RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
MutatedGene model (rodent): PBANKA_1409100; Gene model (P.falciparum): PF3D7_1310600; Gene product: secretory complex protein 61 alpha | ras-related protein Rab-5B (RAB5b; Sec61-alpha; Rab GTPase 5b)
Details mutation: The endogenous Rab5b gene tagged with monomeric Azami Green
PhenotypeNo phenotype has been described
Last modified: 21 June 2016, 12:56
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 27316546
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/ResearcherEbine K; Saito-Nakano Y
Name Group/DepartmentDepartment of Parasitology
Name InstituteNational Institute of Infectious Diseases
Name of the mutant parasite
RMgm numberRMgm-1460
Principal namePbRab5b-mAG
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses Rab5B which is C-terminal tagged with monomeric Azami Green.

Protein (function)
P. falciparum (Pf) has a family of 11 Rab GTPases to regulate its vesicular transport.
This family contains three Rab5 isoforms with PfRab5A (PF3D7_0211200) belonging to a restricted orthology group (OG4_36791) present only in Apicomplexa parasites known to invade erythrocytes (Plasmodia, Babesia and Theileria). However, PfRab5A has an insertion of 30 amino acids between the RabF1 and RabF2 motifs (Rab-effector binding motifs) that is not observed in Rab5A of Theileria and Babesia, suggesting that some PfRab5A effectors might be Plasmodium-specific. Putative PfRab5A-effectors might be involved in the uptake of haemoglobin, as GFP-tagged PfRab5A decorates vesicles containing haemoglobin. The PfRab5B orthology group (OG4_18709) is found more broadly; the C-terminus of PfRab5B (PF3D7_1310600) has no lipid-modification motif, like human Rab8 and Rab23 and ARA6. The PfRab5C (PF3D7_0106800) orthology group (OG4_10168) is the largest. PfRab5C looks like a classical Rab5, but comparison of a 3D-model of PfRab5C with the known 3D-structure of mouse Rab5C showed that these two Rab5s present different amino acids at their effector-interaction surfaces, implying that PfRab5C has the potential to recruit parasite-specific effectors.

PfRab5B is unique in lacking a C-terminal geranyl-geranylation motif, while having N-terminal palmitoylation and myristoylation motifs.

See additional Information. Attempts to delete the Rab5b were unsuccessful indicating an essential function for blood stages (see RMgm-822).

Additional information
In this study the endogenous PbRab5b locus was replaced with the following Rab5 homologues: PbRab5b fused to monomeric Azami Green (RMgm-1460); Rab5b from P. falciparum (PfRab5b, PF3D7_1310600)(RMgm-1461), It was also attempted to replace it with Rab5b from the distantly-related apicomplexan parasite Toxoplasma gondii (TgRab5b, TGME49_207460)(RMgm-1462), and the other conventional Rab5 isoforms of P. berghei, PbRab5a, PBANKA_030800 (RMgm-1463) and PbRab5c PBANKA_020650 (RMgm-1464). Replacement with PfRab5b was successful; Replacement with TgRab5b, PbRab5a and PbRab5c was not successful.

First, the genomic locus encoding PbRab5b was replaced with a fragment encoding PbRab5b fused to monomeric Azami Green (mAG). This transgenic parasite grew as well as the wild type, indicating that the PbRab5b-mAG fusion protein was functional.

The PbRab5b genomic locus was also successfully replaced with PfRab5b fused to mAG (PfRab5b-mAG), indicating that PfRab5b complemented the function of PbRab5b. This result is supported by the presence of a completely conserved effector sequence (HQVTIGAAFL) between PbRab5b [amino acid residues (aa) 60–69] and PfRab5b (aa 63–72) that specifies the function of Rab proteins.

In contrast, P. berghei-transfected with TgRab5b-mAG, which has a similar effector sequence (aa 66–75, HEVTIGAAFL, with the underline indicating different residue) in addition to a characteristic insertion sequence (aa 165–178), did not functionally complement PbRab5b. These results suggest that the molecular function of Rab5b in P. berghei is conserved with P. falciparum, but not with Toxoplasma gondii. After drug selection, parasites transfected with plasmids replacing PbRab5b with conventional Rab5 isoforms, PbRab5a and PbRab5c, were not obtained, suggesting Rab5a and Rab5c were unable to complement endogenous PbRab5b.

To identify the functional regions of PbRab5b, a panel of chimeric constructs, which consisted of PbRab5b with replacements of equivalent regions of PbRab5a or PbRab5c, was expressed. The following chimeric constructs did not complement endogenous PbRab5b; aa 1–34, 1–69, or 1–92 in PbRab5b-mAG proteins were replaced with equivalent regions from PbRab5a, PbRab5b–5a #1, #2, and #3, respectively), and aa 1–69 in PbRab5b-mAG was replaced with corresponding sites of PbRab5c. The only chimera that complemented the PbRab5b locus was a construct that included the entire GTPase motif of Rab5b (aa 1–192, PbRab5b–5a #4). These results indicate that PbRab5b functions differently from PbRab5a or PbRab5c, and that the GTPase activity is required for a proper Rab5b function.

Other mutants
see the link PF3D7_1310600

  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1409100
Gene Model P. falciparum ortholog PF3D7_1310600
Gene productsecretory complex protein 61 alpha | ras-related protein Rab-5B
Gene product: Alternative nameRAB5b; Sec61-alpha; Rab GTPase 5b
Details of the genetic modification
Short description of the mutationThe endogenous Rab5b gene tagged with monomeric Azami Green
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
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
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationpL0006 was obtained from MR4 and was used to make a panel of transfection constructs. To replace the genomic sequence encoding PbRab5b, 500 bp upstream and downstream of the PbRab5b locus were cloned into HindIII–PstI and XhoI–EcoRI sites of pL0006, respectively (PbRab5b-KO plasmid). A series of open reading frames (ORFs) comprising the sequences encoding PbRab5b, mAG (monomeric Azami Green), and the terminator region of P. berghei dihydrofolate reductase (PbDT) were PCR-amplified with overlapping oligonucleotides and were then inserted into the PstI site of the PbRab5b-KO plasmid using an In Fusion HD cloning kit (Clontech, USA) to yield PbRab5b-mAG plasmid. For complementation analysis, each gene-of-interest (GOI) was PCR-amplified, and an In Fusion HD cloning kit was used to replace the PbRab5b ORF in the PbRab5b-mAG plasmid.
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