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A Scherf

Publications and source records attributed to A Scherf.

At least 37 records · Page 2Linked to original sources
PubMed1995

Karyotype analysis of virulent Plasmodium falciparum strains propagated in Saimiri sciureus: strain adaptation leads to deletion of the RESA gene.

The squirrel monkey, Saimiri sciureus, is an important experimental model for the study of the human malaria parasite Plasmodium falciparum. A detailed karyotype analysis of four different P. falciparum strains propagated in S. sciureus was done using various subtelomeric antigen gene probes. We observed deletion of the complete RESA gene from chromosome 1 in all four strains. Interestingly, a loss of RESA was observed immediately upon adaptation to the squirrel monkey, suggesting that this DNA rearrangement is fundamental for the P. falciparum infection of S. sciureus erythrocytes. However, a RESA-specific gene probe hybridized with chromosome 1 of wild isolates from 28 different patients, indicating that this gene is maintained during infection of humans.

Animals↗
PubMed1994

Interchromosomal exchange of a large subtelomeric segment in a Plasmodium falciparum cross.

Duplications and interchromosomal transpositions of chromosome segments are implicated in the genetic variability of Plasmodium falciparum malaria parasites. One parasite clone, HB3, was shown to lack a subtelomeric region of chromosome 13 that normally carries a PfHRPIII gene. We show here that the chromosome 13 segment carrying PfHRPIII was replaced in HB3 by a duplicated terminal segment from chromosome 11. Mapping results indicate that the segment includes at least 100-200 kb of subtelomeric DNA and contains duplicated copies of the Pf332 and RESA-2 genes. We followed inheritance of this duplication in a genetic cross between the HB3 and another P.falciparum clone, Dd2, that is euploid for the Pf332, RESA-2 and PfHRPIII genes. Three types of progeny from the cross showed expected inheritance forms: a Dd2 euploid parent type, an HB3 aneuploid parent type, and a recombinant euploid type that carried PfHRPIII from Dd2 chromosome 13 and Pf332 from HB3 chromosome 11. However, a fourth euploid progeny type was also observed, in which the chromosome 13 segment from HB3 was transposed back to replace the terminus of chromosome 11. Three of 14 individual progeny were of this type. These findings suggest a mechanism of recombination from subtelomeric pairing and exchange between non-homologous chromosomes in meiosis.

Alleles↗
PubMed1994

Plasmodium falciparum: the Pf332 antigen is secreted from the parasite by a brefeldin A-dependent pathway and is translocated to the erythrocyte membrane via the Maurer's clefts.

The transport of the megadalton protein Pf332 was studied during the asexual bloodstage development of Plasmodium falciparum. Four mouse monoclonal antibodies, produced against a recombinant polypeptide derived from the Pf332 protein, were used to analyze the kinetics of synthesis, the subcellular location, and transport of this giant molecule to the erythrocyte membrane. After parasite invasion of a red blood cell, the Pf332 antigen is first detected in young trophozoites at the parasitophorous vacuole membrane or in the cytoplasm of the erythrocyte as large vesicle-like structures. The number of vesicles increases during maturation of the parasite and thus forms a rim-like immunofluorescence pattern between the erythrocyte membrane and the parasitophorous vacuole at very late stages. The various anti-Pf332 antibodies react with the surface of erythrocytes infected with very mature parasites (segmenter stage 42-46 hr postinvasion). Immunoelectron microscopic analysis shows that the Pf332 antigen is transported in association with Maurer's clefts in the cytoplasm of the erythrocyte. This transport could be completely blocked by Brefeldin A, resulting in the accumulation of the antigen within the parasite. These data strongly suggest that the Pf332 antigen is exported to the erythrocyte cytoplasm via the classical Golgi secretory pathway.

Animals↗
PubMed1994

Subtelomeric chromosome instability in Plasmodium falciparum: short telomere-like sequence motifs found frequently at healed chromosome breakpoints.

The stability of chromosome ends of the human malaria parasite P. falciparum was analysed using a polymerase chain reaction (PCR) assay that detects potential chromosome breaks that have been healed by the addition of telomere repeats. The data show that the Pf332 and Pf87 genes located in subtelomeric positions of chromosomes 3 and 11, respectively, represent fragile sites. Breakpoints were observed in different regions of these genes. In the broken genes, the DNA sequences preceding the telomere addition sites generally have complementarity to the predicted RNA template of a P. falciparum telomerase ribonucleoprotein enzyme complex. We propose a model for the creation of new telomeres in P. falciparum adjacent to broken ends containing short telomere-like sequence motifs.

Animals↗
PubMed1993

In vitro phagocytosis inhibition assay for the screening of potential candidate antigens for sub-unit vaccines against the asexual blood stage of Plasmodium falciparum.

We have previously established a direct correlation between immune protection against the asexual blood stage Plasmodium falciparum infection and the presence of opsonizing antibodies promoting phagocytosis of parasitized red blood cells. In the present communication we describe an in vitro assay for measuring phagocytosis inhibition (PIA) specific for P. falciparum-infected erythrocytes. The phagocytosis inhibition assay is a simple procedure for screening potential candidates for sub-unit vaccines against P. falciparum based on the correlation between opsonizing antibodies and immunoprotection. The assay was used to analyse 18 recombinant molecules, corresponding to 11 distinct antigens of P. falciparum. Pre-incubation and selective antibody depletion experiments demonstrate the antigen-antibody specificity of the PIA. The presence of epitopes participating as targets of opsonic antibodies were demonstrated in six distinct polypeptide antigens.

Animals↗
PubMed1993

Immune response in mouse and malaria-exposed humans to peptides derived from Pf11-1, a highly repetitive megadalton protein of Plasmodium falciparum.

We have investigated the immune response against the Plasmodium falciparum gametocyte-specific antigen Pf11-1. This megadalton parasite molecule has been implicated in the process of erythrocyte rupture during gametogenesis. The molecule is composed in great part of degenerated nonapeptide motifs which are tandemly repeated several hundred times. A computer algorithm searching for T sites predicted that the entire repeat region of the Pf11-1 represents potential T cell antigenic major histocompatibility complex class II-binding sites. To test this hypothesis, synthetic peptides corresponding to two nonamer subtype repeats, differing only at two amino acid positions, were used to immunize congenic mouse strains. Both peptides were shown to contain both B and T cell epitopes. The immune response is restricted to the H-2d and H-2k haplotypes. The T cell response against the peptides appeared to be highly specific, clearly discriminating between the two similar nonamer repeat sequences, whereas the humoral response produced cross-reacting antibodies. We also investigated the humoral and T cell reactivities of P. falciparum-primed individuals in West Africa against the synthetic Pf11-1 peptides. Among 51 individuals 35 had antibodies to at least one of the two peptides and a majority of them (28) had antibodies reacting with both peptides. The cellular response was analyzed by [3H]thymidine incorporation or interferon-gamma release. There was considerable variation in the response to the two peptides. Among the human samples 36% responded to one repeat subtype, while only 13% responded to the second subtype. Interestingly, in individual donors the T cell response to both peptides are associated, suggesting that, as shown for mice, the response is restricted by a genetic element. The data obtained on the two subtypes of the nonamer repeat region suggest that the entire Pf11-1 molecule might induce an unusually heterogenous B and T cell response during natural infection in man.

Adolescent↗
PubMed1992

Cloning and characterization of chromosome breakpoints of Plasmodium falciparum: breakage and new telomere formation occurs frequently and randomly in subtelomeric genes.

We analysed the genetic stability of two subtelomeric genes of the human malaria parasite Plasmodium falciparum. A PCR based assay, using a telomere and a target-gene specific primer was used to detect potential chromosome rearrangements. We show that chromosome breakage and the formation of new telomeres occur frequently in the two genes coding for histidine rich proteins (HRP I and HRP II) in laboratory isolates, but remains undetectable in clinical parasite isolates. This finding suggests an essential role of these genes in vivo and that chromosome breakage is rather an accidental process than a programmed chromosome fragmentation. Cloning and sequencing of 8 chromosome breakpoints of the HRP II gene from one parasite isolate shows that the breakage occurs within a broad region in which new telomere formation appear to take place at random sites. Furthermore, this analysis revealed no obvious sequence similarities of sites of telomere addition. Finally, we show that an irregular pattern of heterogeneous telomere repeats is added at each broken end and that each healed chromosome contains a distinct pattern of repeats. We discuss a model for telomere formation in P. falciparum.

Animals↗
PubMed1992

The Pf332 gene of Plasmodium falciparum codes for a giant protein that is translocated from the parasite to the membrane of infected erythrocytes.

We studied the gene structure of the Plasmodium falciparum antigen 332 (Ag332). The gene size was estimated to be approx. 20 kb based on the large size of both the transcript found in mature asexual blood stage parasites and mung bean nuclease fragment generated from genomic DNA. Sequence analysis of genomic and cDNA clones representing different regions of the Pf332 locus showed that the gene product contains a large number of highly degenerated glutamic acid (Glu)-rich repeats (32% Glu). The gene shows dramatic restriction fragment length polymorphism in various P. falciparum isolates and was mapped to the subtelomeric region of chromosome 11. The recombinant 332 fusion protein reacts strongly with the human monoclonal antibody (mAb) 33G2, which is able to inhibit the cytoadherence of parasitized red blood cells on the melanoma cell line C32 and merozoite invasion in in vitro assays. The epitope recognized by this mAb is found frequently in the reported sequence. Ag332 monospecific antibodies were obtained by immunization of mice with a recombinant fusion protein. These antibodies react with a large parasite molecule with an apparent molecular size of 2500 kDa of trophozoite and schizont-infected erythrocytes on Western blot and by immunoprecipitation analysis. Immunofluorescence studies using a confocal microscope showed that Ag332 is exported from the parasite to the infected red blood cell membrane within large vesicle-like structures of about 1 micron diameter.

Amino Acid Sequence↗
PubMed1992

Gene inactivation of Pf11-1 of Plasmodium falciparum by chromosome breakage and healing: identification of a gametocyte-specific protein with a potential role in gametogenesis.

We report the identification of the product of the Plasmodium falciparum Pf11-1 gene and demonstrate that it is a gametocyte-specific protein that has a potential role in the rupture of the host erythrocyte and emergence of the gametes (gametogenesis). The Pf11-1 gene is a large locus (30 kb) whose sequence predicts a glutamic acid-rich polypeptide. Our identification of the Pf11-1 gene product as gametocyte specific was greatly facilitated by the isolation of a mutant parasite clone in which greater than 90% of the Pf11-1 gene was deleted. Molecular analysis of the mutant locus suggests that the underlying genetic mechanism is chromosome breakage and subsequent healing by the addition of telomere repeats. PCR-based analysis showed that similar DNA rearrangements occur commonly in small subpopulations of most laboratory strains, suggesting that the Pf11-1 locus represents a fragile chromosome region. Northern blot analysis demonstrates that a large Pf11-1 gene-specific transcript (much greater than 10 kb) is present in gametocytes but not in asexual blood stage parasites. The Pf11-1 protein was localized by electron microscopy to granules in the cytoplasm of gametocytes adjacent to the membrane of the parasitophorous vacuole. Following in vitro stimulation of gametogenesis, the Pf11-1 protein was found in the membrane of lysed erythrocytes, suggesting a role for Pf11-1 in erythrocyte rupture within the mosquito gut.

Animals↗
PubMed1992

Pfl I-I and Pf332: two giant proteins synthesized in erythrocytes infected with Plasmodium falciparum.

Although the malaria parasite develops within erythrocytes, it has to modify the surrounding red blood cell membrane for its intracellular survival and maturation. These changes include the translocation of proteins across the parasite and the parasitophorous vacuole membranes to the host membrane. In this review, Denise Mattei, Katherine Hinterberg and Artur Scherf focus on two distinct giant parasite molecules of unprecedented size (approximately one MDa), called Pf332 and PflI-I, that are synthesized and exported into the cytoplasm of the host cell in the asexual and sexual blood stages of Plasmodium falciparum, respectively. The corresponding genes are located in genetically unstable subtelomeric chromosome regions.

Journal Article↗
PubMed1992

Involvement of Pf155/RESA and cross-reactive antigens in Plasmodium falciparum merozoite invasion in vitro.

Lines of Plasmodium falciparum FCR3 either expressing or not expressing the blood-stage antigen Pf155/RESA were used to analyze the possible involvement of this antigen in the merozoite invasion process in vitro. Antibodies from human sera, affinity purified on synthetic peptides corresponding to C-terminal repeated sequences in Pf155/RESA, were shown to inhibit merozoite invasion of both types of parasites with similar efficiency. Reversal of the invasion inhibition by fusion proteins containing repeated sequences of Pf155/RESA but not of the cross-reactive antigens Ag332 and Pf11.1 indicated that the inhibitory antibodies had similar target antigens in both Pf155/RESA+ and Pf155/RESA- parasites that involved cross-reacting epitopes present in Pf155/RESA. Rabbit antibodies specific for Pf155/RESA repeats inhibited merozoite invasion of Pf155/RESA expressing parasites efficiently but had no or very small effect on the invasion of Pf155/RESA-deficient parasites. In contrast, rabbit antibodies specific for Ag332 repeats as well as human antibodies affinity purified on synthetic Ag332 peptides inhibited merozoite invasion of both types of parasites with high efficiency. A similar inhibition pattern was seen with the human monoclonal antibody 33G2, which has specificity for Ag332 but also cross-reacts with Pf155/RESA and Pf11.1. Taken together, our data suggest that Pf155/RESA and related cross-reactive antigens as well as Ag332 are involved in the merozoite invasion process and may constitute targets for invasion inhibitory antibodies.

Animals↗
PubMed1992

Characterization of a Plasmodium falciparium mutant that has deleted the majority of the gametocyte-specific Pf11-1 locus.

We identified a gametocyte-specific protein of Plasmodium falciparum called Pf11-1 and provide experimental evidence that this molecule is involved in the emergence of gametes of the infected erythrocyte (gametogenesis). A mutant parasite clone, which has deleted over 90% of the Pf11-1 gene locus, was an important control to establish the gametocyte-specific expression of the Pf11-1. Molecular analysis of the Pf11-1 deletion indicates that it is presumably due to a chromosome breakage with subsequent 'healing' by the addition of telomeric heptanucleotides. Moreover, similar DNA rearrangements are observed in most of the laboratory isolates during asexual propagation in vitro.

Animals↗
PubMed1992

The Pf332 gene codes for a megadalton protein of Plasmodium falciparum asexual blood stages.

We characterized the Plasmodium falciparum antigen 332 (Ag332) which is specifically expressed during the asexual intraerythrocytic cycle of the parasite. The corresponding Pf332 gene has been located in the subtelomeric region of chromosome 11. Furthermore, it is present in all strains so far analyzed and shows marked restriction length fragment polymorphism. Partial sequence and restriction endonuclease digestion of cloned fragments revealed that the Pf332 gene is composed of highly degenerated repeats rich in glutamic acid. Mung been nuclease digestion and Northern blot analysis suggested that the Pf332 gene codes for a protein of about 700 kDa. These data were further confirmed by Western blot and immunoprecipitation of parasites extracts with an antiserum raised against a recombinant clone expressing part of the Ag332. Confocal immunofluorescence showed that Ag332 is translocated from the parasite to the surface of infected red blood cells within vesicle-like structures. In addition, Ag332 was detected on the surface of monkey erythrocytes infected with Plasmodium falciparum.

Amino Acid Sequence↗
PubMed1992

A recombinant hybrid protein as antigen for an anti-blood stage malaria vaccine: a study on the conservation of a protective component.

Recently we have shown that two hybrid proteins expressed in Escherichia coli confer protective immunity to Aotus monkeys against an experimental Plasmodium falciparum infection (Knapp et al., 1992). Both hybrid proteins carry a sequence containing amino acids 631 to 764 of the serine stretch protein SERP (Knapp et al., 1989b). We have studied the diversity of this SERP region in field isolates of P. falciparum. Genomic DNA was extracted from the blood of six donors from different endemic areas of Brazil and West Africa. The SERP region encoding amino acids 630 to 781 was amplified by polymerase chain reaction (PCR) and sequenced. Only conserved amino acid substitutions in maximally two positions of the analyzed SERP fragment could be detected which supports the suitability of this SERP region as a component of an anti-blood stage malaria vaccine.

Amino Acid Sequence↗
PubMed1992

The squirrel monkey as an experimental model for Plasmodium falciparum erythrocyte rosette formation.

Experimental cerebral malaria was recently found to occur in the squirrel monkey Saimiri sciureus when infected with the human malaria parasite Plasmodium falciparum. This report is concerned with the existence of spontaneous rosette formation ex vivo (infected blood samples) and in vitro (cultured parasites) between red blood cells (RBC) infected with squirrel monkey-adapted P. falciparum isolates and normal squirrel monkey RBC. Transfer of P. falciparum with high rosette formation tendencies (90-100 R+) from one donor monkey to several recipients gave rise to parasites that varied extensively in their ex vivo rosette formation capacity (4-96% R+). However, all individual parasites readily form rosettes after 24 hr of in vitro culture (60-95% R+). Host factors may be involved in the modulation of rosette formation, although it is found to occur both in splenectomized and spleen-intact animals. Cross-rosette formation is seen between parasitized human RBC and normal squirrel monkey RBC and vice versa, and rosettes formed by RBC of the two hosts are similarly affected by pH, temperature, EDTA, trypsin, as well as squirrel monkey and African human hyperimmune IgG. These characteristics of rosette formation are preserved after long-term in vitro culture in human RBC. Rosettes formed by some isolates are highly sensitive to heparin while others are not, suggesting at least two distinct mechanisms of rosette formation. This idea is also supported by the observation that specific squirrel monkey antisera to heparin-sensitive strains does not dissociate rosettes formed by a heparin-resistant strain. The results suggest that rosettes and anti-rosette formation antibodies formed by squirrel monkeys and humans exhibited similar characteristics, and that the squirrel monkey is therefore a good experimental model to study erythrocyte rosette formation and cerebral malaria.

Animals↗
PubMed1991

Polymorphism of the alleles of the merozoite surface antigens MSA1 and MSA2 in Plasmodium falciparum wild isolates from Colombia.

The degree of polymorphism and the allelic distribution of 2 major Plasmodium falciparum merozoite surface antigens (MSA1 and MSA2) have been analysed in clinical isolates from Colombia. DNA was prepared directly from patients' blood and used in PCR reactions to amplify block 2 of MSA1 and the central region from MSA2. Thirty one samples were analysed and a marked degree of length polymorphism was detected, especially for MSA2. A high proportion of multiple bands was also observed, most probably resulting from mixed infections. Allele-specific oligonucleotides were used to type both alleles. For MSA1, 26 out of 31 clinical isolates were of the RO33 type, 15 were MAD20 and three were typed as KI. When the MSA2 allele was analysed, 7 isolates hybridised with a CAMP specific probe and 6 hybridised strongly with an FC27-derived oligonucleotide. Two samples, which showed multiple bands, hybridised with both probes. Interestingly, in 14 out of 27 isolates the MSA2 allele remained unassigned by the specific probes. Five of these were cloned and their DNA sequenced; these sequences are discussed.

Adolescent↗