Abstract
BackgroundMalaria parasites form intracellular membranes that separate the parasite from the internal space of erythrocytes, and membrane proteins from the parasites are exported to the host via the membrane. In our previous study, Plasmodium vivax early transcribed membrane protein (PvETRAMP) 11.2, an intracellular membrane protein that is highly expressed in blood-stage parasites, was characterized as a highly immunogenic protein in P. vivax malaria patients. However, the other PvETRAMP family proteins have not yet been investigated. In this study, PvETRAMPs were expressed and evaluated to determine their immunological profiles.MethodsThe protein structure and amino acid alignment were carried out using bioinformatics analysis software. A total of six PvETRAMP family proteins were successfully expressed and purified using a wheat germ cell free protein expression system and the purified proteins were used for protein microarray and immunization of mice. The localization of the protein was determined with serum against PvETRAMP4. IgG subclasses were assessed from the immunized mice.ResultsIn silico analysis showed that P. vivax exhibits nine genes encoding the ETRAMP family. The ETRAMP family proteins are relatively small molecules with conserved structural features. A total of 6 recombinant ETRAMP proteins were successfully expressed and purified. The serum positivity of P. vivax malaria patients and healthy individuals was evaluated using a protein microarray method. Among the PvETRAMPs, ETRAMP4 showed the highest positivity rate of 62%, comparable to that of PvETRAMP11.2, which served as the positive control, and a typical export pattern of PvETRAMP4 was observed in the P. vivax parasite. The assessment of IgG subclasses in mice immunized with PvETRAMP4 showed high levels of IgG1 and IgG2b. PvETRAMP family proteins were identified and characterized as serological markers.ConclusionsThe relatively high antibody responses to PvETRAMP4 as well as the specific IgG subclasses observed in immunized mice suggest that the ETRAMP family is immunogenic in pathogens and can be used as a protein marker and for vaccine development.
Highlights
Malaria parasites form intracellular membranes that separate the parasite from the internal space of erythrocytes, and membrane proteins from the parasites are exported to the host via the membrane
Most early transcribed membrane protein (ETRAMP) are expressed in stage-specific expression patterns during the parasite life cycle, and they mostly localize to the parasitophorous vacuole membrane (PVM), which spatially separates the parasite from the cytosol of erythrocytes in infected red blood cell (RBC) and mediates the free passage of molecules, probably through membranous pores such as the Plasmodium translocon for exported proteins (PTEX) and exported protein 1 (EXP1) [18,19,20,21,22]
Identification of PvETRAMP proteins Nine proteins in the ETRAMP family were found in P. vivax, and the proteins were named according to their orthologues in P. falciparum
Summary
Malaria parasites form intracellular membranes that separate the parasite from the internal space of erythrocytes, and membrane proteins from the parasites are exported to the host via the membrane. Plasmodium vivax early transcribed membrane protein (PvETRAMP) 11.2, an intracellular membrane protein that is highly expressed in blood-stage parasites, was characterized as a highly immunogenic protein in P. vivax malaria patients. The identification and the evaluation of antibodies raised against unknown merozoite antigens involved in parasite survival is necessary for the development of serological markers as well as a vaccine [6,7,8] For this reason, the development of omics techniques and high-throughput screening systems involving cell-free protein synthesis technology has led to the identification of numerous vaccine candidates and has extended the possibilities for investigating serological markers that induce an immune response in endemic areas of Plasmodium falciparum and P. vivax [9,10,11,12]. The P. berghei ETRAMP family member small exported protein 2 (SEP2), localizes to membranous compartments of the ookinete and is released during gliding motility in the sporozoite, indicating that the protein family is involved in blood stage and sexual stage [23]
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