and characterize five T. gondii proteins, including three protein phosphatase 2A subunits, which exhibit a distinct and dynamic localization pattern during parasite division. Individual downregulation of these proteins prevents the accumulation of plasma membrane at the division plane, preventing the completion of cellular abscission. Remarkably, the absence of cytokinetic abscission does not hinder the completion of subsequent division cycles. The resulting progeny are able to egress from the infected cells but fail to glide and invade, except in cases of conjoined twin parasites.
RESUMO
Members of the Apicomplexa phylum possess specialized secretory organelles that discharge, apically and in a timely regulated manner, key factors implicated in parasite motility, host cell invasion, egress and subversion of host cellular functions. The mechanisms regulating trafficking and apical docking of these secretory organelles are only partially elucidated. Here, we characterized two conserved endosomal trafficking regulators known to promote vesicle transport and/or fusion, HOOK and Fused Toes (FTS), in the context of organelle discharge in Toxoplasma gondii. TgHOOK and TgFTS form a complex with a coccidian-specific partner, named HOOK interacting partner (HIP). TgHOOK displays an apically enriched vesicular pattern and concentrates at the parasite apical tip where it colocalizes with TgFTS and TgHIP. Functional investigations revealed that TgHOOK is dispensable but fitness conferring. The protein regulates the apical positioning and secretion of micronemes and contributes to egress, motility, host cell attachment, and invasion. Conditional depletion of TgFTS or TgHIP impacted on the same processes but led to more severe phenotypes. This study provides evidence of endosomal trafficking regulators involved in the apical exocytosis of micronemes and possibly as a consequence or directly on the discharge of the rhoptries. IMPORTANCE Toxoplasma gondii affects between 30 and 80% of the human population, poses a life-threatening risk to immunocompromised individuals, and is a cause of abortion and birth defects following congenital transmission. T. gondii belongs to the phylum of Apicomplexa characterized by a set of unique apical secretory organelles called the micronemes and rhoptries. Upon host cell recognition, this obligatory intracellular parasite secretes specific effectors contained in micronemes and rhoptries to promote parasite invasion of host cells and subsequent persistence. Here, we identified novel T. gondii endosomal trafficking regulators and demonstrated that they regulate microneme organelle apical positioning and exocytosis, thereby strongly contributing to host cell invasion and parasite virulence.
Assuntos
Toxoplasma , Humanos , Toxoplasma/metabolismo , Alta do Paciente , Transporte Biológico , Organelas/genética , Virulência , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Virulence and persistence of the obligate intracellular parasite Toxoplasma gondii involve the secretion of effector proteins belonging to the family of dense granule proteins (GRAs) that act notably as modulators of the host defense mechanisms and participate in cyst wall formation. The subset of GRAs residing in the parasitophorous vacuole (PV) or exported into the host cell, undergo proteolytic cleavage in the Golgi upon the action of the aspartyl protease 5 (ASP5). In tachyzoites, ASP5 substrates play central roles in the morphology of the PV and the export of effectors across the translocon complex MYR1/2/3. Here, we used N-terminal amine isotopic labeling of substrates to identify novel ASP5 cleavage products by comparing the N-terminome of wild-type and Δasp5 lines in tachyzoites and bradyzoites. Validated substrates reside within the PV or PVM in an ASP5-dependent manner. Remarkably, Δasp5 bradyzoites are impaired in the formation of the cyst wall in vitro and exhibit a considerably reduced cyst burden in chronically infected animals. More specifically two-photon serial tomography of infected mouse brains revealed a comparatively reduced number and size of the cysts throughout the establishment of persistence in the absence of ASP5.
Assuntos
Ácido Aspártico Proteases , Toxoplasma , Animais , Camundongos , Toxoplasma/metabolismo , Ácido Aspártico Proteases/metabolismo , Proteínas de Protozoários/metabolismo , Infecção Persistente , Vacúolos/metabolismo , Ácido Aspártico Endopeptidases/metabolismoRESUMO
Toxoplasma gondii possesses sphingolipid synthesis capabilities and is equipped to salvage lipids from its host. The contribution of these two routes of lipid acquisition during parasite development is unclear. As part of a complete ceramide synthesis pathway, T. gondii expresses two serine palmitoyltransferases (TgSPT1 and TgSPT2) and a dihydroceramide desaturase. After deletion of these genes, we determine their role in parasite development in vitro and in vivo during acute and chronic infection. Detailed phenotyping through lipidomic approaches reveal a perturbed sphingolipidome in these mutants, characterized by a drastic reduction in ceramides and ceramide phosphoethanolamines but not sphingomyelins. Critically, parasites lacking TgSPT1 display decreased fitness, marked by reduced growth rates and a selective defect in rhoptry discharge in the form of secretory vesicles, causing an invasion defect. Disruption of de novo ceramide synthesis modestly affects acute infection in vivo but severely reduces cyst burden in the brain of chronically infected mice.
Assuntos
Toxoplasma , Animais , Ceramidas/metabolismo , Camundongos , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismoRESUMO
Regulated microneme secretion governs motility, host cell invasion and egress in the obligate intracellular apicomplexans. Intracellular calcium oscillations and phospholipid dynamics critically regulate microneme exocytosis. Despite its importance for the lytic cycle of these parasites, molecular mechanistic details about exocytosis are still missing. Some members of the P4-ATPases act as flippases, changing the phospholipid distribution by translocation from the outer to the inner leaflet of the membrane. Here, the localization and function of the repertoire of P4-ATPases was investigated across the lytic cycle of Toxoplasma gondii. Of relevance, ATP2B and the non-catalytic subunit cell division control protein 50.4 (CDC50.4) form a stable heterocomplex at the parasite plasma membrane, essential for microneme exocytosis. This complex is responsible for flipping phosphatidylserine, which presumably acts as a lipid mediator for organelle fusion with the plasma membrane. Overall, this study points toward the importance of phosphatidylserine asymmetric distribution at the plasma membrane for microneme exocytosis.
Assuntos
Toxoplasma , Membrana Celular/metabolismo , Exocitose , Micronema , Fosfatidilserinas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismoRESUMO
Toxoplasma gondii infects virtually any nucleated cell and resides inside a non-phagocytic vacuole surrounded by a parasitophorous vacuolar membrane (PVM). Pivotal to the restriction of T. gondii dissemination upon infection in murine cells is the recruitment of immunity regulated GTPases (IRGs) and guanylate binding proteins (GBPs) to the PVM that leads to pathogen elimination. The virulent T. gondii type I RH strain secretes a handful of effectors including the dense granule protein GRA7, the serine-threonine kinases ROP17 and ROP18, and a pseudo-kinase ROP5, that synergistically inhibit the recruitment of IRGs to the PVM. Here, we characterise GRA60, a novel dense granule effector, which localises to the vacuolar space and PVM and contributes to virulence of RH in mice, suggesting a role in the subversion of host cell defence mechanisms. Members of the host cell IRG defence system Irgb10 and Irga6 are recruited to the PVM of RH parasites lacking GRA60 as observed previously for the avirulent RHΔrop5 mutant, with RH preventing such recruitment. Deletion of GRA60 in RHΔrop5 leads to a recruitment of IRGs comparable to the single knockouts. GRA60 therefore represents a novel parasite effector conferring resistance to IRGs in type I parasites, and found associated to ROP18, a member of the virulence complex.
Assuntos
Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Toxoplasma/imunologia , Toxoplasma/metabolismo , Toxoplasmose/imunologia , Toxoplasmose/metabolismo , Animais , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , DNA de Protozoário , Fibroblastos/parasitologia , Prepúcio do Pênis/parasitologia , GTP Fosfo-Hidrolases/imunologia , GTP Fosfo-Hidrolases/metabolismo , Técnicas de Inativação de Genes , Interações Hospedeiro-Parasita , Humanos , Imunidade , Interferon gama/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Toxoplasma/genética , Vacúolos/metabolismo , VirulênciaRESUMO
Toxoplasma gondii and members of the genus Plasmodium are obligate intracellular parasites that leave their infected host cell upon a tightly controlled process of egress. Intracellular replication of the parasites occurs within a parasitophorous vacuole, and its membrane as well as the host plasma membrane need to be disrupted during egress, leading to host cell lysis. While several parasite-derived factors governing egress have been identified, much less is known about host cell factors involved in this process. Previously, RNA interference (RNAi)-based knockdown and antibody-mediated depletion identified a host signaling cascade dependent on guanine nucleotide-binding protein subunit alpha q (GNAQ) to be required for the egress of Toxoplasma tachyzoites and Plasmodium blood stage merozoites. Here, we used CRISPR/Cas9 technology to generate HeLa cells deficient in GNAQ and tested their capacity to support the egress of T. gondii tachyzoites and Plasmodium berghei liver stage parasites. While we were able to confirm the importance of GNAQ for the egress of T. gondii, we found that the egress of P. berghei liver stages was unaffected in the absence of GNAQ. These results may reflect differences between the lytic egress process in apicomplexans and the formation of host cell-derived vesicles termed merosomes by P. berghei liver stages.IMPORTANCE The coordinated release of apicomplexan parasites from infected host cells prior to reinvasion is a critical process for parasite survival and the spread of infection. While Toxoplasma tachyzoites and Plasmodium blood stages induce a fast disruption of their surrounding membranes during their egress from host cells, Plasmodium liver stages keep the host cell membrane intact and leave their host cell in host cell-derived vesicles called merosomes. The knockout of GNAQ, a protein involved in G-protein-coupled receptor signaling, demonstrates the importance of this host factor for the lytic egress of T. gondii tachyzoites. Contrastingly, the egress of P. berghei is independent of GNAQ at the liver stage, indicating the existence of a mechanistically distinct strategy to exit the host cell.
Assuntos
Membrana Celular/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Hepatócitos/parasitologia , Plasmodium berghei/fisiologia , Animais , Sistemas CRISPR-Cas , Células HeLa , Humanos , Malária/parasitologia , Plasmodium berghei/genética , Toxoplasma/metabolismoRESUMO
Apicomplexans are obligate intracellular parasites harboring three sets of unique secretory organelles termed micronemes, rhoptries, and dense granules that are dedicated to the establishment of infection in the host cell. Apicomplexans rely on the endolysosomal system to generate the secretory organelles and to ingest and digest host cell proteins. These parasites also possess a metabolically relevant secondary endosymbiotic organelle, the apicoplast, which relies on vesicular trafficking for correct incorporation of nuclear-encoded proteins into the organelle. Here, we demonstrate that the trafficking and destination of vesicles to the unique and specialized parasite compartments depend on SNARE proteins that interact with tethering factors. Specifically, all secreted proteins depend on the function of SLY1 at the Golgi. In addition to a critical role in trafficking of endocytosed host proteins, TgVps45 is implicated in the biogenesis of the inner membrane complex (alveoli) in both Toxoplasma gondii and Plasmodium falciparum, likely acting in a coordinated manner with Stx16 and Stx6. Finally, Stx12 localizes to the endosomal-like compartment and is involved in the trafficking of proteins to the apical secretory organelles rhoptries and micronemes as well as to the apicoplast.IMPORTANCE The phylum of Apicomplexa groups medically relevant parasites such as those responsible for malaria and toxoplasmosis. As members of the Alveolata superphylum, these protozoans possess specialized organelles in addition to those found in all members of the eukaryotic kingdom. Vesicular trafficking is the major route of communication between membranous organelles. Neither the molecular mechanism that allows communication between organelles nor the vesicular fusion events that underlie it are completely understood in Apicomplexa. Here, we assessed the function of SEC1/Munc18 and SNARE proteins to identify factors involved in the trafficking of vesicles between these various organelles. We show that SEC1/Munc18 in interaction with SNARE proteins allows targeting of vesicles to the inner membrane complex, prerhoptries, micronemes, apicoplast, and vacuolar compartment from the endoplasmic reticulum, Golgi apparatus, or endosomal-like compartment. These data provide an exciting look at the "ZIP code" of vesicular trafficking in apicomplexans, essential for precise organelle biogenesis, homeostasis, and inheritance.
Assuntos
Apicoplastos/metabolismo , Vesículas Citoplasmáticas/metabolismo , Proteínas Munc18/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas SNARE/metabolismo , Toxoplasma/metabolismo , Apicoplastos/genética , Vesículas Citoplasmáticas/genética , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Proteínas Munc18/genética , Plasmodium falciparum/genética , Transporte Proteico , Proteínas de Protozoários/genética , Proteínas SNARE/genética , Toxoplasma/genéticaRESUMO
Toxoplasma gondii aspartyl protease 3 (TgASP3) phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X (PfPMIX, PfPMX). These proteases are essential for parasite survival, acting as key maturases for secreted proteins implicated in invasion and egress. A potent antimalarial peptidomimetic inhibitor (49c) originally developed against Plasmepsin II selectively targets TgASP3, PfPMIX, and PfPMX To unravel the molecular basis for the selectivity of 49c, we constructed homology models of PfPMIX, PfPMX, and TgASP3 that were first validated by identifying the determinants of microneme and rhoptry substrate recognition. The flap and flap-like structures of several reported Plasmepsins are highly flexible and critically modulate the access to the binding cavity. Molecular docking of 49c to TgASP3, PfPMIX, and PfPMX models predicted that the conserved phenylalanine residues in the flap, F344, F291, and F305, respectively, account for the sensitivity toward 49c. Concordantly, phenylalanine mutations in the flap of the three proteases increase twofold to 15-fold the IC50 values of 49c. Compellingly the selection of mutagenized T. gondii resistant strains to 49c reproducibly converted F344 to a cysteine residue.
Assuntos
Antimaláricos/farmacologia , Ácido Aspártico Proteases/antagonistas & inibidores , Ácido Aspártico Proteases/metabolismo , Resistência a Medicamentos/fisiologia , Inibidores de Proteases/farmacologia , Proteínas de Protozoários/química , Antimaláricos/química , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Cisteína , Relação Dose-Resposta a Droga , Resistência a Medicamentos/efeitos dos fármacos , Concentração Inibidora 50 , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutação , Testes de Sensibilidade Parasitária , Fenilalanina/efeitos dos fármacos , Fenilalanina/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Alinhamento de Sequência , Toxoplasma/efeitos dos fármacos , Toxoplasma/genéticaRESUMO
Toxoplasma gondii encodes three protein kinase A catalytic (PKAc1-3) and one regulatory (PKAr) subunits to integrate cAMP-dependent signals. Here, we show that inactive PKAc1 is maintained at the parasite pellicle by interacting with acylated PKAr. Either a conditional knockdown of PKAr or the overexpression of PKAc1 blocks parasite division. Conversely, down-regulation of PKAc1 or stabilisation of a dominant-negative PKAr isoform that does not bind cAMP triggers premature parasite egress from infected cells followed by serial invasion attempts leading to host cell lysis. This untimely egress depends on host cell acidification. A phosphoproteome analysis suggested the interplay between cAMP and cGMP signalling as PKAc1 inactivation changes the phosphorylation profile of a putative cGMP-phosphodiesterase. Concordantly, inhibition of the cGMP-dependent protein kinase G (PKG) blocks egress induced by PKAc1 inactivation or environmental acidification, while a cGMP-phosphodiesterase inhibitor circumvents egress repression by PKAc1 or pH neutralisation. This indicates that pH and PKAc1 act as balancing regulators of cGMP metabolism to control egress. These results reveal a crosstalk between PKA and PKG pathways to govern egress in T. gondii.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/genética , Interações Hospedeiro-Parasita , Proteínas de Protozoários/genética , Toxoplasma/genética , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Acilação , Linhagem Celular Transformada , AMP Cíclico/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Fibroblastos/parasitologia , Regulação da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Estágios do Ciclo de Vida/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/metabolismoRESUMO
The obligate intracellular lifestyle of apicomplexan parasites necessitates an invasive phase underpinned by timely and spatially controlled secretion of apical organelles termed micronemes. In Toxoplasma gondii, extracellular potassium levels and other stimuli trigger a signaling cascade culminating in phosphoinositide-phospholipase C (PLC) activation, which generates the second messengers diacylglycerol (DAG) and IP3 and ultimately results in microneme secretion. Here we show that a delicate balance between DAG and its downstream product, phosphatidic acid (PA), is essential for controlling microneme release. Governing this balance is the apicomplexan-specific DAG-kinase-1, which interconverts PA and DAG, and whose depletion impairs egress and causes parasite death. Additionally, we identify an acylated pleckstrin-homology (PH) domain-containing protein (APH) on the microneme surface that senses PA during microneme secretion and is necessary for microneme exocytosis. As APH is conserved in Apicomplexa, these findings highlight a potentially widely used mechanism in which key lipid mediators regulate microneme exocytosis.
Assuntos
Organelas/efeitos dos fármacos , Organelas/metabolismo , Ácidos Fosfatídicos/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Toxoplasma/fisiologia , Diacilglicerol Quinase/metabolismo , Diglicerídeos/metabolismoRESUMO
Many members of the phylum of Apicomplexa have adopted an obligate intracellular life style and critically depend on active invasion and egress from the infected cells to complete their lytic cycle. Toxoplasma gondii belongs to the coccidian subgroup of the Apicomplexa, and as such, the invasive tachyzoite contains an organelle termed the conoid at its extreme apex. This motile organelle consists of a unique polymer of tubulin fibres and protrudes in both gliding and invading parasites. The class XIV myosin A, which is conserved across the Apicomplexa phylum, is known to critically contribute to motility, invasion and egress from infected cells. The MyoA-glideosome is anchored to the inner membrane complex (IMC) and is assumed to translocate the components of the circular junction secreted by the micronemes and rhoptries, to the rear of the parasite. Here we comprehensively characterise the class XIV myosin H (MyoH) and its associated light chains. We show that the 3 alpha-tubulin suppressor domains, located in MyoH tail, are necessary to anchor this motor to the conoid. Despite the presence of an intact MyoA-glideosome, conditional disruption of TgMyoH severely compromises parasite motility, invasion and egress from infected cells. We demonstrate that MyoH is necessary for the translocation of the circular junction from the tip of the parasite, where secretory organelles exocytosis occurs, to the apical position where the IMC starts. This study attributes for the first time a direct function of the conoid in motility and invasion, and establishes the indispensable role of MyoH in initiating the first step of motility along this unique organelle, which is subsequently relayed by MyoA to enact effective gliding and invasion.
Assuntos
Interações Hospedeiro-Parasita/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose/metabolismo , Animais , Western Blotting , Chlorocebus aethiops , Técnica Indireta de Fluorescência para Anticorpo , Técnicas de Inativação de Genes , Humanos , Imunoprecipitação , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Organelas , Toxoplasma/citologia , Transfecção , Células VeroRESUMO
Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite's ability to modulate host signalling pathways and immune responses.
Assuntos
Ácido Aspártico Proteases/metabolismo , Complexo de Golgi/enzimologia , Interações Hospedeiro-Parasita/fisiologia , Toxoplasma/patogenicidade , Toxoplasmose/enzimologia , Animais , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Transporte Proteico , Reação em Cadeia da Polimerase em Tempo Real , Toxoplasma/enzimologia , TransfecçãoRESUMO
Host cell entry by the Apicomplexa is associated with the sequential secretion of invasion factors from specialized apical organelles. Secretion of micronemal proteins (MICs) complexes by Toxoplasma gondii facilitates parasite gliding motility, host cell attachment and entry, as well as egress from infected cells. The shedding of MICs during these steps is mediated by micronemal protein proteases MPP1, MPP2 and MPP3. The constitutive activity of MPP1 leads to the cleavage of transmembrane MICs and is linked to the surface rhomboid protease 4 (ROM4) and possibly to rhomboid protease 5 (ROM5). To determine their importance and respective contribution to MPP1 activity, in this study ROM4 and ROM5 genes were abrogated using Cre-recombinase and CRISPR-Cas9 nuclease, respectively, and shown to be dispensable for parasite survival. Parasites lacking ROM4 predominantly engage in twirling motility and exhibit enhanced attachment and impaired invasion, whereas intracellular growth and egress is not affected. The substrates MIC2 and MIC6 are not cleaved in rom4-ko parasites, in contrast, intramembrane cleavage of AMA1 is reduced but not completely abolished. Shedding of MICs and invasion are not altered in the absence of ROM5; however, this protease responsible for the residual cleavage of AMA1 is able to cleave other AMA family members and exhibits a detectable contribution to invasion in the absence of ROM4.
Assuntos
Moléculas de Adesão Celular/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Toxoplasma/patogenicidade , Interações Hospedeiro-Parasita , Humanos , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Peptídeo Hidrolases/genética , Proteínas de Protozoários/genética , Toxoplasma/genéticaRESUMO
The glideosome is an actomyosin-based machinery that powers motility in Apicomplexa and participates in host cell invasion and egress from infected cells. The central component of the glideosome, myosin A (MyoA), is a motor recruited at the pellicle by the acylated gliding-associated protein GAP45. In Toxoplasma gondii, GAP45 also contributes to the cohesion of the pellicle, composed of the inner membrane complex (IMC) and the plasma membrane, during motor traction. GAP70 was previously identified as a paralog of GAP45 that is tailored to recruit MyoA at the apical cap in the coccidian subgroup of the Apicomplexa. A third member of this family, GAP80, is demonstrated here to assemble a new glideosome, which recruits the class XIV myosin C (MyoC) at the basal polar ring. MyoC shares the same myosin light chains as MyoA and also interacts with the integral IMC proteins GAP50 and GAP40. Moreover, a central component of this complex, the IMC-associated protein 1 (IAP1), acts as the key determinant for the restricted localization of MyoC to the posterior pole. Deletion of specific components of the MyoC-glideosome underscores the installation of compensatory mechanisms with components of the MyoA-glideosome. Conversely, removal of MyoA leads to the relocalization of MyoC along the pellicle and at the apical cap that accounts for residual invasion. The two glideosomes exhibit a considerable level of plasticity to ensure parasite survival.
Assuntos
Proteínas de Membrana/metabolismo , Miosinas/metabolismo , Toxoplasma/metabolismo , Toxoplasmose/parasitologia , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Sequência de Bases , Membrana Celular/metabolismo , Movimento Celular , Genes Reporter , Interações Hospedeiro-Parasita , Humanos , Proteínas de Membrana/genética , Modelos Biológicos , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Cadeias Leves de Miosina/metabolismo , Miosinas/genética , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIA/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Análise de Sequência de DNA , Deleção de Sequência , Toxoplasma/genéticaRESUMO
Malaria and toxoplasmosis are infectious diseases caused by the apicomplexan parasites Plasmodium and Toxoplasma gondii, respectively. These parasites have developed an invasion mechanism involving the formation of a moving junction (MJ) that anchors the parasite to the host cell and forms a ring through which the parasite penetrates. The composition and the assembly of the MJ, and in particular the presence of protein AMA1 and its interaction with protein RON2 at the MJ, have been the subject of intense controversy. Here, using reverse genetics, we show that AMA1, a vaccine candidate, interacts with RON2 to maintain the MJ structural integrity in T. gondii and is subsequently required for parasite internalization. Moreover, we show that disruption of the AMA1 gene results in upregulation of AMA1 and RON2 homologues that cooperate to support residual invasion. Our study highlights a considerable complexity and molecular plasticity in the architecture of the MJ.
Assuntos
Antígenos de Protozoários/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Toxoplasmose/parasitologia , Antígenos de Protozoários/genética , Deleção de Genes , Humanos , Modelos Moleculares , Ligação Proteica , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasma/patogenicidade , VirulênciaRESUMO
Members of the phylum Apicomplexa possess a highly conserved molecular motor complex anchored in the parasite pellicle and associated with gliding motility, invasion and egress from infected cells. This machinery, called the glideosome, is structured around the acylated gliding-associated protein GAP45 that recruits the motor complex composed of myosin A and two associated myosin light chains (TgMLC1 and TgELC1). This motor is presumably firmly anchored to the inner membrane complex underneath the plasma membrane via an interaction with two integral membrane proteins, GAP50 and GAP40. To determine if the previously mapped phosphorylation sites on TgGAP45 and TgMLC1 have a direct significance for glideosome assembly and function, a series of phospho-mimetic and phospho-null mutants were generated. Neither the overexpression nor the allelic replacement of TgMLC1 with phospho-mutants impacted on glideosome assembly and parasite motility. TgGAP45 phosphorylation mutants were functionally investigated using a complementation strategy in a TgGAP45 inducible knockout background. The loss of interaction with TgGAP50 by one previously reported GAP45-mutant appeared to depend only on the presence of a remaining competing wild type copy of TgGAP45. Accordingly, this mutant displayed no phenotype in complementation experiments. Unexpectedly, GAP45 lacking the region encompassing the cluster of twelve phosphorylation sites did not impact on its dual function in motor recruitment and pellicle integrity. Despite the extensive phosphorylation of TgMLC1 and TgGAP45, this post-translational modification does not appear to be critical for the assembly and function of the glideosome.
Assuntos
Movimento Celular/fisiologia , Proteínas Motores Moleculares/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Membrana Celular , Movimento Celular/genética , Interações Hospedeiro-Parasita , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Motores Moleculares/genética , Fosforilação , Proteínas de Protozoários/genéticaRESUMO
Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.
Assuntos
Galactose/metabolismo , Galectinas/metabolismo , Complexos Multiproteicos/metabolismo , Toxoplasma/metabolismo , Animais , Moléculas de Adesão Celular , Galactose/imunologia , Galectinas/química , Galectinas/genética , Galectinas/imunologia , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/imunologia , Estrutura Terciária de Proteína , Proteínas de Protozoários , Toxoplasma/química , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasmose/genética , Toxoplasmose/imunologia , Toxoplasmose/metabolismoRESUMO
Toxoplasma gondii possesses 11 rather atypical myosin heavy chains. The only myosin light chain described to date is MLC1, associated with myosin A, and contributing to gliding motility. In this study, we examined the repertoire of calmodulin-like proteins in Apicomplexans, identified six putative myosin light chains and determined their subcellular localization in T. gondii and Plasmodium falciparum. MLC2, only found in coccidians, is associated with myosin D via its calmodulin (CaM)-like domain and anchored to the plasma membrane of T. gondii via its N-terminal extension. Molecular modeling suggests that the MyoD-MLC2 complex is more compact than the reported structure of Plasmodium MyoA-myosin A tail-interacting protein (MTIP) complex. Anchorage of this MLC2 to the plasma membrane is likely governed by palmitoylation.
Assuntos
Miosinas Cardíacas/metabolismo , Membrana Celular/metabolismo , Proteínas Motores Moleculares/genética , Proteína MyoD/metabolismo , Cadeias Leves de Miosina/metabolismo , Toxoplasma/metabolismo , Sequência de Aminoácidos , Apicomplexa/classificação , Apicomplexa/metabolismo , Miosinas Cardíacas/química , Miosinas Cardíacas/genética , Lipoilação , Modelos Moleculares , Dados de Sequência Molecular , Proteína MyoD/química , Proteína MyoD/genética , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Filogenia , Estrutura Quaternária de Proteína , Transporte Proteico , Alinhamento de Sequência , Toxoplasma/classificaçãoRESUMO
Arenavirus RNA genomes are initiated by a "prime and realign" mechanism, such that the initiating GTP is found as a single unpaired (overhanging) nucleotide when the complementary genome ends anneal to form double-stranded (ds) RNA panhandle structures. dsRNAs modeled on these structures do not induce interferon (IFN), as opposed to blunt-ended (5' ppp)dsRNA. This study examines whether these viral structures can also act as decoys, by trapping RIG-I in inactive dsRNA complexes. We examined the ability of various dsRNAs to activate the RIG-I ATPase (presumably a measure of helicase translocation on dsRNA) relative to their ability to induce IFN. We found that there is no simple relationship between these two properties, as if RIG-I can translocate on short dsRNAs without inducing IFN. Moreover, we found that (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide can in fact competitively inhibit the ability of blunt-ended (5' ppp)dsRNAs to induce IFN when co-transfected into cells and that this inhibition is strongly dependent on the presence of the 5' ppp. In contrast, (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide does not inhibit poly(I-C)-induced IFN activation, which is independent of the presence of a 5' ppp group.