Summary

RMgm-4678
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0810400; Gene model (P.falciparum): PF3D7_0909200; Gene product: GDP-fucose protein O-fucosyltransferase 2 (PoFUT2)
PhenotypeNo phenotype has been described
Last modified: 2 September 2019, 15:09
  *RMgm-4678
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 31334132
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/ResearcherSanz S, Dinglasan RR
Name Group/DepartmentDepartment of Molecular Microbiology and Immunology
Name InstituteJohns Hopkins Bloomberg School of Public Health
CityBaltimore
CountryUS
Name of the mutant parasite
RMgm numberRMgm-4678
Principal namePbĪ”PoFUT2
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PoFUT2

Protein (function)
Thrombospondin type 1 repeat (TSR) domains are small (50–60 amino acid residues) cysteine-knot motifs with 3 conserved disulfide bonds that play important roles in cell adhesion and motility. Plasmodium parasites express several TSR domain- containing proteins throughout the different stages of their life cycle that are critical for host cell recognition, motility, and invasion. These proteins include circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP) in the sporozoite stage and circumsporozoite and TRAP-related protein (CTRP) in the ookinete stage. TSR domains are O-fucosylated by protein O-fucosyltransferase 2 (PoFUT2). That fucose can be further elongated with a glucose residue, generating an O-linked disaccharide. This modification is important for the secretion of TSR domain- containing proteins. CSP and TRAP TSR domains are also O-fucosylated in the Plasmodium sporozoite stage. A homolog of PoFUT2 is conserved in all the Plasmodium species sequenced, and the parasite synthesizes the GDP-fucose (GDP-Fuc) precursor required for O-fucosylation. It has been shown that the P. falciparum protein O-fucosyltransferase (PoFUT2) is involved in the O-fucosylation of parasite TSR domains. 
It was reported by Lopaticki et al. (2017) that PoFUT2 genetic disruption in P. falciparum resulted in a reduction in the ability of ookinetes to traverse the mosquito midgut to form oocysts and evidence was provided that mosquitoes infected with PoFUT2 KO parasites harbored significantly  fewer sporozoites in the mosquito salivary glands compared to mosquitoes infected with  the wild type P. falciparum parental line, NF54. Finally, they assessed the infectivity of salivary gland sporozoites by analyzing their motility, cell traversal activity, and hepatocyte invasion and by carrying out co-infection experiments using wild type and mutant parasites in a humanized chimeric liver mouse model; these assays revealed an apparent lower fitness of ΔPoFUT2 parasites in completing development in the mosquito and infecting mammalian hepatocytes compared to wild type NF54. Here, we report a robust study that differs from the central results of the previous report and reveals that under laboratory conditions, that PoFUT2 is not essential for murine parasite development and transmission.

Phenotype
No phenotype was detected throughout the life cycle. Normal oocyst and sporozoite production. Normal sporozoite motility and infectivity (in vitro and in vivo).

Additional information
It was reported by Lopaticki et al. (2017) that PoFUT2 genetic disruption in P. falciparum resulted in a reduction in the ability of ookinetes to traverse the mosquito midgut to form oocysts and evidence was provided that mosquitoes infected with PoFUT2 KO parasites harbored significantly  fewer sporozoites in the mosquito salivary glands compared to mosquitoes infected with  the wild type P. falciparum parental line, NF54. Finally, they assessed the infectivity of salivary gland sporozoites by analyzing their motility, cell traversal activity, and hepatocyte invasion and by carrying out co-infection experiments using wild type and mutant parasites in a humanized chimeric liver mouse model; these assays revealed an apparent lower fitness of ΔPoFUT2 parasites in completing development in the mosquito and infecting mammalian hepatocytes compared to wild type NF54. Here, we report a robust study that differs from the central results of the previous report and reveals that under laboratory conditions, that PoFUT2 is not essential for murine parasite development and transmission.

In this study also a P. falciparum mutant that lacks expression of PoFUT2. This mutant shows normal oocyst and sporozoite production in An. gambiae

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0810400
Gene Model P. falciparum ortholog PF3D7_0909200
Gene productGDP-fucose protein O-fucosyltransferase 2
Gene product: Alternative namePoFUT2
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vectorPbGEM-283938
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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