RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1468
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1416700; Gene model (P.falciparum): PF3D7_1318200; Gene product: glycerol-3-phosphate 1-O-acyltransferase (G3PAT; Pb apiG3PAT)
Phenotype Liver stage;
Last modified: 24 June 2016, 13:20
  *RMgm-1468
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) : 27324409
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherShears MJ; McFadden GI
Name Group/DepartmentUniversity of Melbourne, BioSciences
Name InstituteUniversity of Melbourne, BioSciences
CityMelbourne
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-1468
Principal namePb apiG3PAT(-)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageReduced infectivity of sporozoites as shown by infection of mice by mosquito bite or injection of purified sporozoites (prolonged prepatent period and/or reduced number of mice developing blood infections) Pb apiG3PAT (-) and wild type sporozoites did not differ in their ability to invade hepatocytes. No difference in mean parasite size was observed between Pb apiG3PAT (-) and wild type parasites at 24 hours post-infection. However, at 48 and 66 hours post-infection, Pb apiG3PAT (-) parasites were significantly smaller than wild type (with mean areas 22 % and 26 % less than the control).
Wild type parasites produced the majority of detached cells and merosomes at 66 hours post-infection, with smaller numbers of these forms detected at the 72 hours. By contrast, Pb apiG3PAT parasites were never seen to produce detached cells or merosomes at either 66 or 72 hours. Pb apiG3PAT (-) parasites had noticeably smaller apicoplasts and fewer nuclei than wild type at 48 and 66 hours. Normal MSP1 expression at 48h. At 66 hours, with MSP1 staining typically detected around individual merozoites in the wild type, but remaining patchy or indicative of only limited plasma membrane invagination in Pb apiG3PAT (-) parasites.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of G3PAT (Pb apiG3PAT)

Protein (function)
Fatty acids are a major component of the phospholipids that make up all cellular membranes and one hypothesis would be that the fatty acids are required for the enormous amount of membrane biosynthesis necessary late in liver stage development for the formation of tens of thousands of exoerythrocytic merozoites. The precursor of phospholipid biosynthesis is phosphatidic acid. Phosphatidic acid is formed in a three-step enzymatic reaction, the first step of which is the conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate by glycerol 3-phosphate dehydrogenase (G3PDH). Two fatty acid side chains are then transferred to glycerol 3-phosphate, firstly by glycerol 3-phosphate acyltransferase (G3PAT) to form lysophosphatidic acid and then lysophosphatidic acid acyltranferase (LPAAT) to form phosphatidic acid. Analysis of the Plasmodium genome has also uncovered two sets of genes for phosphatidic acid biosynthesis and one set is predicted to target to the apicoplast. In the rodent malaria parasite P. yoelii  G3PDH and G3PAT are indeed ocalized to the apicoplast only during liver stage development, where they are essential for production of viable merozoites (RMgm-977, RMgm-978).

Phenotype
Reduced infectivity of sporozoites as shown by infection of mice by mosquito bite or injection of purified sporozoites (prolonged prepatent period and/or reduced number of mice developing blood infections) Pb apiG3PAT (-) and wild type sporozoites did not differ in their ability to invade hepatocytes. No difference in mean parasite size was observed between Pb apiG3PAT (-) and wild type parasites at 24 hours post-infection. However, at 48 and 66 hours post-infection, Pb apiG3PAT (-) parasites were significantly smaller than wild type (with mean areas 22 % and 26 % less than the control).
Wild type parasites produced the majority of detached cells and merosomes at 66 hours post-infection, with smaller numbers of these forms detected at the 72 hours. By contrast, Pb apiG3PAT parasites were never seen to produce detached cells or merosomes at either 66 or 72 hours. Pb apiG3PAT (-) parasites had noticeably smaller apicoplasts and fewer nuclei than wild type at 48 and 66 hours. Normal MSP1 expression at 48h. At 66 hours, with MSP1 staining typically detected around individual merozoites in the wild type, but remaining patchy or indicative of only limited plasma membrane invagination in Pb apiG3PAT (-) parasites.

Additional information

Other mutants
RMgm-977, RMgm-978 P. yoelii mutants lacking expressing G3PAT or expressing a Myc-tagged version of G3PAT, respectively.
RMgm-1468: a P. berghei mutant expressing a C-terminal HA-GFP tagged version of G3PAT
 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1416700
Gene Model P. falciparum ortholog PF3D7_1318200
Gene productglycerol-3-phosphate 1-O-acyltransferase
Gene product: Alternative nameG3PAT; Pb apiG3PAT
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 usedNo
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
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 15’-AAGAATTCATGGGCTATTTGCATAAATCAGTATC-3’
Additional information primer 1P19
Sequence Primer 25’-AAGCGGCCGCGATGATTACTAAATATAAATACATTGTGTC-3’
Additional information primer 2P20
Sequence Primer 35’-AACCGCGGCCGCCTCATTTAAAAGAAGAAAA-3’
Additional information primer 3P21
Sequence Primer 45’-AAGGGCCCTGTCATAGTCCATTTCCCTTCA-3’
Additional information primer 4P22
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6