alpha-chymotrypsin and Malaria--Falciparum

The malaria parasite Plasmodium falciparum utilizes multiple alternative receptor-ligand interactions for the invasion of human erythrocytes. While some P. falciparum clones make use of sialic acid (SA) residues on the surface of the human glycophorin receptors to invade the erythrocyte, others use alternative receptors independent of sialic acid residues. We hypothesized that over the years, intensified malaria control interventions and declining prevalence in The Gambia have resulted in a selection of parasites with a dominant invasion pathways and ligand expression profiles.. Blood samples were collected from 65 malaria-infected participants with uncomplicated malaria across 3 years (2015, 2016, and 2021). Genetic diversity was determined by genotyping the merozoite surface protein 2 (msp2) polymorphic gene of P. falciparum. Erythrocyte invasion phenotypes were determined using neuraminidase, trypsin, and chymotrypsin enzymes, known to cleave different receptors from the surface of the erythrocyte. Schizont-stage transcript levels were obtained for a panel of 6 P. falciparum invasion ligand genes (eba175, eba181, Rh2b, Rh4, Rh5, and clag2) using 48 successfully cultured isolates.. Though the allelic heterozygosity of msp2 repeat region decreased as expected with reduced transmission, there was an increase in infections with more than a single msp2 allelotype from 2015 to 2021. The invasion phenotypes of these isolates were mostly SA independent with a continuous increase from 2015 to 2021. Isolates from 2021 were highly inhibited by chymotrypsin treatment compared to isolates from 2015 and 2016. Higher invasion inhibition for 2021 isolates was further obtained following erythrocyte treatment with a combination of chymotrypsin and trypsin. The transcript levels of invasion ligand genes varied across years. However, levels of clag2, a rhoptry-associated protein, were higher in 2015 and 2016 isolates than in 2021 isolates, while Rh5 levels were higher in 2021 compared to other years.. Overall, these findings suggest increasing mixed infections with an increase in the use of sialic-acid independent invasion pathways by P. falciparum clinical isolates in the Western part of Gambia.

Topics: Chymotrypsin; Gambia; Humans; Ligands; Malaria, Falciparum; N-Acetylneuraminic Acid; Plasmodium falciparum; Trypsin

The study was planned to screen the marine actinobacterial extract for the protease inhibitor activity and its anti- Pf activity under in vitro and in vivo conditions. Out of 100 isolates, only 3 isolates exhibited moderate to high protease inhibitor activities on trypsin, chymotrypsin and proteinase K. Based on protease inhibitor activity 3 isolates were chosen for further studies. The potential isolate was characterized by polyphasic approach and identified as Streptomyces sp LK3 (JF710608). The lead compound was identified as peptide from Streptomyces sp LK3. The double-reciprocal plot displayed inhibition mode is non-competitive and it confirms the irreversible nature of protease inhibitor. The peptide from Streptomyces sp LK3 extract showed significant anti plasmodial activity (IC50: 25.78 µg/ml). In in vivo model, the highest level of parasitemia suppression (≈ 45%) was observed in 600 mg/kg of the peptide. These analyses revealed no significant changes were observed in the spleen and liver tissue during 8 dpi. The results confirmed up-regulation of TGF-β and down regulation of TNF-α in tissue and serum level in PbA infected peptide treated mice compared to PbA infection. The results obtained infer that the peptide possesses anti- Pf activity activity. It suggests that the extracts have novel metabolites and could be considered as a potential source for drug development.

Topics: Actinobacteria; Animals; Antimalarials; Aquatic Organisms; Base Composition; Chymotrypsin; Cluster Analysis; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Inhibitory Concentration 50; Liver; Malaria, Falciparum; Male; Mice; Models, Biological; Models, Molecular; Parasitic Sensitivity Tests; Plasmodium falciparum; Protease Inhibitors; Protein Conformation; RNA, Ribosomal, 16S; Seawater; Spleen; Trypsin

The malaria parasite, Plasmodium falciparum, invades the human erythrocyte through a complex interaction with erythrocyte receptors characterized by patterns of resistance to various enzymes. As invasion rates are influenced by blood group polymorphisms, we reasoned that the extremely rare rhesus null (Rh(null)) erythrocytes could be informative in characterizing receptors. The aim was to test whether the complete absence of the Rh complex from the cell membrane impacted on parasite invasion. Enzyme treatment patterns for four P. falciparum isolates were first characterised for normal Rh cells. Two isolates showed an enzyme treatment pattern not hitherto described, with resistance to neuraminidase, trypsin and chymotrypsin. In contrast, all isolates had enhanced invasion rates for the Rh(null) cell for all enzyme treatment regimens. The first finding suggests there is another pathway that P. falciparum can utilise to invade the host. We speculate that the Rh null cell membrane exposes a novel ligand defined as Receptor N.

Topics: Animals; Chymotrypsin; Erythrocyte Membrane; Erythrocytes; Host-Parasite Interactions; Humans; Malaria, Falciparum; Neuraminidase; Plasmodium falciparum; Receptors, Cell Surface; Rh-Hr Blood-Group System; Trypsin

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine P. falciparum merozoite surface protein-10 (MSP-10) regions specifically binding to membrane surface receptors on human erythrocytes. Three MSP-10 protein High Activity Binding Peptides (HABPs) were identified, whose binding to erythrocytes became saturable and sensitive on being treated with neuraminidase, trypsin and chymotrypsin. Some of them specifically recognised a 50 kDa erythrocyte membrane protein. Some HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by 70%, suggesting that MSP-10 protein's possible role in the invasion process probably functions by using similar mechanisms to those described for other MSP family antigens. In addition to above results, the high homology in amino-acid sequence and superimposition of both MSP-10, MSP-8 and MSP-1 EGF-like domains and HABPs 31132, 26373 and 5501 suggest that tridimensional structure could be playing an important role in the invasion process and in designing synthetic multi-stage anti-malarial vaccines.

Topics: Amino Acid Sequence; Animals; Binding Sites; Chymotrypsin; Erythrocyte Membrane; Erythrocytes; Humans; Malaria, Falciparum; Molecular Sequence Data; Neuraminidase; Peptide Fragments; Plasmodium falciparum; Protein Binding; Protozoan Proteins; Receptors, Cell Surface; Trypsin

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine Plasmodium falciparum merozoite surface protein-3 (MSP-3) FC27 strain regions that specifically bind to membrane surface receptors on human erythrocytes. Three MSP-3 protein high activity binding peptides (HABPs) were identified; their binding to erythrocytes became saturable, had nanomolar affinity constants, and became sensitive on being treated with neuraminidase and trypsin but were resistant to chymotrypsin treatment. All of them specifically recognized 45-, 55-, and 72-kDa erythrocyte membrane proteins. They all presented alpha-helix structural elements. All HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by ~55%-85%, suggesting that MSP-3 protein's role in the invasion process probably functions by using mechanisms similar to those described for other MSP family antigens.

Topics: Animals; Antigens, Protozoan; Binding Sites; Chymotrypsin; Cross-Linking Reagents; Erythrocyte Membrane; Erythrocytes; Humans; Malaria, Falciparum; Neuraminidase; Peptide Fragments; Plasmodium falciparum; Protein Binding; Protozoan Proteins; Substrate Specificity; Trypsin

The elucidation of digestive processes in the Anopheles gambiae gut leading to the utilization of the blood meal will result in a deeper understanding of the physiology of blood digestion and its impact on parasite-vector interactions. Accordingly, the identification of digestive serine proteases in A. gambiae has implications for the development of alternative strategies for the control of mosquito-borne diseases. We report here on the cDNA and genomic cloning and on the expression analysis of two closely related chymotrypsin genes, Anchym1 and Anchym2. Genomic cloning revealed that Anchym1 and Anchym2, which map on chromosomal division 25D, are clustered in tandem within 6 kb, both genes being interrupted by two short introns. After blood feeding, transcription of Anchym1 and Anchym2 is induced in the midgut epithelium, followed by secretion of the translated products into the midgut lumen where the Anchym1 and Anchym2 zymogens are activated by partial tryptic digestion. The amino-acid residues forming the substrate pocket of Anchym1 and Anchym2 suggested chymotryptic cleavage specificity. This was confirmed by mass spectrometry analysis and Edman degradation sequencing of proteolytic products generated by the recombinant, trypsin-activated Anchym1.

Topics: Amino Acid Sequence; Animals; Anopheles; Blood; Chymotrypsin; Cloning, Molecular; Digestion; DNA, Complementary; Enzyme Induction; Genes, Insect; Insect Vectors; Malaria, Falciparum; Molecular Sequence Data; Sequence Homology, Amino Acid; Substrate Specificity; Tissue Distribution