Summary

RMgm-183
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1126500; Gene model (P.falciparum): PF3D7_0626300; Gene product: 3-oxoacyl-acyl-carrier protein synthase I/II (FABB/F)
Phenotype Sporozoite; Liver stage;
Last modified: 12 March 2009, 18:35
  *RMgm-183
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) : 19068099
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line17XNL is a non-lethal strain of P. yoelii
The mutant parasite was generated by
Name PI/ResearcherA.M. Vaughan; S.H.I. Kappe
Name Group/DepartmentNot applicable
Name InstituteSeattle Biomedical Research Institute
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-183
Principal namefabb/f-
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
SporozoiteNormal numbers of salivary gland sporozoites are produced. Sporozoites are not infectious to mice as shown by the absence of blood infections after inoculation of 50.000 sporozoites.
Liver stageNormal numbers of salivary gland sporozoites are produced. Sporozoites are not infectious to mice as shown by the absence of blood infections after inoculation of 50.000 sporozoites. At 12 and 24 h after injection of sporozoites liver stages showed normal development that was indistinguishable from wild type, i.e. they invaded hepatocytes, formed a parasitophorous vacuole (PV), transformed into trophozoites and initiated schizogony. However, by 44 h the size of the liver stages was significantly less than that of wild type. In addition, abnormal progression of nuclear division became apparent. No expression of MSP1 was detected. No mature merozoites are formed and released, resulting in the absence of blood infection.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of FABB/F.

Protein (function)
FABB/F is an enzyme of the bacterial like type II fatty acid biosynthesis (FAS-II) pathway. In Plasmodium FAS-II enzymes have been localized to the apicoplast, a nonphotosynthetic plastid organelle of cyanobacterial origin.
FAS-II requires acetyl-Coenzyme A (CoA), which can be converted from pyruvate by the pyruvate dehydrogenase complex. Acetyl-CoA carboxylase converts acetyl-CoA to malonyl-CoA, which is tethered to an acyl carrier protein (ACP) by malonyl-CoA:ACP transacylase (FabD). This produces malonyl-ACP, which, in conjunction with acetyl-CoA, is acted upon by β-ketoacyl-ACP synthase III (Fab H) to form β-ketoacyl-ACP. This precursor enters the FAS-II elongation cycle, mediated by FabB/F (β-ketoacyl-acyl-carrier-protein (ACP) synthase), FabG (β-ketoacyl-ACP reductase), FabZ/A (β-hydroxyacyl-ACP dehydratase), and FabI (trans-2-enoyl-ACP reductase). These four FAS-II enzymes iteratively catalyze the addition of two carbon chains to a growing fatty acyl carbon chain via condensation, reduction, dehydration, and reduction steps, respectively.

Phenotype
The phenotype analyses show that FABB/F is not essential for blood stage development, mosquito stage development and initial infection of the liver. The results indicate an essential role in the formation of infective liver stage merozoites demonstrating the importance of FASII pathway for synthesis of fatty acids during late liver stage development.

Additional information

Other mutants
RMgm-180: A mutant expressing myc tagged FABI
RMgm-181: A mutant expressing myc tagged FABZ
RMgm-182: A mutant expressing myc tagged FABG
RMgm-184: A mutant lacking expression of FABz
RMgm-197: A mutant lacking expression of FABI


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1126500
Gene Model P. falciparum ortholog PF3D7_0626300
Gene product3-oxoacyl-acyl-carrier protein synthase I/II
Gene product: Alternative nameFABB/F
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid KpnI/SacII
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
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 1ATggtaccCATTTTAAACAATCAAATGATATAAG
Additional information primer 15’UTR forward primer (KpnI)
Sequence Primer 2ATATaagcttAAGAATATTTTTAAGGGCCATTTC
Additional information primer 25’UTR reverse primer (HindIII)
Sequence Primer 3ATgcggccgcAAGTGCATGTGCAACATCAGG
Additional information primer 33’UTR forward primer (NotI)
Sequence Primer 4ATATccgcggATCAACTAATAAATGATAATATCAAC
Additional information primer 43’UTR reverse primer (SacII)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6