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A Scherf

Publications and source records attributed to A Scherf.

At least 19 recordsLinked to original sources
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

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↗
PubMed1990

Genetic diversity in the major merozoite surface antigen of Plasmodium falciparum: high prevalence of a third polymorphic form detected in strains derived from malaria patients.

We studied the diversity of the polymorphic 195-kDa antigen (p190) of Plasmodium from infected individuals. Genomic parasite DNA was extracted from the blood of 30 donors from different endemic areas of Brazil. The 5' region, encoding the polymorphic N-terminal part of p190 was analysed following polymerase chain reaction (PCR). Multiple infections of genetically distinct parasites could be detected within infected malaria patients. Sequence analysis and oligodeoxyribonucleotide typing of the PCR products demonstrated the prevalence of a third polymorphic form of p190.

Animals↗
PubMed1990

Parasite antigens expressed in Escherichia coli. A refined approach for epidemiological analysis.

A simple method is described to generate carrier-free recombinant antigens following their expression in Escherichia coli. A plasmid, called pMSgt11, has been constructed such that the cleavage site for the protease factor Xa separates the recombinant antigen from an enzymatically active beta-galactosidase. Thus, rapid purification of the active beta-galactosidase recombinant protein, followed by digestion with factor Xa, releases the antigen of interest. The pMSgt11 plasmid is compatible with the phage expression vector, lambda gt11 and the feasibility of applying this system has been demonstrated using malarial recombinant antigens. Inserts from lambda gt11 recombinant Plasmodium falciparum clones have been recloned into the EcoRI site of pMSgt11 and the expressed soluble fusion proteins have been purified from crude extracts using a one step affinity chromatography. After protease digestion, the fusion protein cleavage products were analysed by immunoblot with a panel of different human immune sera. We were able to successfully demonstrate specific antibody titers to the parasite-derived carrier-free antigen, without interference from anti-Escherichia coli-specific antibodies. The general application of this approach to epidemiological analysis is discussed.

Animals↗
PubMed1990

Immunogenicity and antigenicity in rabbits of a repeated sequence of Plasmodium falciparum antigen Pf155/RESA fused to two immunoglobulin G-binding domains of staphylococcal protein A.

A synthetic gene encoding a tetramer of the repeated subunit EENVEHDA of the Plasmodium falciparum antigen Pf155/RESA was expressed in a dual-expression system. The resulting fusion proteins, designated ZZ-M1 and BB-M1, comprised the EENVEHDA repeats and either two immunoglobulin G-binding domains from staphylococcal protein A or the human serum albumin-binding domains from streptococcal protein G, respectively. The soluble fusion proteins were affinity purified to homogeneity in one-step procedures. ZZ-M1 was used for immunization of rabbits. The rabbit antisera reacted with BB-M1 in an enzyme-linked immunosorbent assay and with Pf155/RESA in immunofluorescence of infected erythrocytes and immunoblotting. Inhibition studies revealed that the antibodies mainly recognized epitopes formed by two or more EENVEHDA subunits and were remarkably specific for Pf155/RESA. Importantly, the antibodies also inhibited P. falciparum merozoite reinvasion in vitro efficiently, indicating that they reacted with biologically important epitopes exposed on the native antigen. Immunization with Freund complete adjuvant resulted in high levels of specific immunoglobulin G antibodies over a 1-year period, whereas the antibody response obtained after immunization without adjuvant was generally weaker, immunoglobulin G and M mediated, and not sustained for longer periods. However, these titers were restored after booster injection. Taken together, the results support the usefulness of recombinant gene constructs of this type as immunogens for malaria vaccines.

Amino Acid Sequence↗
PubMed1989

Reactivity of the human monoclonal antibody 33G2 with repeated sequences of three distinct Plasmodium falciparum antigens.

The human mAb 33G2 has high capacity to inhibit in vitro invasion of erythrocytes by Plasmodium falciparum merozoites and, thus, is of special interest with regard to protective immunity against the parasite. In order to obtain more information about asexual blood stage Ag of P. falciparum that are seen by this antibody, material from synchronized P. falciparum cultures was studied by immunofluorescence, immunoelectron microscopy, and immunoblotting. Reactivity was mainly confined to the membrane of infected erythrocytes. Soon after merozoite invasion the antibody stained the erythrocyte membrane. This membrane-associated staining faded during intracellular development of the parasites. Beginning about 18 h after invasion, a dotted pattern appeared which increased in strength with time and persisted to schizont rupture. Pf155/RESA was the major Ag recognized in immunoblots of parasites collected throughout the entire erythrocytic cycle, although other polypeptides also bound the antibody. Among these was a 260-kDa polypeptide found in late trophozoites and schizonts. The specificity of the antibody was analyzed with synthetic peptides corresponding to repeated sequences in the P. falciparum Ag Pf155/RESA, Pf11.1, and Ag332. Synthetic peptides related to Ag332 were the most efficient inhibitors of antibody binding in immunofluorescence studies and cell ELISA. A beta-galactosidase-Ag332 fusion protein was also efficient in reversing reinvasion inhibition caused by 33G2. These results define a family of cross-reactive P. falciparum Ag recognized by mAb 33G2 and suggest that Ag332 was its original target.

Amino Acid Sequence↗
PubMed1989

A versatile phage lambda expression vector system for cloning in Escherichia coli.

By integrating fragments from the expression plasmids pJK2 and pJK4 into a derivative of the bacteriophage lambda, we constructed the phage expression vectors lambda JK2 and lambda JK4, which allow efficient cloning of genomic or cDNA either into the 5' end or the 3' end of the lacZ gene of Escherichia coli. Expression of barrier-free DNA in phase may lead to fusion proteins consisting of active beta-galactosidase (beta Gal) plus an additional polypeptide encoded by the inserted DNA. Analysis of distinct recombinant clones is quick and easy, due to the reversible integration of the plasmid into the genome. As an example, we constructed an expression library of genomic Plasmodium falciparum DNA in lambda JK2. We polymerised (amplified) and expressed a synthetic DNA fragment, which codes for a potential antigenic determinant of the 11-1 gene of Plasmodium falciparum as a fusion to the N terminus of active beta Gal. We demonstrate that such chimeric molecules can be affinity-purified and that polypeptides can be separated from the beta Gal part by cleavage with the protease factor Xa.

Amino Acid Sequence↗