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G Margos

Publications and source records attributed to G Margos.

At least 19 recordsLinked to original sources
PubMed2004

Correlation of structural development and differential expression of invasion-related molecules in schizonts of Plasmodium falciparum.

During asexual development Plasmodium schizonts undergo a series of complex biochemical and structural changes. Using tightly synchronized cultures of 2 P. falciparum lines (clone C10 and strain ITO4) for light microscopy and fluorescence imaging we monitored the timing and sequence of expression of proteins associated with invasion-related organelles. Antibodies to rhoptry, micronemal and dense granule proteins (Rhoptry Associated Protein 1, Apical Membrane Antigen 1, Erythrocyte Binding Antigen 175, Ring-infected Erythrocyte Surface Antigen) and to pellicle-associated proteins (Merozoite Surface Protein 1, PfMyosin-A) were used. Clone C10 developed faster than ITO4; this difference was also found in the timing of protein expression seen by immunofluorescence. Light microscopic data were combined with transmission electron microscopic analysis using serial sectioning of ITO4 schizonts to determine nuclear number and organellar development. Thus a timetable of schizont structural maturation was established. Generally, the timing of organelle-specific antigen expression correlates well with the ultrastructural data. Rhoptries are formed mainly between second and fourth nuclear divisions, micronemes between the end of the fourth nuclear division and merozoite separation from the residual body, while dense granules are generated mainly after the micronemes. PfAMA-1 appears in micronemes before EBA-175, suggesting micronemal heterogeneity.

Animals↗
PubMed2004

Apical membrane antigen 1, a major malaria vaccine candidate, mediates the close attachment of invasive merozoites to host red blood cells.

Apical membrane antigen 1 (AMA-1) of Plasmodium merozoites is established as a candidate molecule for inclusion in a human malaria vaccine and is strongly conserved in the genus. We have investigated its function in merozoite invasion by incubating Plasmodium knowlesi merozoites with red cells in the presence of a previously described rat monoclonal antibody (MAb R31C2) raised against an invasion-inhibitory epitope of P. knowlesi AMA-1 and then fixing the material for ultrastructural analysis. We have found that the random, initial, long-range (12 nm) contact between merozoites and red cells occurs normally in the presence of the antibody, showing that AMA-1 plays no part in this stage of attachment. Instead, inhibited merozoites fail to reorientate, so they do not bring their apices to bear on the red cell surface and do not make close junctional apical contact. We conclude that AMA-1 may be directly responsible for reorientation or that the molecule may initiate the junctional contact, which is then presumably dependent on Duffy binding proteins for its completion.

Animals↗
PubMed2001

P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions.

The ookinete surface proteins (P25 and P28) are proven antimalarial transmission-blocking vaccine targets, yet their biological functions are unknown. By using single (Sko) and double gene knock-out (Dko) Plasmodium berghei parasites, we show that P25 and P28 share multiple functions during ookinete/oocyst development. In the midgut of mosquitoes, the formation of ookinetes lacking both proteins (Dko parasites) is significantly inhibited due to decreased protection against lethal factors, including protease attack. In addition, Dko ookinetes have a much reduced capacity to traverse the midgut epithelium and to transform into the oocyst stage. P25 and P28 are partially redundant in these functions, since the efficiency of ookinete/oocyst development is only mildly compromised in parasites lacking either P25 or P28 (Sko parasites) compared with that of Dko parasites. The fact that Sko parasites are efficiently transmitted by the mosquito is a compelling reason for including both target antigens in transmission-blocking vaccines.

Animals↗
PubMed2001

Interaction between host complement and mosquito-midgut-stage Plasmodium berghei.

After ingestion by mosquitoes, gametocytes of malaria parasites become activated and form extracellular gametes that are no longer protected by the red blood cell membrane against immune effectors of host blood. We have studied the action of complement on Plasmodium developmental stages in the mosquito blood meal using the rodent malaria parasite Plasmodium berghei and rat complement as a model. We have shown that in the mosquito midgut, rat complement components necessary to initiate the alternative pathway (factor B, factor D, and C3) as well as C5 are present for several hours following ingestion of P. berghei-infected rat blood. In culture, 30 to 50% of mosquito midgut stages of P. berghei survived complement exposure during the first 3 h of development. Subsequently, parasites became increasingly sensitive to complement lysis. To investigate the mechanisms involved in their protection, we tested for C3 deposition on parasite surfaces and whether host CD59 (a potent inhibitor of the complement membrane attack complex present on red blood cells) was taken up by gametes while emerging from the host cell. Between 0.5 and 22 h, 90% of Pbs21-positive parasites were positive for C3. While rat red and white blood cells stained positive for CD59, Pbs21-positive parasites were negative for CD59. In addition, exposure of parasites to rat complement in the presence of anti-rat CD59 antibodies did not increase lysis. These data suggest that parasite or host molecules other than CD59 are responsible for the protection of malaria parasites against complement-mediated lysis. Ongoing research aims to identify these molecules.

Animals↗
PubMed2000

Characterisation and expression of pbs25, a sexual and sporogonic stage specific protein of Plasmodium berghei.

Following gametogenesis and fertilisation in the bloodmeal within the mosquito midgut, the newly formed zygotes of the malaria parasite develop into motile invasive ookinetes. During this development, surface molecules are synthesised de novo including molecules of 21-28 kDa from the zygote-ookinete stages. An antiserum recognising a 26 kDa protein of Plasmodium berghei was used to clone the corresponding gene from a cDNA library, which was shown to be identical to the reported Pbs25 gene sequence. We show here that Pbs25 was detectable in preparations of gametes 30 min post-gametocyte activation, expression continued on zygotes, ookinetes and oocysts indicating there is a significant overlap of expression of the two immunogenic zygote-ookinete proteins belonging to the P25/28 protein family of sexual stage antigens. Biochemical analysis of Pbs25 demonstrates the presence of a malaria-specific glycosylphosphatidylinositol (GPI) anchor. Antibodies recognising Pbs25 impaired parasite development in the mosquito.

Animals↗
PubMed2000

Identification of differentially regulated genes of Plasmodium by suppression subtractive hybridization.

Plasmodium, the causative agent of malaria, has many morphologically and functionally distinct developmental stages. In the mosquito host alone, there are five transitions during the development of a gametocyte into a sporozoite. Determining which genes are expressed at the different developmental stages is vital to our understanding of the parasite. There are a growing number of techniques designed to study gene expression, including microarray. Here, Johannes Dessens, Gabrielle Margos, Maria del Carmen Rodriguez and Robert Sinden describe a novel method: suppression subtractive hybridization (SSH) and its successful application in obtaining mosquito midgut stage-specific genes of Plasmodium.

Animals↗
PubMed2000

The roles of the glycosylphosphatidylinositol anchor on the production and immunogenicity of recombinant ookinete surface antigen Pbs21 of Plasmodium berghei when prepared in a baculovirus expression system.

Malarial ookinetes express an immunodominant surface protein (P28) that is a priority candidate for the development of transmission-blocking vaccines. The full length P28 gene from Plasmodium berghei [Pbs21(1-213)] and a deletion construct [Pbs21(1-188)] encoding a protein that lacks the 25 C-terminal amino acids, including the glycosylphosphatidylinositol (GPI) anchor signal, were expressed in insect cells using baculovirus vectors. Pbs21(1-213) protein is strongly hydrophobic, found in the cytoplasm and on the surface of Spodoptera Sf21 cells, and in the culture medium. Pbs21(1-188) protein was largely found in the aqueous phase of the medium and in the cytoplasm of Sf21 cells, but was not detected on the cell surface. The presence of 25 C-terminal amino acids is therefore critical to the attachment of recombinant Pbs21 to the parasite plasma membrane. Mice were immunized subcutaneously or intramuscularly with affinity purified recombinant Pbs21(1-213), Pbs21(1-188) or native Pbs21 proteins. Following two immunizations, native Pbs21 induces higher titres when administered by either route, than the recombinant protein bearing an insect GPI anchor, which in turn is markedly more immunogenic than the recombinant polypeptide lacking a GPI anchor. When specific anti Pbs21 antibody titres exceeded 1 mg/ml all three antigens were capable of inducing transmission blockade > or = 90%, below 1 mg/ml blockade did not correlate with antibody concentration.

Animals↗
PubMed2000

Distinct roles for pbs21 and pbs25 in the in vitro ookinete to oocyst transformation of Plasmodium berghei.

We have developed an in vitro culture system for early sporogonic stages of Plasmodium berghei, which can be used to study developmental events normally taking place in the midgut of an infected mosquito. These include penetration of insect cells by the mature ookinete, transformation into oocysts and the early development of the latter, sustained through several rounds of nuclear division. The system, based upon co-culture of enriched ookinetes with several established insect cell lines, was used to study the development of mutant ookinetes lacking both the Pbs21 and Pbs25 surface proteins. Motility and entry of double knockout and Pbs21 single knockout ookinetes into the insect cells are normal, but the number of ookinetes successfully transforming into oocysts expressing the CSP protein are substantially reduced. Finally, using the yeast two-hybrid system we also show that Pbs25 has the capacity to homodimerise as well as to form heterodimers with Pbs21.

Aedes↗
PubMed1999

The biosynthesis and post-translational modification of Pbs21 an ookinete-surface protein of Plasmodium berghei.

Radiolabelled methionine incorporation into synchronised Plasmodium berghei gametocytes or ookinete cultures, showed that Pbs21 is not synthesised in bloodstage parasites; synthesis was detected within three hours of induction of gametogenesis; synthesis was triggered at gametogenesis, not by fertilisation. We show native Pbs21 to be a hydrophobic membrane protein that was insensitive to cleavage by phosphatidylinositol phospholipase C (PI-PLC), but sensitive to alkaline hydroxylamine, and partially sensitive to glycosylphosphatidylinositol-dependent phospholipase D (GPI-PLD) and HNO2. 3H-myristic and palmitic acid, 3H-glucosamine and mannose incorporation indicated Pbs21 was acylated and glycosylated. Linkage of the acyl group was sensitive to HNO2, which released an acyl-phosphatidylinositol more hydrophobic than that released from P3 of Trypanosoma brucei. All these properties are consistent with the presence of a malaria-specific glycosylphosphatidylinositol (GPI) anchor. In contrast recombinant Pbs21 (rPbs21), expressed in Spodoptera frugiperda cells, was sensitive to both PI-PLC and GPI-PLD, consistent with the protein being modified by a different (S. frugiperda) GPI anchor. Brefeldin A blocked secretion of rPbs21 within a cytoplasmic reticular compartment. Following deletion of the putative GPI anchor addition site (amino acids 189 213), the protein was transported to the cell surface and secreted directly into the aqueous phase of the culture medium. Deletion of amino acids 205-213 disrupted Pbs21 processing, transport through the ER and distribution onto the cell surface. Deletion of amino acids 1-28 prevented transport of Pbs21 into the ER. This suggests that correct processing of the GPI anchor in the ER-Golgi network is essential for the successful secretion of the recombinant protein, which is additionally dependent upon an N-terminal secretory signal sequence.

Acylation↗
PubMed1998

Transgenic expression of a mosquito-stage malarial protein, Pbs21, in blood stages of transformed Plasmodium berghei and induction of an immune response upon infection.

Pbs21 is a surface protein of the ookinete of Plasmodium berghei, which can induce a potent transmission-blocking immune response. Pbs21 is normally expressed only by parasite stages in the mosquito, i.e., female gametes/zygotes, ookinetes, and oocysts. However, the Pbs21 gene is transcribed in female gametocytes which circulate in the bloodstream of the host, where translation of the resulting mRNA is totally repressed. Episomal transfection has been used to investigate whether expression of Pbs21 protein could be achieved in blood stages of the parasite. By using plasmid pMD221, the complete mRNA-encoding region of Pbs21, flanked only by 218 nucleotides (nt) of its promoter region and 438 nt of its 3' region downstream from the polyadenylation site, was introduced into the blood stages of gametocyte-producing and non-gametocyte-producing clones of P. berghei. In both of these transformed parasite lines, Pbs21 protein was expressed in asexual trophozoites, schizonts, and, when present, in both male and female gametocytes. Hence, the flanking regions present are sufficient to allow transcription but lack the elements that exert natural control of sex- and stage-specific transcription. The mRNA and the protein expressed by transformed blood stages were indistinguishable from the wild-type forms by the criteria tested, and the protein was recognized by both conformation-dependent and conformation-independent monoclonal antibodies raised against native Pbs21. In mice infected with transformed non-gametocyte-producing parasites, a Pbs21-specific immune response was induced and characterized with respect to isotype (IgG2a/IgG2b) and quantity (11. 5 +/- 10 microg/ml) of antibody produced. However, as found in previous studies, these antibody levels were insufficient to inhibit development of the parasites in the mosquito. The ability to express mosquito midgut-stage antigens in blood-stage parasites will facilitate further investigations of molecular and immunological properties of these proteins.

Animals↗
PubMed1997

The roles of temperature, pH and mosquito factors as triggers of male and female gametogenesis of Plasmodium berghei in vitro.

Developmentally arrested malarial gametocytes undergo gamete formation in the mosquito midgut immediately after ingestion of the infected bloodmeal. In the rodent malaria parasite Plasmodium berghei male gametogenesis (exflagellation) can be induced in vitro by a temperature decrease (from 39 degrees C in the vertebrate host to 20 degrees C) and a concomitant pH increase (from 7.3 in mouse blood to 8.0). We report the presence of additional Gametocyte Activating Factor(s) (GAF) present in Anopheles stephensi tissue extracts, which induce both male and female gametogenesis at the otherwise nonpermissive pH of 7.3 in vitro but are unable to overcome the low temperature requirement. All constituent cellular events of microgametogeneis studied here are induced by the same triggers in vitro. A temperature decrease is also required for exflagellation in the mosquito midgut. The possible role of GAF as a second obligatory natural trigger of gametogenesis is discussed.

Animals↗
PubMed1995

Expression of the Plasmodium berghei ookinete protein Pbs21 in a baculovirus-insect cell system produces an efficient transmission blocking immunogen.

A surface protein of Plasmodium berghei ookinetes, Pbs21, was expressed in a baculovirus-insect cell system in cell culture and in Heliothis virescens larvae. Groups of BALB/c mice received two intraperitoneal inoculations of either i) Tris-buffer or homogenized H. virescens larvae infected with wild-type baculovirus; ii) enriched, homogenized ookinetes, or iii) homogenized H. virescens larvae expressing recombinant Pbs21 (rPbs21). All animals immunized with ookinetes or with rPbs21 had high titres of antibodies (IgG isotype) that bound to native Pbs21. The large majority of antibodies in immune sera of both groups recognized the antigen under non-reducing but not under reducing conditions. The predominant IgG-subclasses in mice immunized with ookinetes was IgG1 and in mice immunized with rPbs21, the subclasses were IgG1 and IgG2a. Immunization with rPbs21 reduced the infectivity of P.berghei to mosquitoes by 91% compared to a 99% reduction following immunization with ookinetes. This preliminary data indicate that rPbs21 expressed in this eukaryotic system induces a transmission-blocking immunity, which is more effective than that achieved using rPbs21 expressed in Escherichia coli (Matsuoka et al. 1994).

Animals↗
PubMed1994

Serum-free cultivation of several Plasmodium falciparum strains.

Plasmodium falciparum strains from different malaria-endemic regions were grown in continuous culture without human serum. The medium was complemented with Nutridoma-SR instead of serum. All parasite strains developed well in serum-free medium. Two strains were thawed without serum and showed good multiplication in subsequent continuous culture.

Animals↗
PubMed1994

Differential immune responses to Borrelia burgdorferi in European wild rodent species influence spirochete transmission to Ixodes ricinus L. (Acari: Ixodidae).

Immune responses to Borrelia burgdorferi and their influence on spirochete transmission to Ixodes ricinus were analyzed in the natural European reservoir hosts; i.e., the mouse species Apodemus flavicollis (yellow-necked mouse) and Apodemus sylvaticus (wood mouse) and the vole species Clethrionomys glareolus (bank vole), and, in addition, in the laboratory mouse strain NMRI. Naive and preimmunized rodents were infected either by artificially infected I. ricinus larvae or by intradermal injection of spirochetes. Independent of the species, all animals developed antibodies to various spirochetal antigens. However, antibodies to the outer surface proteins A (OspA) and B (OspB) were not found in recipients infected via ticks. Rodents of the genus Apodemus and of the NMRI strain showed higher levels of B. burgdorferi-specific antibodies than those of the species C. glareolus. The rate of spirochete transmission to noninfected ticks correlated with both the quality and quantity of spirochete-specific antibodies generated in the various species: high levels of spirochete-specific immunoglobulins correlated with low transmission rates. Furthermore, lower transmission rates were observed with rodents expressing antibodies to OspA and OspB (i.e., intradermally infected or immunized) than with those lacking these specificities (i.e., infected via ticks). The study provides evidence that transmission of B. burgdorferi from natural hosts to ticks is controlled by the specificity and quantity of spirochete-reactive antibodies and suggests that immunity to B. burgdorferi in natural reservoir hosts is an important regulatory factor in the horizontal transmission of B. burgdorferi in nature.

Animals↗
PubMed1993

Serum-free cultivation of Plasmodium falciparum gametocytes in vitro.

Several components were tested for their ability to replace human serum in cultures of Plasmodium falciparum set up for the development of gametocytes. Besides a serum-free medium, A*I*M*V (Gibco BRL), RPMI medium supplemented with commercially available serum substitutes was used to culture gametocytes. The following substances served as serum replacements: Basal Medium Supplement (Biochrom), Ultroser G (Gibco BRL) and Nutridoma-SR (Boehringer Mannheim). All serum-free additives supported some parasite growth, but only in RPMI supplemented with Nutridoma-SR were morphologically mature gametocytes obtained. The asexual forms developed almost as well as in RPMI with human serum added.

Animals↗
PubMed1993

The effects of hormones on the gametocytogenesis of Plasmodium falciparum in vitro.

To examine possible effects of hormones on the gametocytogenesis of Plasmodium falciparum in vitro, insulin, progesterone, 17-beta-estradiol and testosterone were added in different concentrations to the culture medium. The medium containing RPMI was free of human serum but complemented with BMS (Basal-Medium-Supplement, Biochrom), a semidefined serum replacement. As controls, cultures were grown either with BMS supplemented RPMI without hormones, or with human serum complemented RPMI. Only addition of insulin increased the parasitemia. All tested steroid hormones added to the medium enhanced the number of gametocytes.

Animals↗