alpha-chymotrypsin and Malaria

The ligands that pathogens use to invade their target cells have often proven to be good targets for vaccine development. However, Plasmodium falciparum has redundant ligands that mediate invasion of erythrocytes. The first requirement for the development of a successful ligand-blocking malaria vaccine is the demonstration that antibodies induced to each ligand can block the erythrocyte invasion of parasites with polymorphic sequences. Because of P. falciparum's redundancy in erythrocyte invasion, each ligand needs to be studied under artificial conditions in which parasite invasion is restricted in its use of alternative pathways. Here we investigate the role of erythrocyte-binding antigen 175 (EBA-175), a parasite ligand that binds to sialic acid on glycophorin A, in the invasion of erythrocytes by 10 P. falciparum clones under conditions in which invasion is partially limited to the EBA-175-glycophorin A pathway, using chymotrypsin-treated erythrocytes. We show that the ability to invade erythrocytes for both sialic acid-independent and sialic acid-dependent pathways requires the EBA-175-glycophorin A pathway for erythrocyte invasion. Importantly, antibodies against region II of EBA-175 from the 3D7 clone blocked invasion of chymotrypsin-treated erythrocytes by >50% by all parasite clones studied, including those with multiple different mutations described in the literature. The one exception was FCR3, which had a similar sequence to 3D7 but only 30% inhibition of invasion of chymotrypsin-treated erythrocytes, indicating alternative pathways for invasion of chymotrypsin-treated erythrocytes. Our findings suggest that antibodies to region II of EBA-175, as one component of a ligand-blocking malaria vaccine, are largely unaffected by polymorphism in EBA-175.

Topics: Antibodies, Protozoan; Antigens, Protozoan; Chymotrypsin; Erythrocytes; Glycophorins; Host-Parasite Interactions; Humans; Immunoglobulin G; Malaria; Malaria Vaccines; Protozoan Proteins; Sequence Analysis, DNA

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, uses multiple ligand-receptor interactions to invade host red blood cells (RBCs). We studied the invasion of P falciparum into abnormal RBCs from humans carrying the Southeast Asian ovalocytosis (SAO) trait. One particular parasite line, 3D7-A, invaded these cells efficiently, whereas all other lines studied invaded SAO RBCs to only about 20% of the extent of normal (non-SAO) cells. This result is consistent with the clinical observation that SAO individuals can experience high-density P falciparum infections and provides an explanation for previous discrepant results on invasion of SAO RBCs. Characterization of the invasion phenotype of 3D7-A revealed that efficient invasion of SAO RBCs was paralleled by relatively efficient invasion of normal RBCs treated with either neuraminidase, trypsin, or chymotrypsin and a novel capacity to invade normal RBCs treated sequentially with both neuraminidase and trypsin. Our results suggest that only parasites able to use some particular invasion pathways can invade SAO RBCs efficiently in culture. A similar situation might occur in the field.

Topics: Animals; Asia, Southeastern; Case-Control Studies; Cells, Cultured; Chymotrypsin; Disease Susceptibility; Elliptocytosis, Hereditary; Erythrocyte Membrane; Erythrocytes, Abnormal; Humans; Malaria; Plasmodium falciparum; Receptors, Cell Surface

Erythrocyte invasion by malaria parasites is a complex multistep process involving parasite and erythrocyte receptors. It is a critical stage in the parasite life cycle and, therefore, a logical step in which to intervene to prevent or ameliorate disease. Rodent models of malaria, commonly Plasmodium yoelii, are frequently used for studies of malaria pathogenesis. Little is known, however, about the invasion machinery of rodent malaria parasites. We have found previously that mice congenic for a region of chromosome 1, containing the Duffy antigen/receptor for chemokines (DARC), have different susceptibility to P yoelii infection. Because P vivax, a human parasite, and P knowlesi, a simian parasite, use DARC to enter human erythrocytes, we sought to identify the role of the murine DARC in P yoelii invasion. Using a novel in vivo invasion assay and DARC knock-out mice, we found that DARC knock-out normocytes (mature erythrocytes) had negligible levels of P yoelii invasion compared with wild-type normocytes, demonstrating that DARC is a receptor for invasion of murine erythrocytes. In contrast, DARC knock-out reticulocytes were invaded at a rate similar to that for wild-type reticulocytes. We conclude that there is a DARC- independent pathway for reticulocyte invasion. These findings represent the first identification of a murine malaria receptor on erythrocytes and the first determination that different pathways of invasion exist on normocytes and reticulocytes. Because we show conservation of host-receptor interactions between rodent and human malaria, we can now use this model to identify how immunity can interfere with the invasion process.

Topics: Animals; Antigens, Protozoan; Carrier Proteins; Chymotrypsin; Disease Models, Animal; Duffy Blood-Group System; Erythrocytes; Host-Parasite Interactions; Malaria; Mice; Mice, Knockout; Plasmodium yoelii; Protozoan Proteins; Receptors, Cell Surface; Reticulocytes; Trypsin

A chymotrypsin-like serine protease gene (AgChyL) was cloned from the mosquito Anopheles gambiae by a polymerase chain reaction (PCR)-based subtractive cDNA cloning strategy. AgChyL messenger RNA (mRNA) is abundant in the adult female gut prior to, and for 8 h following, a blood meal. During the peak of digestion, from 12 to 24 h following a blood meal, AgChyL mRNA abundance decreased to barely detectable levels. AgChyL mRNA was abundant again by 48 h following a blood meal. Recombinant pro-AgChyL was expressed in Escherichia coli. The pro-enzyme can be activated by trypsin. Activated AgChyL cleaves the synthetic chymotrypsin substrate succinyl-L-Ala-Ala-Pro-Phe-nitroanilide, but not two other synthetic chymotrypsin substrates or synthetic trypsin and elastase substrates. The potential role of AgChyL in the coordination of An. gambiae digestion is discussed.

Topics: Amino Acid Sequence; Animals; Anopheles; Base Sequence; Blood; Chymotrypsin; DNA, Complementary; Down-Regulation; Feeding Behavior; Female; Gene Expression; Genes, Insect; Insect Proteins; Insect Vectors; Malaria; Molecular Sequence Data; Phylogeny; Recombinant Fusion Proteins; RNA, Messenger; Sequence Analysis; Sequence Homology, Amino Acid; Serine Endopeptidases

Midgut proteases contribute to the success or failure of Plasmodium infection of the mosquito. This paper examines the reciprocal effect of Plasmodium yoelii nigeriensis on midgut trypsin, chymotrypsin, aminopeptidase and carboxypeptidase in the mosquito Anopheles stephensi. The total protein ingested and the rate of protein digestion were unaffected by the parasite, but more protein was ingested at the first than the second bloodmeal. All peptidases were unaffected by the presence of the parasite during the first gonotrophic cycle, when ookinetes were penetrating the midgut. In the second gonotrophic cycle, trypsin and chymotrypsin were unaffected by growing oocysts, but aminopeptidase activity was reduced in the midguts of infected mosquitoes. Chymotrypsin activity was depressed and aminopeptidase activity elevated during the second gonotrophic cycle. Plasmodium infection has a negligible effect on bloodmeal digestion and does not limit the availability of the protein for egg production. The significance of changes in aminopeptidase activity when oocysts are present is discussed.

Topics: Aminopeptidases; Animals; Anopheles; Blood; Carboxypeptidases; Chymotrypsin; Digestion; Feeding Behavior; Female; Malaria; Mice; Parasitemia; Peptide Hydrolases; Plasmodium yoelii; Stomach; Trypsin

Modern protein Fourier transform infrared (FT-IR) spectroscopy has proven to be a versatile and sensitive technique, applicable to many aspects of protein characterization. The major practical drawback for the FT-IR spectroscopy of proteins is the large absorbance band of water, which overlaps the amide I resonances. D2O is often substituted for H2O in infrared experiments. Removal of water from protein samples can be complicated and tedious and potentially lead to denaturation, aggregation, or sample loss. Solvent removal by dialysis is difficult for suspensions and sols. A new method called the D2O dilution technique (Ddt) is described which simplifies the sample preparation step and improves the solvent subtraction. The effect of the D2O concentration on the IR spectrum of aqueous solutions of several model proteins was studied. Dilution of aqueous samples with D2O yields good quality spectra. The Ddt has been evaluated for quantitative analysis using standard proteins and its applicability to solutions and suspensions of a genetically engineered malaria antigen is demonstrated. Use of resolution-enhancement techniques with spectra in mixed solvents has also been investigated.

Topics: Adjuvants, Pharmaceutic; Antigens, Protozoan; Chymotrypsin; Deuterium; Feasibility Studies; Fourier Analysis; Humans; Kinetics; Malaria; Muramidase; Myoglobin; Protein Structure, Secondary; Proteins; Reproducibility of Results; Sensitivity and Specificity; Serum Albumin; Solutions; Spectrophotometry, Infrared; Vaccines; Water

A short-term in vitro culture system that allows for significant re-invasion of target erythrocytes by Plasmodium vivax was used to study the role of the Duffy blood group antigen as a ligand for merozoite invasion by this human malaria species. Using human Duffy-positive and -negative erythrocytes, various primate erythrocytes, enzymatic modification of erythrocytes, and mAb that defines a new Duffy determinant (Fy6) we conclude that the erythrocyte glycoprotein carrying Duffy determinants is required as a ligand for the invasion of human erythrocytes by P. vivax merozoites. Blockade of invasion by Fab fragments of the anti-Fy6 mAb equal to that of the intact molecule and the correlation of P. vivax susceptibility with the presence of the Fy6 determinant suggests this epitope or a nearby domain may be an active site on the Duffy glycoprotein. However, as for P. knowlesi, there is evidence that an alternate pathway for P. vivax invasion of simian erythrocytes may exist.

Topics: Animals; Antibodies, Monoclonal; Blood Group Antigens; Cells, Cultured; Chymotrypsin; Duffy Blood-Group System; Epitopes; Erythrocytes; Humans; Immunoglobulin Fab Fragments; Macaca mulatta; Malaria; Neuraminidase; Plasmodium; Plasmodium vivax; Saimiri; Trypsin

A murine monoclonal antibody, named anti-Fy6, which agglutinates all human red cells except those of Fy(a-b) phenotype was used for purification and characterization of Duffy antigens. Duffy antigens are multimeric red cell membrane proteins composed of different subunits of which only one, designated pD protein, reacts in immunoblots with the murine monoclonal antibody anti-Fy6. Affinity-purified detergent-soluble antigen-antibody complex obtained from red cells, surface-labeled with 125I yielded a complex pattern of bands when separated by polyacrylamide gel electrophoresis. Proteins that react with anti-Fy6 in immunoblots are: pA and pB (greater than 100 kDa) and pD (36-46 kDa). Electroeluted pD protein aggregates and generates bands of similar molecular mass to pA and pB proteins. Electroeluted pA and pB proteins disaggregate yielding pD protein. Oligomers and monomers of pD protein are present in red cells carrying Duffy antigens and absent in Fy(a-b-) cells. Six other proteins of molecular weight ranging from 68 to 21 kDa either associate or co-purify with pD protein. These proteins are only present in Duffy antigen positive cells. The pD protein is different in Fy(a+b-) and Fy(a-b+) cells by fingerprint analysis. Human antisera identify the same proteins in red cell carrying Duffy antigens as the murine monoclonal antibody anti-Fy6.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Blood Group Antigens; Chymotrypsin; Duffy Blood-Group System; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Humans; Malaria; Membrane Proteins; Molecular Weight; Peptide Fragments; Peptide Mapping; Plasmodium; Plasmodium vivax; Receptors, Immunologic

Plasmodium fragile infection of the toque monkey is a natural host-parasite association in which parasite sequestration occurs as during P. falciparum infection of humans. We have studied parasite sequestration of P. fragile and demonstrated the existence of a new property of cytoadherence of infected erythrocytes, "rosetting," which is defined as the agglutination of uninfected erythrocytes around parasitized erythrocytes. Rosetting in vitro and sequestration in vivo appear simultaneously as the parasite matures. The spleen plays a role in modulating cytoadherence; both sequestration and rosetting, which occur with cloned parasites from spleen-intact animals, are markedly reduced in splenectomized animals infected with parasites derived from the same clone. Sequestration and rosetting can be reversed by immune serum. Protease treatment of infected blood abolishes rosetting; however, if treatment is performed at an early stage of schizogony, rosetting reappears if parasites are allowed to further develop in the absence of protease. These results indicate that with P. fragile in its natural primate host, rosetting and sequestration are related to the presence on the infected erythrocyte surface of a parasite-derived antigenic component, the expression of which is modulated by the spleen.

Topics: Animals; Antibodies, Protozoan; Chymotrypsin; Erythrocytes; Female; Macaca; Malaria; Male; Neuraminidase; Plasmodium; Rosette Formation; Spleen; Splenectomy; Trypsin

Plasmodium berghei sporozoites were observed to react with human hepatoma (HepG2) target cells which had been fixed with methanol, formaldehyde, or glutaraldehyde. The reaction consisted of attachment of sporozoites to the fixed target cells and the release of circumsporozoite protein which bound to target cell areas adjacent to the attachment sites. Treatment of fixed target cells with 0.1 N H2SO4 at 80 C, neuraminidases, neuraminidase plus galactose oxidase or inclusion of transferrin, orosomucoid, their asialo forms, or various monosaccharides in the incubation medium had no significant effect on target cell reactivity with sporozoites. Fixed cells oxidized with periodate or cells extracted with methanol or chloroform-methanol were reactive but lost activity if allowed to air dry after treatment. Treatment with papain or chymotrypsin at levels producing heavy cell structure damage caused a major loss of activity.

Topics: Carcinoma, Hepatocellular; Cells, Cultured; Chymotrypsin; Formaldehyde; Galactose Oxidase; Glutaral; Host-Parasite Interactions; Liver Neoplasms; Malaria; Methanol; Neuraminidase; Papain; Plasmodium berghei; Transferrin

Serum protease inhibitors were determined in paired sera from 7 patients with cerebral malaria and 2 patients with acute malaria showing high and low growth inhibition activity in the initial and follow-up sera respectively. Alpha-1 antichymotrypsin and alpha-1 antitrypsin but not alpha-2 macroglobulin showed direct correlation with the growth inhibition activity. When alpha-1 antitrypsin was deliberately added to the malarial culture no growth inhibition occurred indicating that the alpha-1 antichymotrypsin was the most likely factor responsible for inhibition of growth of malarial parasites in vitro.

Topics: alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; alpha-Macroglobulins; Chymotrypsin; Growth Inhibitors; Humans; Malaria; Plasmodium falciparum; Protease Inhibitors

The serum proteins supposed to be indicative of the nutritional status, albumin, prealbumin and transferrin, as well as the serum proteinase inhibitors alpha 1-protease inhibitor (alpha 1-antichymotrypsin (Ach) and alpha 2-macroglobulin (alpha 2M) were measured in 14 Thai males suffering from uncomplicated falciparum malaria on the day of admission and after treatment with mefloquin on the 2nd, 28th and 63rd day. The same serum proteins had been determined from 31 healthy Thai males. Upon admission albumin and prealbumin concentrations had been lower and Ach higher in malaria patients compared with healthy Thai males. A significantly higher alpha 1 PI value was observed on the day of admission compared with the 28th day of the malaria patients. Only on the day of admission and only for the patients was a statistically significant negative linear regression found for albumin and prealbumin with Ach and a positive correlation for prealbumin with alpha 2 M as well as for albumin and transferrin correlated with alpha 1 PI. In well-nourished malaria patients the synthesis of the "acute phase reactants", alpha 1 PI and Ach, might be enhanced and in a reverse relationship the synthesis of albumin, pre-albumin and transferrin depressed.

Topics: alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; alpha-Macroglobulins; Animals; Blood Proteins; Chymotrypsin; Humans; Malaria; Male; Nutrition Disorders; Nutritional Physiological Phenomena; Plasmodium falciparum; Protease Inhibitors; Serum Albumin; Transferrin

Invasion of erythrocytes by malaria parasites is known to be blocked by proteolytic digestion of merozoite receptors allegedly present in red cell membranes. This information was used in the present work to develop a simple and convenient assay for parasite invasion into red blood cells and for evaluating the role played by red cell membrane components in this process. Synchronized in vitro cultures of Plasmodium falciparum containing only ring stages were subjected to either trypsin or pronase digestion, a treatment that neither affected ring development into schizonts nor mature merozoite release. Cells from this culture were not invaded by the released merozoites. However, upon addition of untreated human red blood cells, marked invasion was observed, either microscopically or as [3H]isoleucine incorporation. The new assay circumvents the need for separating schizonts from uninfected cells and provides a convenient means for assessing how chemical and biochemical manipulation of red blood cells affects their invasiveness by parasites. Using this assay, we verified that sheep and rabbit erythrocytes were resistant to invasion, as were human erythrocytes which had been treated with trypsin, pronase or neuraminidase. Chymotrypsin digestion of human erythrocytes was without effect on invasion. Human erythrocytes which were chemically modified with the impermeant amino reactive reagent H2DIDS, or with the crosslinker of spectrin, TCEA, were found to resist invasion. The results underscore the involvement of surface membrane components as well as of elements of the cytoskeleton in the process of parasite invasion into erythrocytes.

Topics: Animals; Chymotrypsin; Erythrocyte Membrane; Erythrocytes; Humans; Malaria; Neuraminidase; Plasmodium falciparum; Rabbits; Sheep; Sialic Acids; Species Specificity; Trypsin

The importance of the red cell membrane sialoglycoproteins in the invasion of P. falciparum merozoites has been assessed. Human erythrocytes deficient in glycophorin A (En(a-)cells) or B (S-s-U-, S-s-U+ cells) showed significant resistance to invasion. Treatment of normal erythrocytes with trypsin and chymotrypsin also reduced invasion. These results indicate that determinants carried on glycophorins A, B and C play an essential role in the successful invasion into human red cells. Sugar components present on glycophorin, in particular N-acetyl glucosamine and N-acetyl galactosamine, as shown by specific sugar and antibody inhibition studies, appear to act as important determinants for attachment to the erythrocyte. This implicates a protein(s) on the merozoite surface membrane which has the properties of a lectin.

Topics: Chymotrypsin; Erythrocytes; Glycophorins; Humans; Malaria; Plasmodium falciparum; Sialoglycoproteins; Trypsin

Topics: Animals; Bone Marrow; Chymotrypsin; Liver; Malaria; Male; Mice; Mononuclear Phagocyte System; Plasmodium berghei; Spleen

Duffy blood group negative human erythrocytes (FyFy) are resistant to infection by Plasmodium knowlesi, a simian malaria that infects Duffy positive human erythrocytes. The P. knowlesi resistance factor, Duffy negative erythrocytes, occurs in high frequency in West Africa, where the people are resistant to vivax malaria. This suggests that Duffy blood group determinants (Fya or Fyb) may be erythrocyte receptors for P. vivax.

Topics: Binding Sites; Blood Group Antigens; Chymotrypsin; Erythrocytes; Genotype; Homozygote; Humans; Malaria; Plasmodium; Pronase; Videotape Recording

Topics: Animals; Antigens; Autoradiography; Bicarbonates; Blood; Carbon Radioisotopes; Chymotrypsin; Culture Media; Deoxyribonucleases; Freeze Drying; Glucose; Haplorhini; Humans; Immunodiffusion; Isoleucine; Malaria; Methionine; Muramidase; Oxidation-Reduction; Papain; Pepsin A; Plasmodium falciparum; Pronase; Protein Hydrolysates; Ribonucleases; Solubility; Tritium; Trypsin

Chymotrypsin- and Pronase-treated human erythrocytes were refractory to invasion by P. knowlesi merozoites; invasion was not inhibited by trypsin or neurammidase treatment. These data implicate a surface protein other than sialoglycoprotein as the receptor site for merozoites. Invasion of rhesus erythrocytes was unaffected by pretreatment with these enzymes. Differences in membrane structure of erythrocytes from various species may explain the absence of an enzyme effect on rhesus erythrocytes.

Topics: Animals; Binding Sites; Cell Membrane; Chymotrypsin; Erythrocytes; Haplorhini; Macaca; Malaria; Neuraminidase; Plasmodium; Pronase; Trypsin

Topics: Animals; Bradykinin; Chymotrypsin; Guinea Pigs; Haplorhini; Malaria; Rats; Trypsin