Toxoplasma gondii infection in pregnant women may result in abortion and foetal abnormalities, and may be life-threatening in immunocompromised hosts. To identify the potential infection markers of this disease, 2-DE and Western blot methods were employed to study the parasite circulating antigens and host-specific proteins in the sera of T. gondii-infected individuals. The comparisons were made between serum protein profiles of infected (n=31) and normal (n=10) subjects. Antigenic proteins were identified by immunoblotting using pooled sera and monoclonal anti-human IgM-HRP. Selected protein spots were characterised using mass spectrometry. Prominent differences were observed when serum samples of T. gondii-infected individuals and normal controls were compared. A significant up-regulation of host-specific proteins, α(2)-HS glycoprotein and α(1)-B glycoprotein, was also observed in the silver-stained gels of both active and chronic infections. However, only α(2)-HS glycoprotein and α(1)-B glycoprotein in the active infection showed immunoreactivity in Western blots. In addition, three spots of T. gondii proteins were detected, namely (i) hypothetical protein chrXII: 3984434-3 TGME 49, (ii) dual specificity protein phosphatase, catalytic domain TGME 49 and (iii) NADPH-cytochrome p450 reductase TGME 49. Thus, 2-DE approach followed by Western blotting has enabled the identification of five potential infection markers for the diagnosis of toxoplasmosis: three are parasite-specific proteins and two are host-specific proteins.
Plasmodium knowlesi contributes to the majority of human malaria incidences in Malaysia. Its uncontrollable passage among the natural monkey hosts can potentially lead to zoonotic outbreaks. The merozoite of this parasite invades host erythrocytes through interaction between its erythrocyte-binding proteins (EBPs) and their respective receptor on the erythrocytes. The regionII of P. knowlesi EBP, P. knowlesi beta (PkβII) protein is found to be mediating merozoite invasion into monkey erythrocytes by interacting with sialic acid receptors. Hence, the objective of this study was to investigate the genetic diversity, natural selection and haplotype grouping of PkβII of P. knowlesi isolates in Malaysia. Polymerase chain reaction amplifications of PkβII were performed on archived blood samples from Malaysia and 64 PkβII sequences were obtained. Sequence analysis revealed length polymorphism, and its amino acids at critical residues indicate the ability of PkβII to mediate P. knowlesi invasion into monkey erythrocytes. Low genetic diversity (π = 0.007) was observed in the PkβII of Malaysia Borneo compared to Peninsular Malaysia (π = 0.015). The PkβII was found to be under strong purifying selection to retain infectivity in monkeys and it plays a limited role in the zoonotic potential of P. knowlesi. Its haplotypes could be clustered into Peninsular Malaysia and Malaysia Borneo groups, indicating the existence of two distinct P. knowlesi parasites in Malaysia as reported in an earlier study.
This study was performed to identify circulating Plasmodium falciparum proteins in patient serum, which may be useful as diagnostic markers. Depletion of highly abundant proteins from each pooled serum sample obtained from P. falciparum-infected patients and healthy individuals was performed using the Proteoseek Antibody-Based Albumin/IgG Removal Kit (Thermo Scientific, Rockford, IL). In analysis 1, the depleted serum was analyzed directly by NanoLC-MS/MS. In analysis 2, the depleted serum was separated by two-dimensional electrophoresis followed by western blot analysis. Subsequently, the selected band was analyzed by NanoLC-MS/MS. The result of analysis 1 revealed the presence of two mature erythrocyte surface antigen (MESA) proteins and chloroquine resistance transporter protein (PfCRT). In addition, analysis 2 revealed an antigenic 75-kDa band when the membrane was probed with purified IgG from the pooled serum obtained from P. falciparum-infected patients. MS/MS analysis of this protein band revealed fragments of P. falciparum MESA proteins. Thus, in this study, two different analyses revealed the presence of Plasmodium MESA protein in pooled serum from malaria patients; thus, this protein should be further investigated to determine its usefulness as a diagnostic marker.
Plasmodium knowlesi is a simian malaria parasite that has been identified to cause malaria in humans. To date, several thousand cases of human knowlesi malaria have been reported around Southeast Asia. Thus far, there is no detailed study on genetic diversity and natural selection of P. knowlesi circumsporozoite protein (CSP), a prominent surface antigen on the sporozoite of the parasite. In the present study, the genetic diversity and natural selection acting on the nonrepeat regions of the gene encoding P. knowlesi CSP were investigated, focusing on the T-cell epitope regions at the C-terminal of the protein.