Summary

RMgm-1585
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0608000; Gene model (P.falciparum): PF3D7_1209600; Gene product: porphobilinogen deaminase (PBGD)
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_1340000; Gene product: dihydrofolate synthase/folylpolyglutamate synthase, putative (PbDHFS-FPGS)
Replacement locus: Gene model: PBANKA_1459200; Gene product: delta-aminolevulinic acid synthetase (ALAS)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 12 September 2016, 19:01
  *RMgm-1585
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 27600503
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/ResearcherRizopoulos Z, Matuschewski K, Haussig J.
Name Group/DepartmentParasitology Unit
Name InstituteMax Planck Institute for Infection Biology
CityBerlin
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-1585
Principal namepbgd(-)
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
OocystNormal numbers of oocysts are formed. However, day 10 and 14 oocysts had a reduced size (`60%) compared to wild type oocysts. A large (~10-20-fold) reduction in the numbers of midgut-associated sporozoites was observed. No hemocoel or salivary gland sporozoites were detected.
SporozoiteNormal numbers of oocysts are formed. However, day 10 and 14 oocysts had a reduced size (`60%) compared to wild type oocysts. A large (~10-20-fold) reduction in the numbers of midgut-associated sporozoites was observed. No hemocoel or salivary gland sporozoites were detected.
Liver stageA large (~10-20-fold) reduction in the numbers of midgut-associated sporozoites was observed. No hemocoel or salivary gland sporozoites were detected. No mosquito transmission.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PBGD and expresses GFP under control of the strong hsp70 promoter

Protein (function)
The malaria parasite is capable of de novo heme biosynthesis despite its ability to acquire heme from red blood cell (RBC) hemoglobin. The first enzyme of the de novo heme-biosynthetic pathway, δ-aminolevulinate synthase (ALAS) and the last two enzymes, Protoporphyrinogen IX oxidase (PPO) and Ferrochelatase (FC) localize to the mitochondrion. The enzymes that catalyze the intermediate steps: ALA dehydratase (ALAD), Porphobilinogen deaminase (PBGD) and Uroporphyrinogen III decarboxylase (UROD) localize to the apicoplast.

Phenotype
Mutant parasites showed normal blood stage development, indicating a non-essential role of PBGD during blood stage development. Normal numbers of oocysts are formed. However, day 10 and 14 oocysts had a reduced size (`60%) compared to wild type oocysts. A large (~10-20-fold) reduction in the numbers of midgut-associated sporozoites was observed. No hemocoel or salivary gland sporozoites were detected.

Additional information
In this study, the essentiality of heme biosynthesis was systematically profiled by targeted gene deletion of enzymes in early steps of this pathway. It was shown that disruption of endogenous heme biosynthesis leads to a first detectable defect in oocyst maturation and sporogony in the Anopheles vector, whereas blood stage propagation, colonization of mosquito midguts or initiation of oocyst development occurs indistinguishable from wild-type parasites. Although sporozoites are produced by parasites lacking an intact pathway for heme biosynthesis, they are absent from mosquito salivary glands, indicative of a vital role for heme biosynthesis only in sporozoite maturation.

Rescue of the first defect in sporogony permitted analysis of potential roles in liver stages (see RMgm-1582). It was shown that liver stage parasites benefit from, but do not strictly depend upon their own aminolevulinic acid synthase and that they can scavenge aminolevulinic acid from the host environment.

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0608000
Gene Model P. falciparum ortholog PF3D7_1209600
Gene productporphobilinogen deaminase
Gene product: Alternative namePBGD
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/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFragments of the 3’ and 5’ UTR were amplified from gDNA using gene-specific primers. First, 3’ fragments were cloned into the pBAT vector, following restriction digestion with HindIII and KpnI to generate an intermediate construct (pHEME-im). Then, the 5’ UTR fragment was digested with SacII and EcoRV and cloned into the respective SacII- and PvuII-linearized pHEME-im construct, thus removing the mCherry-3xMyc tag. Finally, the pHEME-ko vector was linearized with ScaI and SalI before transfection into P. berghei strain ANKA (wt) parasites.
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
Details of the genetic modification
Inducable system usedNo
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/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_1340000
Gene productdihydrofolate synthase/folylpolyglutamate synthase, putative
Gene product: Alternative namePbDHFS-FPGS
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_1459200
Gene productdelta-aminolevulinic acid synthetase
Gene product: Alternative nameALAS
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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