RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_0910300; Gene model (P.falciparum): PF3D7_1138400; Gene product: guanylyl cyclase alpha (GCα)
Name tag: triple-HA
Phenotype Gametocyte/Gamete;
Last modified: 18 February 2024, 18:05
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 37327340
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherKuehnel RM, Brochet M
Name Group/DepartmentDepartment of Microbiology and Molecular Medicine, Faculty of Medicine
Name InstituteUniversity of Geneva
Name of the mutant parasite
RMgm numberRMgm-5385
Principal nameGCα::3xHA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot tested
Gametocyte/GameteSee additional information
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a 3xHA-tagged version of GCα

Protein (function)
See additional information

See additional information

Additional information
Plasmodium parasites encode two transmembrane cGMP-producing guanylyl cyclases (GCs; GCα and GCβ). Calcium mobilization requires the rapid elevation of cGMP levels. We thus set out to investigate the relative roles of PDEs in the timely activation of gametocytes. We first individually knocked out each of the three genes coding for PDEα, PDEγ (RMgm-5381), and PDEδ (RMgm-5382), which were previously shown to be dispensable for asexual proliferation in erythrocytes.
Knockout (KO) clonal lines were obtained, and no defects in exflagellation were observed upon stimulation by XA and pH elevation, suggesting that these PDEs are not directly regulated by XA or pH. However, the PDEδ-KO line showed a significantly different calcium response to XA and pH at 20°C with a slower decay (and a larger AUC, 'area under the curve' =), indicating that PDEδ is involved in lowering calcium levels following initial mobilization.
All three lines showed a similar response to the calcium ionophore A23187, indicating that calcium available for mobilization was not affected.

As PDEβ was previously shown to be essential for the growth of asexual blood stages, we tagged the endogenous PDEβ with hemagglutinin (HA) epitope tag and an auxin-inducible degron (AID)(RMgm-5383) to degrade the fusion protein in the presence of auxin in a strain expressing the Tir1 protein. Western blot analysis indicated depletion of PDEβ-AID/HA upon auxin [or indole acetic acid (IAA)] treatment, and yet, PDEβ-depleted gametocytes exflagellated upon stimulation by XA and elevated pH, suggesting that PDEβ is not important to respond to XA or a rise in extracellular pH at a permissive temperature. It is however possible that lower levels of PDEβ-AID/HA may be sufficient to achieve its function in gametocytes.

As both PDEα and PDEδ were found to be active at 37°C, we wondered whether simultaneous deletion of both enzymes would lead to premature and abortive activation of gametocytes. A PDEα/δ-KO clonal line (RMgm-5384) did not show any obvious growth defect of asexual blood stages or growth defect in the formation of male and female gametocytes. However, activation of PDEα/δ-KO gametocytes with XA at 20°C did not lead to the formation of active exflagellation centers, and male gametogenesis was found to be blocked at an early stage as no DNA replication could be observed upon stimulation with XA and a rise in pH.

Together, these results demonstrate that neither PDEα nor PDEδ directly contribute to the elevation of cGMP levels in response to XA or pH. Instead, both enzymes are active  at 37°C to prevent premature activation of gametocytes in the vertebrate host. Simultaneous deletion of PDEα and PDEδ likely leadsto prolonged premature and abortive activation of gametogenesis due to sustained elevated cGMP levels at a non-permissive temperature that leads to an early block between calcium mobilization and DNA replication in microgametocytes. This emphasizes an important and unexpected role of temperature in the efficient progression of DNA replication in microgametocytes.

As synthesis of cGMP seems to be a limiting factor in the activation of gametocytes in response to XA or a rise in pH, we turned our attention to the two cGMP-producing GCs. GCβ was previously shown to be redundant for gamete formation. Analysis of a GCβ-disrupted mutant showed no calcium defect in response to XA, or PDE inhibitors in gametocytes both at 20° and 37°C. Consistent with this observation, knocking down GCα in developing gametocytes previously revealed the crucial role of this GC in mediating XA response in P. yoelii. To study the role of GCα in P. berghei gametocytes, an AID/HA tag was added to the GCα C-terminus), which imposed a fitness cost, with low exflagellation rates even in the absence of auxin. However, a 1-hour treatment with auxin of gametocytes before activation led to a further significant reduction in exflagellation and calcium mobilization upon XA, pH, or BIPPO treatments. Calcium response to the calcium ionophore A23187 was however normal, suggesting that the parasite calcium stores are not affected upon GCα-AID/HA depletion. Together, these results confirm that, in gametocytes, GCα is the main GC responsible for basal or induced cGMP synthesis in response to XA or a rise in extracellular pH.

The basal activity of GCα in P. yoelii gametocytes was recently shown to require a GCα-interacting protein named GEP1. To look for other GCα partners, we used affinity purification of 3xHA-tagged GCα (
RMgm-5385) from nonactivated gametocytes. This identified GEP1 and (PBANKA_111510; PF3D7_0515500), cell division control 50 (CDC50B; PBANKA_0617000; PF3D7_0719500) that were recently shown to interact with GCα in P. falciparum schizonts.
In addition, four peptides were detected mapping on the unique GC organizer (UGO; PBANKA_1201400), a multipass membrane protein. To confirm the interaction between UGO and GCα in P. berghei gametocytes, UGO was tagged with 3xHA (
RMgm-5386). Affinity purification of UGO3xHA recovered GCα as expected, but neither GEP1 nor CDC50B.
In both GCα-HA and UGO-HA immunoprecipitates, we additionally detected PBANKA_0306700, the predicted ortholog of the signaling linking factor (SLF), another multipass membrane protein.

To investigate the role of CDC50B in gametocytes, a CDC50B-KO clonal line was generated (RMgm-5387), and no obvious defects in exflagellation was observed, suggesting that CDC50B is dispensable for GCα activity.
No SLF-KO line could be generated (RMgm-5388), suggesting an essential role in asexual blood stages. We tagged the endogenous slf with an AID/HA epitope tag (RMgm-5389) to degrade the fusion protein in the presence of auxin in a strain expressing the Tir1 protein. Following a 1-hour auxin treatment of gametocytes, depletion of SLF-AID/HA was observed by Western blot, associated with a twofold diminution of the exflagellation rate in response to XA and a rise in pH.
Evidence is presented for a role of SLF in maintaining GCα basal activity and an an additional role of SLF in basal calcium homeostasis.

No UGO-KO line could be generated (RMgm-5390), and we therefore fused UGO to an AID/HA tag at the endogenous locus (RMgm-5391). induction of UGO-AID/HA depletion led to a 10-fold reduction in calcium mobilization upon stimulation by XA or a rise in pH. Evidence is presented that UGO is not required for GCα, PDEα, and PDEδ basal activities but is essential to up-regulate GCα activity in response to XA or pH

Other mutants

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0910300
Gene Model P. falciparum ortholog PF3D7_1138400
Gene productguanylyl cyclase alpha
Gene product: Alternative nameGCα
Details of the genetic modification
Name of the tagtriple-HA
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe gene deletion–targeting vector for pdeα was constructed using the pAB022 plasmid, which contains polylinker sites flanking an hdhfr expression cassette conferring resistance to pyrimethamine. Polymerase chain reaction (PCR) primers MB763 and MB764 were used to generate a fragment of pdeα 5′ upstream sequence from genomic DNA, which was inserted into Hind III and Pst I restriction sites upstream of the hdhfr cassette. A fragment generated with primers MB765 and MB766 from the 3′ flanking region of pdeα was then inserted downstream of the hdhfr cassette using Kpn I and Not I restriction sites. The linear targeting sequence was released using Hind III and Not I, and the construct was transfected into the ANKA line 2.34 or PDEδ-KO.

3xHA, KO, or AID/HA tagging of GCα, UGO, and PDEγ was generated using phage recombineering in Escherichia coli TSA strain with PlasmoGEM vectors (https://plasmogem.umu.se/pbgem/). For final targeting vectors not available in the PlasmoGEM repository, generation of KO and tagging constructs was performed using sequential recombineering and gateway steps as previously described. For each gene of interest (goi), the Zeocin resistance/Phe sensitivity cassette was introduced using oligonucleotides goi HA-F × goi HA-R and goi KO-F × goi KO-R for 3xHA, AID/HA tagging, and KO targeting vectors. Insertion of the gateway (GW) cassette following gateway reaction was confirmed using primer pairs GW1 × goi QCR1 and GW2 × goi QCR2. The modified library inserts were then released from the plasmid backbone using Not I. The UGO-AID/HA and GCα-AID/HA targeting vectors were transfected into the 615 parasite line, and the PDEγ-KO, UGO-3xHA, and GCα-3xHA vectors into the 2.34 line
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6