RESUMEN
Apical membrane antigen-1 (AMA1) is a conserved malarial vaccine candidate essential for the formation of tight junctions with the rhoptry neck protein (RON) complex, enabling Plasmodium parasites to invade human erythrocytes, hepatocytes, and mosquito salivary glands. Despite its critical role, extensive surface polymorphisms in AMA1 have led to strain-specific protection, limiting the success of AMA1-based interventions beyond initial clinical trials. Here, we identify an i-body, a humanised single-domain antibody-like molecule that recognises a conserved pan-species conformational epitope in AMA1 with low nanomolar affinity and inhibits the binding of the RON2 ligand to AMA1. Structural characterisation indicates that the WD34 i-body epitope spans the centre of the conserved hydrophobic cleft in AMA1, where interacting residues are highly conserved among all Plasmodium species. Furthermore, we show that WD34 inhibits merozoite invasion of erythrocytes by multiple Plasmodium species and hepatocyte invasion by P. falciparum sporozoites. Despite a short half-life in mouse serum, we demonstrate that WD34 transiently suppressed P. berghei infections in female BALB/c mice. Our work describes the first pan-species AMA1 biologic with inhibitory activity against multiple life-cycle stages of Plasmodium. With improved pharmacokinetic characteristics, WD34 could be a potential immunotherapy against multiple species of Plasmodium.
Asunto(s)
Antígenos de Protozoos , Eritrocitos , Hígado , Proteínas de la Membrana , Ratones Endogámicos BALB C , Proteínas Protozoarias , Animales , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Femenino , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Humanos , Eritrocitos/parasitología , Eritrocitos/inmunología , Hígado/parasitología , Hígado/inmunología , Hígado/metabolismo , Vacunas contra la Malaria/inmunología , Malaria/inmunología , Malaria/parasitología , Malaria/prevención & control , Reacciones Cruzadas/inmunología , Plasmodium falciparum/inmunología , Plasmodium berghei/inmunología , Epítopos/inmunología , Hepatocitos/parasitología , Hepatocitos/inmunología , Hepatocitos/metabolismo , Plasmodium/inmunología , Merozoítos/inmunología , Merozoítos/metabolismoRESUMEN
Plasmodium falciparum causes severe malaria and assembles a protein translocon (PTEX) complex at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported to facilitate growth. The preceding liver stage of infection involves growth in a hepatocyte-derived PVM; however, the importance of protein export during P. falciparum liver infection remains unexplored. Here, we use the FlpL/FRT system to conditionally excise genes in P. falciparum sporozoites for functional liver-stage studies. Disruption of PTEX members ptex150 and exp2 did not affect sporozoite development in mosquitoes or infectivity for hepatocytes but attenuated liver-stage growth in humanized mice. While PTEX150 deficiency reduced fitness on day 6 postinfection by 40%, EXP2 deficiency caused 100% loss of liver parasites, demonstrating that PTEX components are required for growth in hepatocytes to differing degrees. To characterize PTEX loss-of-function mutations, we localized four liver-stage Plasmodium export element (PEXEL) proteins. P. falciparum liver specific protein 2 (LISP2), liver-stage antigen 3 (LSA3), circumsporozoite protein (CSP), and a Plasmodium berghei LISP2 reporter all localized to the periphery of P. falciparum liver stages but were not exported beyond the PVM. Expression of LISP2 and CSP but not LSA3 was reduced in ptex150-FRT and exp2-FRT liver stages, suggesting that expression of some PEXEL proteins is affected directly or indirectly by PTEX disruption. These results show that PTEX150 and EXP2 are important for P. falciparum development in hepatocytes and emphasize the emerging complexity of PEXEL protein trafficking.
Asunto(s)
Hepatocitos , Hígado , Malaria Falciparum , Plasmodium falciparum , Proteínas Protozoarias , Esporozoítos , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Animales , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Esporozoítos/metabolismo , Esporozoítos/crecimiento & desarrollo , Ratones , Hígado/parasitología , Hígado/metabolismo , Humanos , Hepatocitos/parasitología , Hepatocitos/metabolismo , Malaria Falciparum/parasitologíaRESUMEN
Apicomplexan parasites are aetiological agents of numerous diseases in humans and livestock. Functional genomics studies in these parasites enable the identification of biological mechanisms and protein functions that can be targeted for therapeutic intervention. Recent improvements in forward genetics and whole-genome screens utilising CRISPR/Cas technology have revolutionised the functional analysis of genes during Apicomplexan infection of host cells. Here, we highlight key discoveries from CRISPR/Cas9 screens in Apicomplexa or their infected host cells and discuss remaining challenges to maximise this technology that may help answer fundamental questions about parasite-host interactions.
Asunto(s)
Apicomplexa , Parásitos , Humanos , Animales , Sistemas CRISPR-Cas , Genoma , Apicomplexa/genética , Parásitos/genética , Interacciones Huésped-ParásitosRESUMEN
BACKGROUND & AIMS: Necroptosis is a highly inflammatory mode of cell death that has been implicated in causing hepatic injury including steatohepatitis/ nonalcoholic steatohepatitis (NASH); however, the evidence supporting these claims has been controversial. A comprehensive, fundamental understanding of cell death pathways involved in liver disease critically underpins rational strategies for therapeutic intervention. We sought to define the role and relevance of necroptosis in liver pathology. METHODS: Several animal models of human liver pathology, including diet-induced steatohepatitis in male mice and diverse infections in both male and female mice, were used to dissect the relevance of necroptosis in liver pathobiology. We applied necroptotic stimuli to primary mouse and human hepatocytes to measure their susceptibility to necroptosis. Paired liver biospecimens from patients with NASH, before and after intervention, were analyzed. DNA methylation sequencing was also performed to investigate the epigenetic regulation of RIPK3 expression in primary human and mouse hepatocytes. RESULTS: Identical infection kinetics and pathologic outcomes were observed in mice deficient in an essential necroptotic effector protein, MLKL, compared with control animals. Mice lacking MLKL were indistinguishable from wild-type mice when fed a high-fat diet to induce NASH. Under all conditions tested, we were unable to induce necroptosis in hepatocytes. We confirmed that a critical activator of necroptosis, RIPK3, was epigenetically silenced in mouse and human primary hepatocytes and rendered them unable to undergo necroptosis. CONCLUSIONS: We have provided compelling evidence that necroptosis is disabled in hepatocytes during homeostasis and in the pathologic conditions tested in this study.
Asunto(s)
Necroptosis , Enfermedad del Hígado Graso no Alcohólico , Humanos , Femenino , Masculino , Ratones , Animales , Epigénesis Genética , Enfermedad del Hígado Graso no Alcohólico/genética , Hepatocitos , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteínas Quinasas/genéticaRESUMEN
Tryptophan C-mannosylation stabilizes proteins bearing a thrombospondin repeat (TSR) domain in metazoans. Here we show that Plasmodium falciparum expresses a DPY19 tryptophan C-mannosyltransferase in the endoplasmic reticulum and that DPY19-deficiency abolishes C-glycosylation, destabilizes members of the TRAP adhesin family and inhibits transmission to mosquitoes. Imaging P. falciparum gametogenesis in its entirety in four dimensions using lattice light-sheet microscopy reveals defects in ΔDPY19 gametocyte egress and exflagellation. While egress is diminished, ΔDPY19 microgametes still fertilize macrogametes, forming ookinetes, but these are abrogated for mosquito infection. The gametogenesis defects correspond with destabilization of MTRAP, which we show is C-mannosylated in P. falciparum, and the ookinete defect is concordant with defective CTRP secretion on the ΔDPY19 background. Genetic complementation of DPY19 restores ookinete infectivity, sporozoite production and C-mannosylation activity. Therefore, tryptophan C-mannosylation by DPY19 ensures TSR protein quality control at two lifecycle stages for successful transmission of the human malaria parasite.
Asunto(s)
Culicidae , Malaria Falciparum , Animales , Culicidae/metabolismo , Glicosilación , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trombospondinas/metabolismo , Triptófano/metabolismoRESUMEN
Repeated Plasmodium falciparum infections drive the development of clinical immunity to malaria in humans; however, the immunological mechanisms that underpin this response are only partially understood. We investigated the impact of repeated P. falciparum infections on human γδ T cells in the context of natural infection in Malian children and adults, as well as serial controlled human malaria infection (CHMI) of U.S. adults, some of whom became clinically immune to malaria. In contrast to the predominant Vδ2+ T cell population in malaria-naïve Australian individuals, clonally expanded cytotoxic Vδ1effector T cells were enriched in the γδ T cell compartment of Malian subjects. Malaria-naïve U.S. adults exposed to four sequential CHMIs defined the precise impact of P. falciparum on the γδ T cell repertoire. Specifically, innate-like Vδ2+ T cells exhibited an initial robust polyclonal response to P. falciparum infection that was not sustained with repeated infections, whereas Vδ1+ T cells increased in frequency with repeated infections. Moreover, repeated P. falciparum infection drove waves of clonal selection in the Vδ1+ T cell receptor repertoire that coincided with the differentiation of Vδ1naïve T cells into cytotoxic Vδ1effector T cells. Vδ1+ T cells of malaria-exposed Malian and U.S. individuals were licensed for reactivity to P. falciparum parasites in vitro. Together, our study indicates that repeated P. falciparum infection drives the clonal expansion of an adaptive γδ T cell repertoire and establishes a role for Vδ1+ T cells in the human immune response to malaria.
Asunto(s)
Malaria Falciparum , Plasmodium falciparum , Adulto , Australia , Niño , Humanos , Malaria Falciparum/parasitología , Receptores de Antígenos de Linfocitos T gamma-delta , Linfocitos TRESUMEN
The current Malaria RTS,S vaccine is based on virus-like particles (VLPs) comprising the NANP repetitive epitopes from the cicumsporozoite protein (CSP) of Plasmodium falciparum. This vaccine has limited efficacy, only preventing severe disease in about 30% of vaccinated individuals. A more efficacious vaccine is urgently needed to combat malaria. Here we developed a particulate malaria vaccine based on the same CSP epitopes but using biopolymer particles (BPs) as an antigen carrier system. Specific B- and T-cell epitope-coated BPs were assembled in vivo inside an engineered endotoxin-free mutant of Escherichia coli. A high-yield production process leading to ~27% BP vaccine weight over biomass was established. The epitope-coated BPs were purified and their composition, i.e., the polymer core and epitope identity, was confirmed. Epitope-coated BPs were used alongside soluble peptide epitopes and empty BPs to vaccinate sheep. Epitope-coated BPs showed enhanced immunogenicity by inducing anti-NANP antibody titre of EC50 > 150,000 that were at least 20 times higher than induced by the soluble peptides. We concluded that the additional T-cell epitope was not required as it did not enhance immunogenicity when compared with the B-cell epitope-coated BPs. Antibodies specifically bound to the surface of Plasmodium falciparum sporozoites and efficiently inhibited sporozoite motility and traversal of human hepatocytes. This study demonstrated the utility of biologically self-assembled epitope-coated BPs as an epitope carrier for inclusion in next-generation malaria vaccines.
RESUMEN
Malaria is a devastating disease caused by Plasmodium parasites. Emerging resistance against current antimalarial therapeutics has engendered the need to develop antimalarials with novel structural classes. We recently described the identification and initial optimization of the 2-anilino quinazoline antimalarial class. Here, we refine the physicochemical properties of this antimalarial class with the aim to improve aqueous solubility and metabolism and to reduce adverse promiscuity. We show the physicochemical properties of this class are intricately balanced with asexual parasite activity and human cell cytotoxicity. Structural modifications we have implemented improved LipE, aqueous solubility and in vitro metabolism while preserving fast acting P. falciparum asexual stage activity. The lead compounds demonstrated equipotent activity against P. knowlesi parasites and were not predisposed to resistance mechanisms of clinically used antimalarials. The optimized compounds exhibited modest activity against early-stage gametocytes, but no activity against pre-erythrocytic liver parasites. Confoundingly, the refined physicochemical properties installed in the compounds did not engender improved oral efficacy in a P. berghei mouse model of malaria compared to earlier studies on the 2-anilino quinazoline class. This study provides the framework for further development of this antimalarial class.
Asunto(s)
Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Antimaláricos/química , Antimaláricos/farmacología , Malaria/tratamiento farmacológico , Plasmodium/efectos de los fármacos , Quinazolinas/química , Quinazolinas/farmacología , Aminación , Compuestos de Anilina/uso terapéutico , Animales , Antimaláricos/uso terapéutico , Femenino , Humanos , Malaria/parasitología , Ratones , Plasmodium/fisiología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/fisiología , Quinazolinas/uso terapéuticoRESUMEN
Twenty years ago the Molecular Approaches to Malaria conference was conceived as a forum to present the very latest advances in malaria research and to consolidate and forge new collaborative links between international researchers. The 6th MAM conference, held in February 2020 in Australia, provided 5 days of stimulating scientific exchange and highlighted the incredible malaria research conducted globally that is providing the critical knowledge and cutting-edge technological tools needed to control and ultimately eliminate malaria.
Asunto(s)
Malaria , Plasmodium , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Desarrollo de Medicamentos , Resistencia a Medicamentos , Humanos , Inmunogenicidad Vacunal , Malaria/tratamiento farmacológico , Malaria/inmunología , Malaria/parasitología , Malaria/prevención & control , Vacunas contra la Malaria/inmunología , Plasmodium/efectos de los fármacos , Plasmodium/genética , Plasmodium/patogenicidad , Plasmodium/fisiologíaRESUMEN
Plasmodium sporozoites infect the liver and develop into exoerythrocytic merozoites that initiate blood-stage disease. The hepatocyte molecular pathways that permit or abrogate parasite replication and merozoite formation have not been thoroughly explored, and a deeper understanding may identify therapeutic strategies to mitigate malaria. Cellular inhibitor of apoptosis (cIAP) proteins regulate cell survival and are co-opted by intracellular pathogens to support development. Here, we show that cIAP1 levels are upregulated during Plasmodium liver infection and that genetic or pharmacological targeting of cIAPs using clinical-stage antagonists preferentially kills infected hepatocytes and promotes immunity. Using gene-targeted mice, the mechanism was defined as TNF-TNFR1-mediated apoptosis via caspases 3 and 8 to clear parasites. This study reveals the importance of cIAPs to Plasmodium infection and demonstrates that host-directed antimalarial drugs can eliminate liver parasites and induce immunity while likely providing a high barrier to resistance in the parasite.
Asunto(s)
Apoptosis , Hepatocitos/patología , Hígado/patología , Hígado/parasitología , Malaria/patología , Malaria/parasitología , Administración Oral , Animales , Apoptosis/efectos de los fármacos , Disponibilidad Biológica , Caspasa 3/metabolismo , Culicidae/parasitología , Dipéptidos/administración & dosificación , Dipéptidos/farmacología , Hepatocitos/efectos de los fármacos , Inmunidad/efectos de los fármacos , Indoles/administración & dosificación , Indoles/farmacología , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/metabolismo , Estadios del Ciclo de Vida/efectos de los fármacos , Malaria/inmunología , Plasmodium/efectos de los fármacos , Plasmodium/crecimiento & desarrollo , Plasmodium/metabolismo , Proteínas Protozoarias/metabolismo , Esporozoítos/efectos de los fármacos , Esporozoítos/fisiología , Tiazoles/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Artemisin combination therapy (ACT) is the main treatment option for malaria, which is caused by the intracellular parasite Plasmodium. However, increased resistance to ACT highlights the importance of finding new drugs. Recently, the aspartic proteases Plasmepsin IX and X (PMIX and PMX) were identified as promising drug targets. In this study, we describe dual inhibitors of PMIX and PMX, including WM382, that block multiple stages of the Plasmodium life cycle. We demonstrate that PMX is a master modulator of merozoite invasion and direct maturation of proteins required for invasion, parasite development, and egress. Oral administration of WM382 cured mice of P. berghei and prevented blood infection from the liver. In addition, WM382 was efficacious against P. falciparum asexual infection in humanized mice and prevented transmission to mosquitoes. Selection of resistant P. falciparum in vitro was not achievable. Together, these show that dual PMIX and PMX inhibitors are promising candidates for malaria treatment and prevention.
Asunto(s)
Antimaláricos/farmacología , Ácido Aspártico Endopeptidasas/efectos de los fármacos , Malaria/tratamiento farmacológico , Animales , Transmisión de Enfermedad Infecciosa/prevención & control , Estadios del Ciclo de Vida/efectos de los fármacos , Merozoítos/efectos de los fármacos , Ratones , Ratones Transgénicos , Plasmodium berghei/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacosRESUMEN
Plasmodium parasites cause malaria and are maintained between Anopheles mosquitoes and mammalian hosts in a complex life cycle. Malaria parasites occupy tissue niches that can be difficult to access, and models to study them can be challenging to recapitulate experimentally, particularly for Plasmodium species that infect humans. 2D culture models provide extremely beneficial tools to investigate Plasmodium biology but they have limitations. More complex 3D structural networks, such as organoids, have unveiled new avenues for developing more physiological tissue models, and their application to malaria research offers great promise. Here, we review current models for studying Plasmodium infection with a key focus on the obligate pre-erythrocytic stage that culminates in blood infection, causing malaria, and discuss how organoids should fulfil an important and unmet need.
Asunto(s)
Interacciones Huésped-Parásitos/fisiología , Hígado/parasitología , Malaria , Organoides/parasitología , Parasitología/tendencias , Plasmodium/fisiología , Animales , HumanosRESUMEN
Plasmodium falciparum gametocytes infect mosquitoes and are responsible for malaria transmission. New interventions that block transmission could accelerate malaria elimination. Gametocytes develop within erythrocytes and activate protein export pathways that remodel the host cell. Plasmepsin V (PMV) is an aspartyl protease that is required for protein export in asexual parasites, but its function and essentiality in gametocytes has not been definitively proven, nor has PMV been assessed as a transmission-blocking drug target. Here, we show that PMV is expressed and can be inhibited specifically in P. falciparum stage I-II gametocytes. PMV inhibitors block processing and export of gametocyte effector proteins and inhibit development of stage II-V gametocytes. Gametocytogenesis in the presence of sublethal inhibitor concentrations results in stage V gametocytes that fail to infect mosquitoes. Therefore, PMV primes gametocyte effectors for export, which is essential for the development and fitness of gametocytes for transmission to mosquitoes.
Asunto(s)
Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Culicidae/crecimiento & desarrollo , Inhibidores Enzimáticos/farmacología , Gametogénesis/efectos de los fármacos , Malaria Falciparum/prevención & control , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Culicidae/efectos de los fármacos , Culicidae/parasitología , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Humanos , Estadios del Ciclo de Vida , Malaria Falciparum/enzimología , Malaria Falciparum/parasitología , Malaria Falciparum/transmisión , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Proteínas Protozoarias/metabolismoRESUMEN
BACKGROUND: Malaria is the most important vector-borne disease in the world. Epidemiological and ecological studies of malaria traditionally utilize detection of Plasmodium sporozoites in whole mosquitoes or salivary glands by microscopy or serological or molecular assays. However, these methods are labor-intensive, and can over- or underestimate mosquito transmission potential. To overcome these limitations, alternative sample types have been evaluated for the study of malaria. It was recently shown that Plasmodium could be detected in saliva expectorated on honey-soaked cards by Anopheles stephensi, providing a better estimate of transmission risk. We evaluated whether excretion of Plasmodium falciparum nucleic acid by An. stephensi correlates with expectoration of parasites in saliva, thus providing an additional sample type for estimating transmission potential. Mosquitoes were exposed to infectious blood meals containing cultured gametocytes, and excreta collected at different time points post-exposure. Saliva was collected on honey-soaked filter paper cards, and salivary glands were dissected and examined microscopically for sporozoites. Excreta and saliva samples were tested by real time polymerase chain reaction (RT-rtPCR). RESULTS: Plasmodium falciparum RNA was detected in mosquito excreta as early as four days after ingesting a bloodmeal containing gametocytes. Once sporogony (the development of sporozoites) occurred, P. falciparum RNA was detected concurrently in both excreta and saliva samples. In the majority of cases, no difference was observed between the Ct values obtained from matched excreta and saliva samples, suggesting that both samples provide equally sensitive results. A positive association was observed between the molecular detection of the parasites in both samples and the proportion of mosquitoes with sporozoites in their salivary glands from each container. No distinguishable parasites were observed when excreta samples were stained and microscopically analyzed. CONCLUSIONS: Mosquito saliva and excreta are easily collected and are promising for surveillance of malaria-causing parasites, especially in low transmission settings or in places where arboviruses co-circulate.
Asunto(s)
Anopheles/parasitología , Heces/parasitología , Malaria/transmisión , Mosquitos Vectores/parasitología , Plasmodium/aislamiento & purificación , Saliva/parasitología , Animales , ADN Protozoario/genética , Femenino , Malaria Falciparum/transmisión , Masculino , Plasmodium/genética , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , Plasmodium vivax/genética , Plasmodium vivax/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Esporozoítos/genética , Esporozoítos/aislamiento & purificaciónRESUMEN
Toxoplasma gondii infects approximately 30% of the world's population, causing disease primarily during pregnancy and in individuals with weakened immune systems. Toxoplasma secretes and exports effector proteins that modulate the host during infection, and several of these proteins are processed by the Golgi-associated aspartyl protease 5 (ASP5). Here, we identify ASP5 substrates by selectively enriching N-terminally derived peptides from wild-type and Δasp5 parasites. We reveal more than 2,000 unique Toxoplasma N-terminal peptides, mapping to both natural N termini and protease cleavage sites. Several of these peptides mapped directly downstream of the characterized ASP5 cleavage site, arginine-arginine-leucine (RRL). We validate candidates as true ASP5 substrates, revealing they are not processed in parasites lacking ASP5 or in wild-type parasites following mutation of the motif from RRL to ARL. All identified ASP5 substrates are dense granule proteins, and interestingly, none appear to be exported, thus differing from the analogous system in related Plasmodium spp. Instead we show that the majority of substrates reside within the parasitophorous vacuole (PV), and its membrane (the PVM), including two kinases and one phosphatase. We show that genetic deletion of WNG2 leads to attenuation in a mouse model, suggesting that this putative kinase is a new virulence factor in Toxoplasma Collectively, these data constitute the first in-depth analyses of ASP5 substrates and shed new light on the role of ASP5 as a maturase of dense granule proteins during the Toxoplasma lytic cycle.IMPORTANCEToxoplasma gondii is one of the most successful human parasites. Central to its success is the arsenal of virulence proteins introduced into the infected host cell. Several of these virulence proteins require direct maturation by the aspartyl protease ASP5, and all require ASP5 for translocation into the host cell, yet the true number of ASP5 substrates and complete repertoire of effectors is currently unknown. Here we selectively enrich N-terminally derived peptides using Terminal Amine Isotopic Labeling of Substrates (TAILS) and use quantitative proteomics to reveal novel ASP5 substrates. We identify, using two different enrichment techniques, new ASP5 substrates and their specific cleavage sites. ASP5 substrates include two kinases and one phosphatase that reside at the host-parasite interface, which are important for infection.
Asunto(s)
Proteasas de Ácido Aspártico/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/metabolismo , Toxoplasma/enzimología , Toxoplasma/metabolismo , Proteasas de Ácido Aspártico/genética , Células Cultivadas , Fibroblastos/parasitología , Eliminación de Gen , Humanos , Membranas Intracelulares/metabolismo , Proteínas Protozoarias/genética , Toxoplasma/genética , Vacuolas/metabolismo , Vacuolas/parasitologíaRESUMEN
Plasmodium falciparum exports hundreds of virulence proteins within infected erythrocytes, a process that requires cleavage of a pentameric motif called Plasmodium export element or vacuolar transport signal by the endoplasmic reticulum (ER)-resident protease plasmepsin V. We identified plasmepsin V-binding proteins that form a unique interactome required for the translocation of effector cargo into the parasite ER. These interactions are functionally distinct from the Sec61-signal peptidase complex required for the translocation of proteins destined for the classical secretory pathway. This interactome does not involve the signal peptidase (SPC21) and consists of PfSec61, PfSPC25, plasmepsin V and PfSec62, which is an essential component of the post-translational ER translocon. Together, they form a distinct portal for the recognition and translocation of a large subset of Plasmodium export element effector proteins into the ER, thereby remodelling the infected erythrocyte that is required for parasite survival and pathogenesis.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , Eritrocitos/metabolismo , Plasmodium falciparum/metabolismo , Canales de Translocación SEC/metabolismo , Ácido Aspártico Endopeptidasas/genética , Transporte Biológico/genética , Transporte Biológico/fisiología , Membrana Celular/fisiología , Malaria Falciparum/patología , Plasmodium falciparum/patogenicidad , Canales de Translocación SEC/genética , Factores de VirulenciaRESUMEN
Transmission of the malaria parasite Plasmodium falciparum involves infection of Anopheles mosquitoes. Here we characterize SOPT, a protein expressed in P. falciparum ookinetes that facilitates infection of the mosquito midgut. SOPT was identified on the basis that it contains a signal peptide, a PEXEL-like sequence and is expressed in asexual, ookinete and sporozoite stages, suggesting it is involved in infecting the human or mosquito host. SOPT is predicted to contain a subtilisin-like fold with a non-canonical catalytic triad and is orthologous to P. berghei PIMMS2. Localization studies reveal that SOPT is not exported to the erythrocyte but is expressed in ookinetes at the parasite periphery. SOPT-deficient parasites develop normally through the asexual and sexual stages and produce equivalent numbers of ookinetes to NF54 controls, however, they form fewer oocysts and sporozoites in mosquitoes. SOPT-deficient parasites were also unable to activate the immune-responsive midgut invasion marker SRPN6 after mosquito ingestion, suggesting they are defective for entry into the midgut. Disruption of SOPT in P. berghei (PIMMS2) did not affect other lifecycle stages or ookinete development but again resulted in fewer oocysts and sporozoites in mosquitoes. Collectively, this study shows that SOPT/PIMMS2 plays a conserved role in ookinetes of different Plasmodium species.