Disruption of Plasmodium falciparum histidine-rich protein 2 may affect haem metabolism in the blood stage

Abstract

Background Haem 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. Methods The 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. Results Pfhrp2- 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). Conclusion Our 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.

Failure of rapid diagnostic tests in Plasmodium falciparum malaria cases among travelers to the UK and Ireland: Identification and characterisation of the parasites

“…It is not clear whether poor growth was due to a loss of fitness associated with the deletion of pfhrp2 / 3 , the delay in establishing the culture, or any other technical factors. Disruption of pfhrp2 in the 3D7 laboratory line has been reported to have no effect on parasite growth ( Yang et al, 2020 ); however, the pfhrp2 locus alone was disrupted by gene replacement in this study, and so any fitness impact due to loss of flanking genes was not examined. The exact biological function of HRP2 is still unclear, but it has been linked with hemozoin formation in several studies ( Schneider and Marletta, 2005 , Sullivan et al, 1996 ).…”

“…The exact biological function of HRP2 is still unclear, but it has been linked with hemozoin formation in several studies ( Schneider and Marletta, 2005 , Sullivan et al, 1996 ). The pfhrp2 knock-out 3D7 parasite line has been shown to transcriptionally downregulate enzymes required for heme metabolism in the food vacuole, but to upregulate transcripts for proteins required for heme biosynthesis in the apicoplast, potentially maintaining a relatively unchanged heme level in the parasite ( Yang et al, 2020 ). This might indicate that pfhrp2 deletion can impact parasite survival in the mosquito and liver stages, as de novo heme biosynthesis in the apicoplast has been reported to be essential in the mosquito ( Ke et al, 2014 ) and liver stages ( Nagaraj et al, 2013 ).…”

Fitness Costs ofpfhrp2andpfhrp3Deletions Underlying Diagnostic Evasion in Malaria Parasites

“…Strangely, while these genes are among the most highly expressed in the blood stage malaria parasites, we currently know rather little about their function (1). Some results suggest that HRP2 is involved in heme metabolism (21,22): the HRP2 protein has a heme binding site (23), and deletions of pfhrp2 results in significant under or over expression of several genes involved in hemozoin conversion (24). This feature of pfhrp2 biology seems most likely to impact fitness in pfhrp2 deleted parasites.…”

Fitness costs of pfhrp2 and pfhrp3 deletions underlying diagnostic evasion in malaria parasites

“…We used a chloroquine sensitive African parasite for these experiments, although in South American and African countries pfhrp2 /3 deletions have arisen in CQ-resistant parasites populations. There is some evidence that CQ-resistance and pfhrp2 may be functionally linked (1), because Pfhrp2 has a heme binding domain (23) and is thought to mediate conversion of heme to hemozoin in the vacuole (24). CQ resistant parasites have modified heme metabolism pathways (30), altered food vacuole pH (31), and HRP-2 mediated hemozoin conversion is pH-dependent (21).…”