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AGIA Tag System for Ultrastructural Protein Localization Analysis in Blood-Stage Plasmodium falciparum.

ABSTRACT: Precise subcellular localization of proteins is the key to elucidating the physiological role of these molecules in malaria parasite development, understanding of pathogenesis, and protective immunity. In Plasmodium falciparum, however, detection of proteins in the blood-stage parasites is greatly hampered by the lack of versatile protein tags which can intrinsically label such molecules. Thus, in this study, to develop a novel system that can be used to evaluate subcellular localization of known and novel proteins, we assessed the application of AGIA tag, consisting of 9 amino acids (EEAAGIARP), in P. falciparum blood-stage parasites. Specifically, AGIA-tagged ring-infected erythrocyte surface antigen (RESA-AGIA) was episomally expressed in P. falciparum 3D7 strain. The RESA-AGIA protein was detected by Western blotting and immunofluorescence assay (IFA) using recombinant rabbit anti-AGIA tag monoclonal antibody (mAb) with a high signal/noise ratio. Similarly, AGIA-tagged multidrug resistance protein 1 (MDR1-AGIA), as an example of polyptic transmembrane protein, was endogenously expressed and detected by Western blotting and IFA with anti-AGIA tag mAb. Immunoelectron microscopy of the RESA-AGIA transfected merozoites revealed that mouse anti-RESA and the rabbit anti-AGIA mAb signals could definitively co-localize to the dense granules. Put together, this study demonstrates AGIA tag/anti-AGIA rabbit mAb system as a potentially useful tool for elucidating the subcellular localization of new and understudied proteins in blood-stage malaria parasites at the nanometer-level resolution.

SUBMITTER: Morita M

PROVIDER: S-EPMC8714843 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHaem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo on its own. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages.MethodsThe CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and PCR, Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-sequencing and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2--3D7 parasites.ResultsPfhrp2- transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512 and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, respectively, with 373, 520, 1499, 353, 1253 and 742 of these DEGs upregulated and 591, 741, 1639, 711, 1259 and 1036 of them downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups at six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding delta-aminolevulinic acid synthetase and ferrochelatase, both related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and those encoding haem oxygenase, stromal-processing peptidase and porphobilinogen deaminase were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value = 1).ConclusionOur data revealed changes in the transcriptome expression profile of the Pfhrp2--3D7 parasite during the intraerythrocytic stages. The findings provide insight at the gene transcript level that will facilitate further research on and development of anti-malaria drugs.

Project description:BackgroundRed blood cell (RBC) polymorphisms are suggested to influence the course of Plasmodium falciparum malaria. Whereas some variants have been found to be protective, others have been found to enhance parasite development. This study evaluated the effect of variant haemoglobin (Hb) and ABO blood groups on P. falciparum merozoite invasion, multiplication rates as well as gametocyte development.MethodsApproximately 2.5 mL of venous blood was collected from each participant. Flow cytometry was used to determine the in vitro merozoite invasion rates of NF54 parasites into the blood of 66 non-parasitaemic individuals with variant Hb genotypes (HbSS, HbSC) and blood groups (A, B, O), which were then compared with invasion into HbAA blood. The ex vivo asexual parasite multiplication and gametocyte production rates of parasites from 79 uncomplicated malaria patients with varying Hb genotypes (HbAS, HbAC and HbAA) were also estimated using microscopy.ResultsMerozoite invasion rates were significantly reduced by about 50% in RBCs containing HbSS and HbSC relative to HbAA cells. The presence of blood group O and B reduced the invasion rates of HbSS by about 50% and 60%, respectively, relative to HbSC but the presence of blood group A removed the inhibitory effect of HbSS. The initial parasite densities in uncomplicated malaria patients with Hb genotypes HbAS and HbAC cells were similar but significantly lower than those with genotype HbAA. The ex vivo parasite multiplication rate, gametocytaemia and gametocyte conversion rates followed a similar trend but did not reach statistical significance (p?>?0.05).ConclusionsParasite invasion rate into erythrocytes is dependent on both erythrocyte blood group antigen and haemoglobin genotype as blood group O and B provided protection via reduced merozoite invasion in RBCs containing HbSS relative to HbSC. Regardless of haemoglobin type, greater than 70% malaria patients had circulating ring stage parasites that differentiated into stage II gametocytes in 4 days.

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AGIA Tag System for Ultrastructural Protein Localization Analysis in Blood-Stage Plasmodium falciparum.

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