RESUMEN
Plasmodium, the malaria parasite, undergoes a complex life cycle alternating between a vertebrate host and a mosquito vector of the genus Anopheles In red blood cells of the vertebrate host, Plasmodium multiplies asexually or differentiates into gamete precursors, the male and female gametocytes, responsible for parasite transmission. Sexual stage maturation occurs in the midgut of the mosquito vector, where male and female gametes egress from the host erythrocytes to fuse and form a zygote. Gamete egress entails the successive rupture of two membranes surrounding the parasite, the parasitophorous vacuole membrane and the erythrocyte plasma membrane. In this study, we used the rodent model parasite Plasmodium berghei to design a label-free quantitative proteomic approach aimed at identifying gender-related proteins differentially released/secreted by purified mature gametocytes when activated to form gametes. We compared the abundance of molecules secreted by wild type gametocytes of both genders with that of a transgenic line defective in male gamete maturation and egress. This enabled us to provide a comprehensive data set of egress-related molecules and their gender specificity. Using specific antibodies, we validated eleven candidate molecules, predicted as either gender-specific or common to both male and female gametocytes. All of them localize to punctuate, vesicle-like structures that relocate to cell periphery upon activation, but only three of them localize to the gametocyte-specific secretory vesicles named osmiophilic bodies. Our results confirm that the egress process involves a tightly coordinated secretory apparatus that includes different types of vesicles and may put the basis for functional studies aimed at designing novel transmission-blocking molecules.
Asunto(s)
Estadios del Ciclo de Vida/fisiología , Plasmodium berghei/crecimiento & desarrollo , Plasmodium berghei/metabolismo , Proteoma/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Eritrocitos/metabolismo , Eritrocitos/parasitología , Femenino , Gametogénesis , Células Germinativas/metabolismo , Masculino , Ratones , Proteómica , Fracciones Subcelulares/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
Male and female Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal egress from the red blood cells by rupturing the two surrounding membranes, the parasitophorous vacuole and the red blood cell membranes. Proteins of the so-called osmiophilic bodies, (OBs), secretory organelles resident in the cytoplasm, are important players in this process. Once gametes emerge, the female is ready to be fertilized while the male develops into motile flagellar gametes. Here, we describe the function(s) of PBANKA_1115200, which we named Gamete Egress Protein (GEP), a protein specific to malaria parasites. GEP is restricted to gametocytes, expressed in gametocytes of both genders and partly localizes to the OBs. A mutant lacking the protein shows aberrant rupture of the two surrounding membranes, while OBs discharge is delayed but not aborted. Moreover, we identified a second function of GEP during exflagellation since the axonemes of the male flagellar gametes were not motile. Genetic crossing experiments reveal that both genders are unable to establish infections in mosquitoes and thus the lack of GEP leads to a complete block in Plasmodium transmission from mice to mosquitoes. The combination of our results reveals essential and pleiotropic functions of GEP in Plasmodium gametogenesis.
Asunto(s)
Gametogénesis/genética , Células Germinativas/crecimiento & desarrollo , Malaria/transmisión , Plasmodium berghei/crecimiento & desarrollo , Proteínas Protozoarias/genética , Animales , Anopheles/parasitología , Eritrocitos/parasitología , Femenino , Técnicas de Inactivación de Genes , Malaria/parasitología , Masculino , Ratones , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
BACKGROUND: Aedes mosquitoes are vectors of arboviral diseases of great relevance for public health. The recent outbreaks of dengue, Zika, chikungunya and the rapid worldwide spreading of Aedes albopictus emphasize the need for improvement of vector surveillance and control. Host antibody response to mosquito salivary antigens is emerging as a relevant additional tool to directly assess vector-host contact, monitor efficacy of control interventions and evaluate risk of arboviral transmission. METHODOLOGY/PRINCIPAL FINDINGS: Groups of four BALB/c mice were immunized by exposure to bites of either Aedes albopictus or Aedes aegypti. The 34k2 salivary proteins from Ae. albopictus (al34k2) and Ae. aegypti (ae34k2) were expressed in recombinant form and Ae. albopictus salivary peptides were designed through B-cell epitopes prediction software. IgG responses to salivary gland extracts, peptides, al34k2 and ae34k2 were measured in exposed mice. Both al34k2 and ae34k2, with some individual and antigen-specific variation, elicited a clearly detectable antibody response in immunized mice. Remarkably, the two orthologous proteins showed very low level of immune cross-reactivity, suggesting they may eventually be developed as species-specific markers of host exposure. The al34k2 immunogenicity and the limited immune cross-reactivity to ae34k2 were confirmed in a single human donor hyperimmune to Ae. albopictus saliva. CONCLUSIONS/SIGNIFICANCE: Our study shows that exposure to bites of Ae. albopictus or Ae. aegypti evokes in mice species-specific IgG responses to al34k2 or ae34k2, respectively. Deeper understanding of duration of antibody response and validation in natural conditions of human exposure to Aedes mosquitoes are certainly needed. However, our findings point to the al34k2 salivary protein as a promising potential candidate for the development of immunoassays to evaluate human exposure to Ae. albopictus. This would be a step forward in the establishment of a serological toolbox for the simultaneous assessment of human exposure to Aedes vectors and the pathogens they transmit.
Asunto(s)
Aedes/inmunología , Inmunoglobulina G/inmunología , Saliva/inmunología , Proteínas y Péptidos Salivales/inmunología , Proteínas y Péptidos Salivales/metabolismo , Aedes/fisiología , Aedes/virología , Animales , Formación de Anticuerpos , Arbovirus/inmunología , Biomarcadores , Reacciones Cruzadas , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunización , Insectos Vectores , Ratones , Ratones Endogámicos BALB C , Saliva/metabolismo , Glándulas Salivales/metabolismo , Proteínas y Péptidos Salivales/genética , Especificidad de la EspecieRESUMEN
The malaria parasite Plasmodium falciparum is exposed, during its development, to major changes of ionic composition in its surrounding medium. We demonstrate that the P. falciparum serpentine-like receptor PfSR25 is a monovalent cation sensor capable of modulating Ca2+ signaling in the parasites. Changing from high (140 mM) to low (5.4 mM) KCl concentration triggers [Ca2+]cyt increase in isolated parasites and this Ca2+ rise is blocked either by phospholipase C (PLC) inhibition or by depleting the parasite's internal Ca2+ pools. This response persists even in the absence of free extracellular Ca2+ and cannot be elicited by addition of Na+, Mg2+ or Ca2+. However, when the PfSR25 gene was deleted, no effect on [Ca2+]cyt was observed in response to changing KCl concentration in the knocked out (PfSR25 -) parasite. Finally, we also demonstrate that: i) PfSR25 plays a role in parasite volume regulation, as hyperosmotic stress induces a significant decrease in parasite volume in wild type (wt), but not in PfSR25 - parasites; ii) parasites lacking PfSR25 show decreased parasitemia and metacaspase gene expression on exposure to the nitric oxide donor sodium nitroprusside (SNP) and iii), compared to PfSR25 - parasites, wt parasites showed a better survival in albumax-deprived condition.
Asunto(s)
Señalización del Calcio , Malaria Falciparum/parasitología , Plasmodium falciparum/fisiología , Potasio/metabolismo , Proteínas Protozoarias/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Estrés Fisiológico , Eritrocitos/parasitología , Regulación de la Expresión Génica , Carga de Parásitos , Proteínas Protozoarias/genética , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Membrane microdomains that include lipid rafts, are involved in key physiological and pathological processes and participate in the entry of endocellular pathogens. These assemblies, enriched in cholesterol and sphingolipids, form highly dynamic, liquid-ordered phases that can be separated from the bulk membranes thanks to their resistance to solubilization by nonionic detergents. To characterize complexity and dynamics of detergent-resistant membranes of sexual stages of the rodent malaria parasite Plasmodium berghei, here we propose an integrated study of raft components based on proteomics, lipid analysis and bioinformatics. This analysis revealed unexpected heterogeneity and unexplored pathways associated with these specialized assemblies. Protein-protein relationships and protein-lipid co-occurrence were described through multi-component networks. The proposed approach can be widely applied to virtually every cell type in different contexts and perturbations, under physiological and/or pathological conditions.
Asunto(s)
Estadios del Ciclo de Vida/fisiología , Malaria/parasitología , Microdominios de Membrana/metabolismo , Plasmodium berghei/crecimiento & desarrollo , Plasmodium berghei/metabolismo , Animales , Colesterol/química , Colesterol/metabolismo , Simulación por Computador , Detergentes/química , Modelos Animales de Enfermedad , Gametogénesis/fisiología , Humanos , Lípidos/análisis , Microdominios de Membrana/química , Ratones , Ratones Endogámicos BALB C , Proteómica , Esfingolípidos/química , Esfingolípidos/metabolismoRESUMEN
The sporozoite, the stage of the malaria parasite transmitted by the mosquito, first develops for â¼2 weeks in an oocyst. Rupture of the oocyst capsule is required for release of sporozoites, which then transfer to the salivary gland where they are injected into a new host. Here we identify two parasite proteins that we call oocyst rupture proteins 1 (ORP1) and ORP2. These proteins have a histone-fold domain (HFD) that promotes heterodimer formation in the oocyst capsule at the time of rupture. Oocyst rupture is prevented in mutants lacking either protein. Mutational analysis confirms the HFD as essential for ORP1 and ORP2 function, and heterodimer formation was verified in vitro. These two proteins are potential targets for blocking transmission of the parasite in the mosquito.
Asunto(s)
Plasmodium berghei/fisiología , Proteínas Protozoarias/metabolismo , Esporozoítos/fisiología , Secuencia de Aminoácidos , Animales , Femenino , Malaria/parasitología , Masculino , Ratones , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Pliegue de Proteína , Proteínas Protozoarias/genéticaRESUMEN
The early transcribed membrane proteins ETRAMPs belong to a family of small, transmembrane molecules unique to Plasmodium parasite, which share a signal peptide followed by a short lysine-rich stretch, a transmembrane domain and a variable, highly charged C-terminal region. ETRAMPs are usually expressed in a stage-specific manner. In the blood stages they localize to the parasitophorous vacuole membrane and, in described cases, to vesicle-like structures exported to the host erythrocyte cytosol. Two family members of the rodent parasite Plasmodium berghei, uis3 and uis4, localize to secretory organelles of sporozoites and to the parasitophorous membrane vacuole of the liver stages. By the use of specific antibodies and the generation of transgenic lines, we showed that the P. berghei ETRAMP family member SEP2 is abundantly expressed in gametocytes as well as in mosquito and liver stages. In intracellular parasite stages, SEP2 is routed to the parasitophorous vacuole membrane while, in invasive ookinete and sporozoite stages, it localizes to the parasite surface. To date SEP2 is the only ETRAMP protein detected throughout the parasite life cycle. Furthermore, SEP2 is also released during gliding motility of salivary gland sporozoites. A limited number of proteins are known to be involved in this key function and the best characterized, the CSP and TRAP, are both promising transmission-blocking candidates. Our results suggest that ETRAMP members may be viewed as new potential candidates for malaria control.
Asunto(s)
Proteínas de la Membrana/metabolismo , Plasmodium berghei/metabolismo , Proteínas Protozoarias/metabolismo , Esporozoítos/metabolismo , Regiones no Traducidas 3' , Animales , Anopheles/parasitología , Línea Celular Tumoral , Expresión Génica , Regulación de la Expresión Génica , Humanos , Hígado/parasitología , Proteínas de la Membrana/genética , Ratones , Plasmodium berghei/citología , Transporte de Proteínas , Proteínas Protozoarias/genética , Secuencias Reguladoras de Ácidos Nucleicos , Esporozoítos/citologíaRESUMEN
The malaria parasite Plasmodium largely modifies the infected erythrocyte through the export of proteins to multiple sites within the host cell. This remodeling is crucial for pathology and translocation of virulence factors to the erythrocyte surface. In this study, we investigated localization and export of small exported proteins/early transcribed membrane proteins (SEP/ETRAMPs), conserved within Plasmodium genus. This protein family is characterized by a predicted signal peptide, a short lysine-rich stretch, an internal transmembrane domain and a highly charged C-terminal region of variable length. We show here that members of the rodent Plasmodium berghei family are components of the parasitophorous vacuole membrane (PVM), which surrounds the parasite throughout the erythrocytic cycle. During P. berghei development, vesicle-like structures containing these proteins detach from the PVM en route to the host cytosol. These SEP-containing vesicles remain associated with the infected erythrocyte ghosts most probably anchored to the membrane skeleton. Transgenic lines expressing the green fluorescent protein appended to different portions of sep-coding region allowed us to define motifs required for protein export. The highly charged terminal region appears to be involved in protein-protein interactions.
Asunto(s)
Eritrocitos/fisiología , Malaria/patología , Plasmodium berghei , Proteínas Protozoarias/metabolismo , Animales , Deformación Eritrocítica/genética , Membrana Eritrocítica/genética , Membrana Eritrocítica/metabolismo , Immunoblotting , Ratones , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Transporte de Proteínas , Proteínas Protozoarias/genéticaRESUMEN
Plasmodium parasites, the causal agents of malaria, dramatically modify the infected erythrocyte by exporting parasite proteins into one or multiple erythrocyte compartments, the cytoplasm and the plasma membrane or beyond. Despite advances in defining signals and specific cellular compartments implicated in protein trafficking in Plasmodium-infected erythrocytes, the contribution of lipid-mediated sorting to this cellular process has been poorly investigated. In this study, we examined the proteome of cholesterol-rich membrane microdomains or lipid rafts, purified from erythrocytes infected by the rodent parasite Plasmodium berghei. Besides structural proteins associated with invasive forms, we detected chaperones, proteins implicated in vesicular trafficking, membrane fusion events and signalling. Interestingly, the raft proteome of mixed P. berghei blood stages included proteins encoded by members of a large family (bir) of putative variant antigens potentially implicated in host immune system interactions and targeted to the surface of the host erythrocytes. The generation of transgenic parasites expressing BIR/GFP fusions confirmed the dynamic association of members of this protein family with membrane microdomains. Our results indicated that lipid rafts in Plasmodium-infected erythrocytes might constitute a route to sort and fold parasite proteins directed to various host cell compartments including the cell surface.
Asunto(s)
Antígenos/inmunología , Microdominios de Membrana/química , Plasmodium/fisiología , Proteínas Protozoarias/metabolismo , Transducción de Señal , Vesículas Transportadoras/metabolismo , Animales , Antígenos/genética , Eritrocitos/metabolismo , Eritrocitos/parasitología , Interacciones Huésped-Parásitos , Humanos , Estadios del Ciclo de Vida , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Plasmodium/química , Plasmodium/genética , Plasmodium/metabolismo , Plasmodium berghei/genética , Plasmodium berghei/crecimiento & desarrollo , Plasmodium berghei/parasitología , Transporte de Proteínas , Proteómica/métodos , Proteínas Protozoarias/análisis , Tripsina/farmacologíaRESUMEN
Transmission of the malaria parasite depends on specialized gamete precursors (gametocytes) that develop in the bloodstream of a vertebrate host. Gametocyte/gamete differentiation requires controlled patterns of gene expression and regulation not only of stage and gender-specific genes but also of genes associated with DNA replication and mitosis. Once taken up by mosquito, male gametocytes undergo three mitotic cycles within few minutes to produce eight motile gametes. Here we analysed, in two Plasmodium species, the expression of SET, a conserved nuclear protein involved in chromatin dynamics. SET is expressed in both asexual and sexual blood stages but strongly accumulates in male gametocytes. We demonstrated functionally the presence of two distinct promoters upstream of the set open reading frame, the one active in all blood stage parasites while the other active only in gametocytes and in a fraction of schizonts possibly committed to sexual differentiation. In ookinetes both promoters exhibit a basal activity, while in the oocysts the gametocyte-specific promoter is silent and the reporter gene is only transcribed from the constitutive promoter. This transcriptional control, described for the first time in Plasmodium, provides a mechanism by which single-copy genes can be differently modulated during parasite development. In male gametocytes an overexpression of SET might contribute to a prompt entry and execution of S/M phases within mosquito vector.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/metabolismo , Plasmodium berghei/crecimiento & desarrollo , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Factores de Transcripción/genética , Animales , Animales Modificados Genéticamente , Núcleo Celular/química , Núcleo Celular/metabolismo , Proteínas Cromosómicas no Histona/análisis , Eritrocitos/parasitología , Dosificación de Gen , Células Germinativas/química , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Masculino , Plasmodium berghei/citología , Plasmodium berghei/genética , Plasmodium falciparum/citología , Plasmodium falciparum/genética , Regiones Promotoras Genéticas , Proteínas Protozoarias/análisis , Reproducción Asexuada , Diferenciación Sexual/genética , Factores de Transcripción/análisis , Transcripción GenéticaRESUMEN
A genome-wide expression analysis was undertaken to identify novel genes specifically activated from early stages of gametocytogenesis in Plasmodium falciparum. A comparative analysis was conducted on sexually induced cultures of reference parasite clone 3D7 and its gametocyteless derivative clone F12. Competitive hybridisations on long-oligomer microarrays representing 4488 P. falciparum genes identified a remarkably small number of transcripts differentially produced in the two clones. Upregulation of the mRNAs for the early gametocyte markers Pfs16 and Pfg27 was however readily detected in 3D7, and such genes were used as reference transcripts in a comparative time course analysis of 3D7 and F12 parasites between 30 and 40 h post-invasion in cultures induced to enter gametocytogenesis. One hundred and seventeen genes had expression profiles which correlated to those of pfs16 and pfg27, and Northern blot analysis and published proteomic data identified those whose expression was gametocyte-specific. Immunofluorescence analysis with antibodies against two of these gene products identified two novel parasite membrane associated, sexual stage-specific proteins. One was produced from stage I gametocytes and the second showed peak production in stage II gametocytes. The two proteins were named Pfpeg-3 and Pfpeg-4, for P. falciparum proteins of early gametocytes.
Asunto(s)
Genoma de Protozoos , Células Germinativas/fisiología , Plasmodium falciparum/genética , Transcripción Genética , Animales , Perfilación de la Expresión Génica , Plasmodium falciparum/crecimiento & desarrollo , ARN Mensajero/genética , ARN Protozoario/genéticaRESUMEN
A gene-family, named sep, encoding small exported proteins conserved across Plasmodium species has been identified. SEP proteins (13-16 kDa) contain a predicted signal peptide at the NH(2)-terminus, an internal hydrophobic region and a polymorphic, low-complexity region at the carboxy-terminus. One member of the Plasmodium berghei family, Pbsep1, encodes an integral membrane protein expressed along the entire erythrocytic cycle. Immunolocalisation results indicated that PbSEP1 is targeted to the membrane of the parasitophorous vacuole up to the early phases of schizogony, while, in late schizonts, it re-locates in structures within the syncitium. After erythrocyte rupture, PbSEP1 is still detectable in free merozoites thus suggesting its involvement in the early steps of parasite invasion. Seven members of the sep-family in Plasmodium falciparum have been identified. Two of them correspond to previously reported gene sequences included in a family of early transcribed membrane proteins (etramp). Structural, functional and phylogenetic features of the sep family, shown in the present work, supercede this previous classification. PfSEP proteins are exported beyond the parasite membrane and translocated, early after invasion, to the host cell compartment in association with vesicle-like structures. Colocalisation results indicated that PfSEP-specific fluorescence overlaps, at the stage of trophozoite, with that of Pf332, a protein associated with Maurer's clefts, membranous structures in the cytosol of parasitised red blood cells, most probably involved in trafficking of parasite proteins. The specific signals necessary to direct SEP proteins to the vacuolar membrane in P. berghei or to the host cell compartment in P. falciparum remain to be determined.