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M F Wiser

Publications and source records attributed to M F Wiser.

At least 19 recordsLinked to original sources
PubMed1999

An alternative secretory pathway in Plasmodium: more questions than answers.

The malaria parasite extensively modifies the host erythrocyte. Many of these modifications are mediated by proteins exported from the parasite and targeted to specific locations within the infected erythrocyte. However, little is known about how the parasite targets proteins to specific locations beyond its own plasma membrane. Treatment of infected erythrocytes with brefeldin A results in the accumulation of many exported Plasmodium proteins into a compartment distinct from the ER. Proteins destined for the host erythrocyte membrane, the parasitophorous vacuole or inclusions within the erythrocyte cytoplasm accumulate in this novel compartment, and co-localization studies indicate that there is a single compartment per parasite. Exported proteins only accumulate in this novel compartment if brefeldin A treatment is concurrent with their synthesis. This novel compartment is probably a membrane-bound organelle located at the parasite periphery, and may be the first step in an alternative secretory pathway that specializes in the export of proteins into the host cell. Such an alternative secretory pathway raises questions about how exported proteins are differentially targeted to this novel organelle versus the ER and the fate of exported proteins after this novel organelle.

Amino Acid Sequence↗
PubMed1999

Characterization of a Plasmodium chabaudi gene encoding a protein with glutamate-rich tandem repeats.

Several highly antigenic proteins containing tandem repeats rich in glutamic acid residues have been described in Plasmodium falciparum. However, relatively little information is available about analogous genes in rodent parasites. This report describes a 4.2-kb genomic DNA fragment from P. chabaudi with a deduced amino acid sequence that is predominantly glutamate-rich tandem repeats. Several different monoclonal antibodies raised against a 93-kDa P. chabaudi protein, which does not correspond to the cloned DNA fragment, recognize a recombinant protein expressed from the 4.2-kb DNA fragment. The only sequence similarities between these two genes are tandem repeats with a predominance of glutamate pairs followed by a hydrophobic residue. This repetitious-sequence motif may be the basis for the observed cross-reactivity. A similar motif has been demonstrated to be the basis for antibody cross-reactivity between glutamate-rich proteins of P. falciparum. The expression of multiple glutamate-rich proteins with cross-reacting epitopes may be a general phenomenon in Plasmodium species.

Amino Acid Sequence↗
PubMed1999

Characterization of the parasitophorous vacuole membrane from Plasmodium chabaudi and implications about its role in the export of parasite proteins.

Little is known about how the malaria parasite transports and targets proteins into the host erythrocyte. Parasite proteins exported into the host cell not only have to cross the parasite plasma membrane but also must traverse the parasitophorous vacuolar membrane (PVM) that surrounds the parasite. The PVM of Plasmodium chabaudi-infected erythrocytes was analyzed by immunofluorescence using an antibody against a known PVM protein, a fluorescent lipid probe, and electron microscopy. These analyses reveal qualitatively different membranous projections from the PVM. Some PVM projections are uniformly labeled with the antibody and with lipid probes and probably correspond to the Maurer's clefts. In contrast to this uniform labeling of the PVM and projections, a 93-kDa P. chabaudi erythrocyte membrane-associated protein is occasionally detected in vesicle-like structures adjacent to the parasite. These vesicle-like structures are found only coincident with protein synthesis and are located at discrete sites on the PVM. These observations suggest that the 93-kDa protein does not move along the membranous projections of the PVM toward the erythrocyte membrane. It is proposed that the 93-kDa protein is secreted directly into the erythrocyte cytoplasm at discrete PVM domains and then binds to the cytoplasmic face of the erythrocyte membrane.

Animals↗
PubMed1999

Export of proteins via a novel secretory pathway.

The intraerythrocytic location of the malaria parasite necessitates modification of the host cell. These alterations are mediated either directly or indirectly by parasite proteins exported to specific compartments within the host cell. However, little is known about how the parasite specifically targets proteins to locations beyond its plasma membrane. Mark Wiser, Norbert Lanners and Richard Bafford here propose an alternative secretory pathway for the export of parasite proteins into the host erythrocyte. The first step of this pathway is probably an endoplasmic reticulum (ER)-like organelle that is distinct from the normal ER. Possible mechanisms of protein trafficking in the infected erythrocyte are also discussed. The proposed ER-like organelle and alternative secretory pathway raise many questions about the cell biology of protein export and trafficking in Plasmodium.

Animals↗
PubMed1998

The complete sequence of Plasmodium berghei merozoite surface protein-1 and its inter- and intra-species variability.

The complete gene for merozoite surface protein-1 (MSP-1) from Plasmodium berghei has been cloned and sequenced. Comparison of the P. berghei MSP-1 sequence with MSP-1 from other rodent parasites reveals five conserved domains interrupted by four variable blocks. These variable blocks exhibit no sequence homology but do have similar amino acid compositions. Primary proteolytic processing sites are located near the boundaries between the conserved domains and the variable blocks. Sequencing of the variable blocks from several P. berghei isolates shows that the predominant intra-species difference is in the number of tandem repeats. The inter- and intra-species differences suggest that the variable blocks are localized areas with relatively high levels of slipped-strand mispairing, unequal crossing-over, or other intragenic recombination activity. MSP-1 from P. berghei exhibits more repetitiveness than MSP-1 from other species suggesting that P. berghei experiences a higher intrinsic level of events producing variable numbers of tandem repeats or a lower level of events leading to the degeneration of tandem repeats.

Amino Acid Sequence↗
PubMed1997

A cryptic protease activity from Trypanosoma cruzi revealed by preincubation with kininogen at low temperatures.

Cysteine proteases have been identified in parasitic protozoa including the causative agent of Chagas' disease Trypanosoma cruzi. T. cruzi lysates subjected to substrate-containing SDS-polyacrylamide gel electrophoresis exhibit major bands of proteolytic activity in the 45-55 kDa molecular mass range (cruzipain activity). Paradoxically, addition of kininogen (a cystatin-like protease inhibitor) to the lysates before electrophoresis results in the appearance of additional bands of proteolytic activity in the 160-190 kDa molecular mass range. This inhibitor-activated protease activity depends upon the reaction conditions and exhibits novel properties. For example, a 24-48 hour preincubation at low temperature (-20 degrees C optimum) greatly enhances the proteolytic activity. The results suggest that a metastable complex forms between kininogen and a cryptic 30 kDa cysteine protease from T. cruzi and that this complex participates in the activation of proteolytic activity.

Animals↗
PubMed1997

A novel alternate secretory pathway for the export of Plasmodium proteins into the host erythrocyte.

The malarial parasite dramatically alters its host cell by exporting and targeting proteins to specific locations within the erythrocyte. Little is known about the mechanisms by which the parasite is able to carry out this extraparasite transport. The fungal metabolite brefeldin A (BFA) has been used to study the secretory pathway in eukaryotes. BFA treatment of infected erythrocytes inhibits protein export and results in the accumulation of exported Plasmodium proteins into a compartment that is at the parasite periphery. Parasite proteins that are normally localized to the erythrocyte membrane, to nonmembrane bound inclusions in the erythrocyte cytoplasm, or to the parasitophorous vacuolar membrane accumulate in this BFA-induced compartment. A single BFA-induced compartment is detected per parasite and the various exported proteins colocalize to this compartment regardless of their final destinations. Parasite membrane proteins do not accumulate in this novel compartment, but accumulate in the endoplasmic reticulum (ER), suggesting that the parasite has two secretory pathways. This alternate secretory pathway is established immediately after merozoite invasion and at least some dense granule proteins also use the alternate pathway. The BFA-induced compartment exhibits properties that are similar to the ER, but it is clearly distinct from the ER. We propose to call this new organelle the secondary ER of apicomplexa. This ER-like organelle is an early, if not the first, step in the export of Plasmodium proteins into the host erythrocyte.

Animals↗
PubMed1997

Plasmodium chabaudi: immunogenicity of a highly antigenic glutamate-rich protein.

The immunogenicity of a 93-kDa Plasmodium chabaudi protein that contains glutamate-rich tandem repeats was investigated in this study. Immunoblotting with various monoclonal antibodies indicates that this 93-kDa protein is equivalent to a potential P. chabaudi RESA analogue. However, the sequence of the P. chabaudi protein does not exhibit any significant homology to Pf155/RESA. Antibodies against the 93-kDa protein appear early during P. chabaudi infection and reach high titers. The highest antibody titers are found when the parasitemia is descending, suggesting that this protein may play some role in immunity. Immunization of mice with the recombinant protein also results in high antibody titers, indicating that the protein is quite immunogenic. However, mice immunized with recombinant protein and challenged with P. chabaudi do not exhibit a delayed appearance of parasitemia, a reduced parasitemia, or a shortened duration of parasitemia. Glutamate-rich P. falciparum proteins such as Pf155/RESA, are being considered as vaccine candidates. The studies with P. chabaudi suggest that interpretation of serological data using glutamate-rich proteins should proceed with caution. The glutamate-rich repeats, although highly immunogenic, may not be important in host immunity against malaria. However, antibodies that appear late in the P. chabaudi infection do appear to play a role in anti-malarial immunity.

Animals↗
PubMed1997

Evaluation of immunogenicity of an oral Salmonella vaccine expressing recombinant Plasmodium berghei merozoite surface protein-1.

A repetitive region of Plasmodium berghei merozoite surface protein-1 (PbMSP-1) was expressed as a fusion protein with either maltose binding protein or the B subunit of heat-labile enterotoxin from Escherichia coli. Vaccination of mice with the fusion proteins indicates that this region of PbMSP-1 is antigenic as evidenced by an antibody response. The fusion proteins were also expressed in Salmonella and mice were orally immunized with the recombinant Salmonella. Some of the vaccinated mice survived a challenge with P. berghei blood-stage parasites without developing parasitemia. All control mice became patent and succumbed to the challenge infection. This partial protection was also observed with purified recombinant protein and was independent of the adjuvant used. Mice immunized with recombinant Salmonella showed either extremely low or no antibody response to PbMSP-1, suggesting that cell-mediated immunity is important for the observed protection. These studies show that it is feasible to develop a cost effective oral vaccine against the blood stage of the malarial parasite.

Administration, Oral↗
PubMed1996

Further characterization of a 58 kDa Plasmodium berghei phosphoprotein as a cochaperone.

Molecular chaperones are important for proper protein folding during protein biogenesis. This report describes a protein from Plasmodium berghei which is 30% identical and 40% similar to a recently described mammalian cochaperone, or heat shock protein 70 interacting protein. The P. berghei cochaperone accumulates throughout the trophozoite stage and decreases during the schizont stage. The stage specific expression is consistent with its presumed role in protein folding or protein-protein interactions. The largest difference between the Plasmodium and mammalian sequences is a more extensive domain of imperfect glycine-glycine-methionine-proline (GGMP) tandem repeats in the parasite's cochaperone sequence. Immunofluorescence studies show that the protein is an abundant cytosolic protein of the parasite. However, antibodies raised against the GGMP repeat domain, which is also found in other parasite chaperones, react with both the parasite and host erythrocyte membrane. The reactivity with the host membrane suggests that the parasite exports molecular chaperones into the infected erythrocyte.

Amino Acid Sequence↗
PubMed1995

Sequence and mapping of galectin-5, a beta-galactoside-binding lectin, found in rat erythrocytes.

A monomeric rat beta-galactoside-binding lectin previously purified from extracts of rat lung has been localized to erythrocytes, and the cDNA encoding it has been isolated from a rat reticulocyte cDNA library. The deduced amino acid sequence of the cDNA predicts a protein with a M(r) of 16,199, with no evidence of a signal peptide. The deduced sequence is identical to the sequences of seven proteolytic peptides derived from the purified lectin. Peptide analysis by mass spectrometry indicates that the N-terminal methionine is cleaved and that serine 2 is acetylated. The lectin shares all the strictly conserved amino acid residues of other members of the mammalian galectin family and is designated galectin-5 (GenBank accession number L36862). Galectin-5 is a weak agglutinin of rat erythrocytes, despite its monomeric structure. The gene encoding galectin-5 (LGALS5) has been mapped in mouse to chromosome 11, approximately 50 centimorgans from the centromere and 1.8 +/- 1.8 centimorgans from the polymorphic marker D11Mit34n, a region syntenic with human chromosome 17q11.

Amino Acid Sequence↗
PubMed1994

Effect of pyrimethamine resistance on sporogony in a Plasmodium berghei/Anopheles stephensi model.

A pyrimethamine-resistant line of Plasmodium berghei was derived by treating infected mice with high doses of pyrimethamine and selecting for recrudescence. This resistant line was compared with the parental pyrimethamine-sensitive line in order to ascertain whether drug resistance is associated with a biological advantage. Overall, the pyrimethamine-resistant line is quite similar to the sensitive line, except that it proceeds through sporogonic development more slowly than the pyrimethamine-sensitive parental line. However, under pyrimethamine pressure the sensitive line is unable to undergo the sporogonic cycle, whereas the resistant line is unaffected. These results indicate that the transmission of pyrimethamine resistance in this model is favored only under conditions of drug pressure.

Animals↗
PubMed1993

Accessibility and distribution of intraerythrocytic antigens of Plasmodium-infected erythrocytes following mild glutaraldehyde fixation and detergent extraction.

Malarial antigens on the surface of infected erythrocytes have been described by many investigators. However, few of these antigens have been unambiguously demonstrated to be exposed on the surface of erythrocytes. This study demonstrates that mild glutaraldehyde fixation results in the cytoplasmic face of the host membrane becoming accessible to antibody under conditions that normally do not expose the cytoplasmic face of uninfected erythrocytes. These results indicate that caution should be used in interpreting data on the membrane disposition of malarial antigens. Detergent extraction of the glutaraldehyde-fixed erythrocytes results in an increased permeabilization such that malarial antigens on the parasite surface and within the cytoplasm of the infected erythrocyte are accessible to antibody. The accessibility of these antigens was demonstrated by both immunofluorescence and two-color flow cytometry. The antigens within the host cytoplasm were not diffuse but associated with patchy aggregates. Analysis of the antigens associated with the cytoplasmic aggregates by immunoelectron microscopy indicated that they were not associated with membrane-bound compartments. The fixation and permeabilization protocol described herein will have useful applications for the characterization and analysis of malarial antigens.

Animals↗
PubMed1992

Rapid transport of the acidic phosphoproteins of Plasmodium berghei and P. chabaudi from the intraerythrocytic parasite to the host membrane using a miniaturized fractionation procedure.

A miniaturized procedure for the separation of the host erythrocyte membrane from malarial parasites based on saponin lysis and density-gradient centrifugation with Percoll is described. The procedure requires only 20-35 microliters packed infected erythrocytes, is simple to perform, needs no sophisticated equipment, and can be completed in less than 2 h. Analysis of the isolated erythrocyte membranes and parasites using marker enzymes and electron microscopy revealed that both the purity and the yield of these fractions were relatively high. Erythrocyte membrane proteins, including spectrin, ankyrin, and band 4.1, were not found on the parasitophorous vacuolar membrane, which remained associated with some but not all of the isolated parasites. Application of this method to pulse-chase experiments indicated that the acidic phosphoproteins of Plasmodium berghei and P. chabaudi were rapidly transported from the parasite to the erythrocyte membrane immediately after their synthesis. The rapid export of these acidic phosphoproteins from the parasite distinguishes them from other proteins exported by the malarial parasite.

Animals↗