Disruption of Plasmodium Falciparum Histidine-Rich Protein II can affect Heme Metabolism in the Blood Stage

Background. Heme is a key metabolic factor in the life of malaria parasite. In the blood stage it acquires host hemoglobin to generate amino acids for its own protein synthesis and by-product heme for metabolic use. The malaria parasite also can de novo synthesize heme by itself. Plasmodium falciparum-specic histidine-rich protein 2 (PfHRP2) is a histidine-rich protein and has a heme-binding site to mediate hemozoin formation, a bio-crystallized form of heme-aggregates. It is interesting to investigate the vibration of hemoglobin-derived heme metabolism and de novo heme-biosynthetic pathway in the Pfhrp2 disrupted parasites during the intraerythrocytic stages. Methods. A CRISPR-Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasites and polymerase chain reaction (PCR), western blotting and southern blotting were used to manifest the successful establishment of transgenic parasites. RNA-seq and comparative transcriptome analysis were performed to identify the difference in gene expression between 3D7 and Pfhrp2 - 3D7 parasites. Results. Pfhrp2 - transgenic parasites were successfully established by the CRISPR/Cas9 system. The disruption of the exon 2 of Pfhrp2 can down-regulate the gene expression of Pfhrp3 which involved in hemoglobin-derived heme metabolism. It also up-regulates the gene expression level of enzymes of heme biosynthesis. Conclusion: These data support that although Pfhrp2 is a dispensable gene for intraerythrocytic stages parasite but heme metabolism’s stabilizing is very important. The disruption of Pfhrp2 can both affect the pathway of heme metabolism and biosynthesis. A co-operation mechanism may exist between the heme biosynthesis and metabolism pathways for parasite growth in blood stage. The pUF1-BSD-Cas9 plasmid was constructed from the pUF1-Cas9 plasmid. It offered Cas9 endonuclease and blasticidin S deaminase (BSD) by changing the drug selection marker from yDHODH into BSD. The plasmid which provide donor DNAs and sgRNA were constructed from the pL6cs plasmid by two steps. First, gene-specic DNA cassettes expressing sgRNAs were chosen from gene sequences and inserted into pL6cs vector. Secondly, two ~ 550 bp genomic DNA sequences from the upstream and downstream of the Pfhrp2 gene were chosen as the left and the right homologous arms. The construct was screened with enzyme digestion and DNA sequencing to ensure its correct. After that, P. falciparum 3D7 strain was subsequently transfected with 50 µg pL6cs-hDHFR-hrp2 (donor DNA) and 50 µg pUF1-BSD-Cas9 plasmids via electroporation. To select the successfully transfected parasite, BSD and WR99210 were added to the culture medium one day after electroporation.


Introduction
Malaria remains one of the most signi cant health challenges for human beings. Plasmodium falciparum, as one of the most deadly human malaria diseases, is the most burdensome form and causes about 228 million cases of malaria and result in 405 000 deaths in 2018 worldwide [1] .
Heme is a crucial metabolic factor and it derives primarily from the parasite's heme biosynthesis pathway [2] at the early ring stage and from hemoglobin digestion at the latter stages [3] . Malaria parasite ingests more than 75% of its host cell hemoglobin in a short period for its nutritional requirements in the blood stage [3,4] . It was digested in the food vacuole to generate amino acids, releasing the toxic heme moiety [5] . Since heme is toxic, it stores the excess heme as hemozoin pigment, a bio-crystallized form of hemeaggregates [6] .
Plasmodium falciparum-speci c histidine-rich protein 2 (PfHRP2, PlasmoDB: PF3D7_0831800, www.plasmodb.org) constitutes two exons and it is a water-soluble protein and was released from infected erythrocytes and circulated in the malaria-infected patient [7,8] . HRPII and HRP III as a homologous protein could bind heme in the digestive vacuole and play a role in hemozoin formation [9][10][11] . Antimalarial drugs chloroquine, it binds to toxic heme metabolites and thereby prevents their conversion and deposition to the inert hemozoin [12] . However, the malaria parasite also has a hemebiosynthetic pathway. Studies with Plasmodium berghei-infected mice and Plasmodium falciparum in cultures using knockout (KO) parasites generated for δ-aminolevulinate synthease (ALAS) and ferrochelatase (FC) have indicated that the heme biosynthetic pathway is nonessential for parasite survival in the blood stages [13,14] .
The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has been successfully using in genome-editing for human malaria parasite Pf and Pv [15][16][17] . Brie y, a single guide RNA (sgRNA) guides the Cas9 endonuclease to cause double-strand breaks (DSBs), and DSBs can be repaired by homologous recombination using donor DNA. Transgenic parasites can be obtained after 3-6 weeks [15,16] . It has exhibited as highly precise and e cient in genome editing.
The present study aims to speci c disruption of the gene locus of Pfhrp2 from wild type 3D7 parasites and investigates how explicit gene disruption affects the hemoglobin-derived heme metabolism and heme-biosynthetic pathway.

Methods
Parasite culture, Synchronization, and Pellets collection. P. falciparum asexual stages (3D7 strain) were cultured in vitro in human erythrocytes (blood group O+) obtained from the Beijing Red Cross Blood Center. It was grown under 5% O 2 , and 5% CO 2 in RPMI-1640 media supplemented with 5 g/L Albumax II (Life Technologies), 2 g/L sodium bicarbonate, 25 mM HEPES pH7.4 (pH adjusted with potassium hydroxide), 1 mM hypoxanthine and 50 mg/L gentamicin as previously described [18] .
For synchronization, parasites were cultured to at least 10% parasitemia in T-75 asks containing 50 ml medium at 1% hematocrit. Then it was moved from a ask to a 50 mL tube, centrifuged for 5 min at 500x g and removed supernatant. 15 mL 5% D-Sorbitol solution was added to the pellet and incubated at 37℃ for 10 min, centrifuged, and removed supernatant. The culture was synchronized with three rounds of sorbitol treatments. Then after the invasion at 8 h, 16 h, 24 h, 32 h, 40 h, 46/0 h collected and centrifuged culture solution, pellets were stored at -80℃ until to use.
Brie y, pUF1-BSD-Cas9 expresses Cas9 nuclease and blasticidin S deaminase (BSD). The pL6CS-hDHFR-hrp2 plasmid, which offers donor DNAs and sgRNAs, targeting the Pfhrp2 gene (guide hrp2 ). The left and right homologous arms were ampli ed separately by PCR from the genomic DNA of P. falciparum 3D7 (primers P1/P2 for the left arm and P3/P4 for right arm). The construct of transitional-pL6CS-hDHFR-hrp2 was transformed into competent cells, and plasmids were extracted and checked with restriction enzyme and sequencing. After the correct transitional-pL6CS-hDHFR-hrp2 was obtained, this transitional plasmid was linearized with AvrII & XhoI. The sgRNAs of hrp2 were annealed and inserted into linearized transitional-pL6CS-hDHFR-hrp2 plasmids using the in-fusion kit. The construct was transformed into competent cells again and extracted. The nal plasmid was con rmed by restriction enzyme digestion and DNA sequencing. The con rmed plasmid was isolated and used for electroporation to generate P. falciparum transgenic strains.
At the electroporation step, the two plasmids were carried out by the spontaneous uptake method using ~ 50 µg of maxi-prepped plasmid DNA, and 8 square wave electroporation pulses of 356 V for 1 ms each, separated by 0.1 seconds. Drugs ( nal concentration is 2.5 mg/L blasticidin S and 250 mg/L G418) were added into complete medium post-transfection to kill those parasites without episomal pUF1-BSD-Cas9 and pL6CS-hDHFR-hrp2. All primers and sgRNA sequences used for constructing plasmids can be seen in Additional le 1. Con rmation of transgene success via PCR. Twenty days after electroporation, live P. falciparum appeared, and genomic DNA was extracted from harvest parasite pellets using the Qiagen DNA extraction kit (QIAGEN, Valencia, California USA). A PCR was performed in 20 µl total volume consisting of 10 × buffer with 15 nM MgCl2, 200 µM dNTPs, 15 µM forward and reverse primers (P1/P2, as indicated in Fig. 2), 0.69 units of Taq DNA Polymerase, and 2 µl of DNA template. An in vitro cultured P. falciparum parasite 3D7 was used as a positive control for Pfhrp2 gene ampli cation experiments. All PCR products were separated and visualized on agarose gels, and products with the expected size were sent for sequencing to con rm.
Western blotting analysis. Successfully transgenic parasites were cultured in asks. For the analysis of Pfhrp2 expression, when parasitemia passed 5%, iRBCs were collected and incubated with 0.15% saponin lysis solution on ice for 7 min. After centrifugation at 500 × g for 5 min at room temperature (RT), the supernatants was collected and added an appropriate amount of SDS-PAGE sample buffer, and denatured at 95℃ for 8 min, and resolved by electrophoresis in a 12.5% polyacrylamide gel (Life Technologies) and transferred onto a 0.2-um polyvinylidene di uoride (PVDF) membrane (Hybond LFP; GE Healthcare). HRP2 was speci cally detected by using the Anti-Plasmodium falciparum monoclonal antibody [MPFG-55P] (HRP) (Abcam, Massachusetts, USA).
Southern Blot Analysis. The genomic DNA from transgenic parasites was isolated, as described above. For parasites, 5 µg of genomic DNA was digested overnight using the PstI or SacI restriction endonucleases (TaKaRa Bio companies). The DNAs were separated on a 1.0% agarose gel and transferred to Hybond™ -N + membrane (GE Healthcare Amersham™) using the high salt capillary transfer method. Probes were PCR-ampli ed, cleaned, and labeled with DIG-dUTP using a PCR DIG Probe Synthesis Kit (Roche). The blots were hybridized with the labeled probes, washed, and exposed to lm and detected by in a cassette.

Successful construct plasmids for gene knockdown
The pUF1-BSD-Cas9 plasmid was constructed from the pUF1-Cas9 plasmid. It offered Cas9 endonuclease and blasticidin S deaminase (BSD) by changing the drug selection marker from yDHODH into BSD. The plasmid which provide donor DNAs and sgRNA were constructed from the pL6cs plasmid by two steps. First, gene-speci c DNA cassettes expressing sgRNAs were chosen from gene sequences and inserted into pL6cs vector. Secondly, two ~ 550 bp genomic DNA sequences from the upstream and downstream of the Pfhrp2 gene were chosen as the left and the right homologous arms. The construct was screened with enzyme digestion and DNA sequencing to ensure its correct. After that, P. falciparum 3D7 strain was subsequently transfected with 50 µg pL6cs-hDHFR-hrp2 (donor DNA) and 50 µg pUF1-BSD-Cas9 plasmids via electroporation. To select the successfully transfected parasite, BSD and WR99210 were added to the culture medium one day after electroporation.
Total RNA extraction. Total RNA was extracted using TRIzol® according to the manufacturer's protocol. Brie y, different time collected pellets from transgenic parasites were ground into powder by liquid nitrogen and transferred into a new tube with Trizol reagent. The mix was shaken and kept for 5 min at room temperature, then centrifuged at 10,000 × g for 5 min at 4℃. The supernatant was added chloroform/isoamyl alcohol (24:1) with lysis reagents. After centrifuged at 10,000 × g for 10 min at 4℃, the supernatant was transferred into a new tube with an equal volume of isopropanol and kept at -20℃ for 1 h. After centrifuged at 13600 × g for 20 min at 4℃, the supernatant was precipitated by ethanol and dry for 3 min. The RNA pellet was dissolved with RNase-free water.
mRNA Library Construction. Oligo(dT)-attached magnetic beads were used to puri ed mRNA from parasite pellets. Puri ed mRNA was fragmented into small pieces with buffer at the appropriate temperature. First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by second-strand cDNA synthesis. Afterward, A-Tailing Mic and RNA Index Adapters were added by incubating to end repair. The cDNA fragments were ampli ed by PCR, and puri ed by Ampure XP Beads, then dissolved in EB solution. The double-stranded PCR products were heated to denature and circularized by the splint oligo sequence to get the nal library. The single-strand circle DNA (ssCir DNA) was formatted as the nal library. The nal library was ampli ed with phi29 to make DNA nanoball (DNB), which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray, and pair-end 100 bases reads were generated on BGISEQ500 platform (BGI-Shenzhen, China).
Primally, differential expression analysis was performed using the DESeq2(v1.4.5) [24] with Q value ≤ 0.05. Figure 1 Schematic illustration of the hrp2 gene deletion principle using CRISPR/Cas9. The hrp2 gene was replaced by hDHFR sequences through homologous recombination that happened at the left and right arms. Primers for PCR to check the hDHFR are labeled as P1 and P2.

Successful checked by PCR and DNA sequencing
Around 20 days after electroporation, live parasites could be seen in the culture under selection with the above described two drugs. A portion of the live parasite population was collected for genomic DNA isolation, and a PCR was performed to validate the modi cation of the Pfhrp2 gene. In these PCRs, two primers were designed at the genomic DNA sequences beyond the left and right homologous arms (P1/P2, Fig. 2), to prevent contamination from the episomal plasmid template. PCR products were analyzed by agarose electrophoresis and sequenced to con rm. Therefore, CRISPR/Cas9 system was successfully used to knock out the Pfhrp2 gene.

Successful checked by western blot
Transgenic parasites that had been checked by PCR and DNA sequencing were further con rmed by Western blot. Theoretical molecular weights of the proteins were 32.41KD (Fig. 3a). Western blot result showed that the molecular weight of HRP2 was not the same as its theoretical molecular weight. The band corresponding to HRP2 appeared to be slightly larger than its theoretical molecular weight, which may be caused by post-translational modi cation.

Successful checked by Southern blot
Transgenic parasites that had been checked by PCR, DNA sequencing, and western blot were further con rmed by Southern blot. Two experiments were performed to verify that the gene of Pfhrp2 was replaced by drug resistance gene hDHFR. The one experiment is to prove that wild strain 3D7 parasite still owns the gene of Pfhrp2, while transgenic parasite does not (Fig. 3b). The other experiment is to prove that the gene of Pfhrp2 was replaced by the gene of hDHFR (Fig. 3c). The theoretical molecular weights of the proteins were 39KD. Southern blot results showed that only the gene of hDHFR was detected in the transgenic parasite. Therefore, the gene of Pfhrp2 was successfully knocked out from P. falciparum 3D7 strain using CRISPR/Cas9 system. Figure 3 (a) Western blot to con rm the expression of PfHRP2 protein. The supernatants of parasite culture medium were separated on SDS-PAGE, and mouse monoclonal [MFPG-55P] to Plasmodium falciparum (HRP) was 1st antibody used for the Western Blot to con rm the HRP2 protein express. HRPgoat anti-mouse was the second antibodies. (b) Southern blot to con rm the gene disruption of Pfhrp2. The genomic DNA was digested overnight using the SacI restriction endonucleases. The DNAs were separated on agarose gel and transferred to membrane. The blots were hybridized with the labeled Pfhrp2 probe and exposure 10 min. (c) Southern blot to con rm the drug resistance gene of hDHFR. The genomic DNA was digested overnight using the PstI restriction endonucleases. The DNAs were separated on agarose gel and transferred to membrane. The blots were hybridized with the labeled hDHFR probe and exposure 40 min. L2 was the transgenic parasites which only disruption of exon 2 of Pfhrp2.

An overview of the RNA-Seq data
To test the possible role for Pfhrp2 in transcriptional regulation, we compared the global transcript levels in transgenic parasites to wild type 3D7 at six stages during the intraerythrocytic developmental cycle (IDC). Three biological replicates were used for each stage and P. falciparum species. After RNA sequencing, the quality of the data was assessed. An overview of the sequencing and assembly is shown in Supplementary Material Table 1.
After ltering the low-quality reads, clean reads were obtained. Total clean reads from the RNA-Seq data were mapped uniquely to the reference genome in all the samples. More than 97% of the total clean reads had Phred-like quality scores at the Q20 level. Via comparative transcriptome analysis, this RNA-Seq data provided a solid foundation for identifying the genes participating in heme metabolism ( Table 1).

Analyses of differentially expressed genes
The gene of enzymes participated in hemoglobin-derived heme metabolism, and the de novo hemebiosynthesis pathway was selected and analyzed. Most of their gene expression was affected by the de ciency of the gene of Pfhrp2. Transcripts of these enzymes uctuated at different time points. It is notable that the rst enzyme,δ-aminolevulinate synthease (ALAS, PlasmoDB:PF3D7_1246100), and the last enzyme, ferrochelatase (FC, PlasmoDB:PF3D7_1364900), in the heme-biosynthetic pathway were upregulated in the trophozoite and schizont stages. Most of the proteins that participated in hemoglobinderived heme metabolism were up-regulated in the ring and trophozoite stages. The gene expression of Pfhrp3 (PlasmoDB:PF3D7_1372200) was down-regulated in all the time points (Fig. 4). Figure 4 The transcription pattern of the genes involved in heme metabolism. Heat map representation of the relative transcription activity of 21 genes at six points during the asexual erythrocytic growth. Horizontal axis is the expression level of each gene and calculated by Log2(FPKM + 1). Its value from − 4 (lowest, blue) to + 4 (highest, red).

Discussion
The traditional method to edit P. falciparum genes is very ine cient and it always required several months to knock in/out target genes. It greatly limits molecular studies in malaria parasite. Recently, CRISPR/Cas9 has been used for gene editing in various organisms including Plasmodium [15,16,25] . The Cas9 endonuclease is guided to target DNA site by a sgRNA and induces DSBs at this site. Then the induced DSBs is repaired by homologous recombination using donor DNAs.
Our CRISPR/Cas9 system contains homologous arms (donor DNA fragments), sgRNA, and a selectable marker in one plasmid while Cas9 nuclease with selectable marker in another plasmid. Twenty days after electroporation, speci c gene disruption parasites appeared. In this study, we successfully applied this CRISPR/Cas9-based genome editing system to disrupt the gene of Pfhrp2 from P. falciparum 3D7.
The growth of the parasite in red cells seems not be affected by the deletion of Pfhrp2 (data not shown). Furthermore, multiple genetic origins of histidine-rich protein 2 gene deletion in P. falciparum parasites were previously found in South America, Asia, and Africa [26][27][28][29] . These all proved that Pfhrp2 is not an essential gene for the survival of parasite during intraerythrocytic stages.
Malaria parasite owns hemoglobin-derived heme metabolism and de novo heme-biosynthesis pathway. During the intraerythrocytic stages, parasite ingests host cell hemoglobin within the food vacuole to supply amino acids for its growth and release the toxic heme. The released by-product heme could bind with HRPII and HRP III to become hemozoin [12] . Plasmodium Heme Detoxi cation Protein (HDP) has also been proved the extremely potent in converting heme into hemozoin [30] . According to the RNA-Seq data, with disruption of Pfhrp2 the transcript level of Pfhrp3 was also completely down-regulated, the process of heme converted into hemozoin was in uenced, however the transcript level of HDP was up-regulated at the schizont stage could partially take place the role of Pfhrp2 and Pfhrp3. The vast majority of enzymes relate to hemoglobin-derived heme metabolism in P. falciparum 3D7, such as falcipain (FP), plasmepsin (PM) and falcilysin (FLN) up-regulate their gene transcript level in at least one time point to offset the impact and provide heme for the growth of parasite. So heme is de nitely needed by intraerythrocytic stage parasites.
Chloroquine was discovered and derived from quinine in 1934 [31] . It is effective against the malarial parasite during its intraerythrocytic stages. It could inhibit the HRP-mediated synthesis of hemozoin and breakdown the heme pathway that occurs within the acidic digestive vacuole of the parasite [12] .From our research data, because parasites can acquire heme from different pathway only disruption of Pfhrp2 cannot exterminate the heme metabolism completely. The comprehensive regulatory mechanism may exist in the heme metabolism and synthesis. We believe this network will be bene cial for understanding of heme acquisition and the drug resistance during intraerythrocytic stages.

Declarations
Author Contributions YY and QC designed research. YY, TT, BF, SL, and NH performed research. XM, XX, and LJ analyzed data; YY wrote the paper. All authors read and approved the nal manuscript. Figure 1 Schematic illustration of the hrp2 gene deletion principle using CRISPR/Cas9.

Figure 2
Genomic DNA PCR to con rm the gene disruption. Western blot and Southern blot to con rm the gene disruption.

Figure 4
The transcription pattern of the genes involved in heme metabolism.

Supplementary Files
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