Summary

RMgm-1349
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0108300; Gene model (P.falciparum): PF3D7_0609800; Gene product: palmitoyltransferase DHHC2, putative (DHHC2)
Details mutation: 'promoter-swap' mutant; the promoter of DHHC2 replaced by the promoter of PBANKA_140060 (clag)
Transgene
Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 5 April 2016, 09:51
  *RMgm-1349
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 26526684
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 676m1cl1 (RMgm-29)
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherSantos JM; Janse CJ; Mair GR
Name Group/DepartmentInstituto de Medicina Molecular
Name InstituteFaculdade de Medicina da Universidade de Lisboa
CityLisbon
CountryPortugal
Name of the mutant parasite
RMgm numberRMgm-1349
Principal name2593cl1,2
Alternative nameclag::dhhc2
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 ookineteThe promoter-swap mutant produced normal numbers of female and male gametocytes, gametes. In addition, the mutant showed normal fertilisation rates and production of (diploid and tetraploid) zygotes. However zygotes failed to develop into mature ookinetes.
OocystNo oocysts are formed.
The promoter-swap mutant produced normal numbers of female and male gametocytes and gametes. In addition, the mutant showed normal fertilisation rates and production of (diploid and tetraploid) zygotes. However zygotes failed to develop into mature ookinetes.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
In the 'promoter-swap' mutant the promoter of DHHC2 replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes (the promoter of PBANKA_140060; clag; cytoadherence linked asexual protein). In addition, the mutant expresses the fusion protein GFP-Luciferase under control of the constitutive eef1a promoter.

Protein (function)
Many proteins are post-translationally modified by the addition of lipids. Palmitoylation results in the addition of a C-16 fatty acid to a cysteine residue within a given protein. Palmitoylation is reversible and thus can dynamically regulate a protein’s subcellular localization, gene expression and activity.
Blocking palmitoylation in P. falciparum with 2-bromopalmitate (2-BMP) results in a complete failure to develop merozoites during the blood stage of the life cycle. Preventing palmitoylation of proteins through targeted mutagenesis of cysteine residues within the modification target results in the mis-localization of proteins found in the inner membrane complex (IMC).
The palmitoylation reaction is catalysed by TM-spanning enzymes called palmitoyl-S-acyl-transferases (PAT). One family of PATs is characterised by the presence of a conserved DH(H/Y)C motif, and certain apicomplexan organisms express more than 10 individual S-acyltransferases. They differ in localisation and timing of expression, and therefore are likely to modify distinct protein populations and biological functions.
The global extent of palmitoylation in asexual blood stages of P. falciparum comprises several hundred proteins; they include factors involved in gliding motility, invasion, adhesion, IMC function, signalling, protein transport and proteolytic activity. Of 11 PATs known from rodent malaria parasites five have been detected in blood stage parasites of P. berghei using an HA-tagging approach: they are DHHC3 (IMC), DHHC5 (ER), DHHC7 (rhoptry), DHHC8 (punctate_not_Golgi), and DHHC9 (IMC). Seven DHHC-PATs were found to be redundant for P. berghei blood stage development in a reverse genetic screen: they are DHHC 3, 5, 6, 7, 9, 10 and 11.
Three PATs are under putative translational control in the female P. berghei gametocyte: dhhc2, dhhc3 and dhhc10.

Phenotype
Attempts to disrupt the dhhc2 gene in P. berghei were unsuccessful (see RMgm-1350) indicating an essential role of DHCC2 for blood stage development/multiplication.

Phenotype analyses of the promoter-swap mutant indicate that DHHC2 plays an important role in the development of zygotes into mature ookinetes (see also mutant RMgm-1351 for a comparable promoter swap mutant).

The promoter-swap mutant produced normal numbers of female and male gametocytes and gametes. In addition, the mutant showed normal fertilisation rates and production of (diploid and tetraploid) zygotes. However zygotes failed to develop into mature ookinetes. No oocysts are formed.

Additional information
Overnight ookinete cultures of mutant parasites stained with Hoechst showed signs of fertilisation. Image analyses of ploidy confirmed that fertilisation had not been affected with parasite DNA content of these forms being approximately 4N; the zygotes have thus completed meiotic DNA replication to tetraploidy. However, none of these zygotes developed into mature ookinetes and presented morphogenetic defects. In these cultures only clusters of round zygotes or zygotes with small, thin protrusions were present indicating initiated, but failed morphogenesis.

Other mutants
RMgm-1350: Unsuccessful attempts to disrupt the dhhc2 gene
RMgm-1351: A comparable promoter swap mutant as described here. The dhhc2 gene is tagged with GFP.
RMgm-1352, RMgm-1353: Mutants expressing a C-terminal GFP-tagged version of DHHC2


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0108300
Gene Model P. falciparum ortholog PF3D7_0609800
Gene productpalmitoyltransferase DHHC2, putative
Gene product: Alternative nameDHHC2
Details of the genetic modification
Short description of the mutation'promoter-swap' mutant; the promoter of DHHC2 replaced by the promoter of PBANKA_140060 (clag)
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) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationThe construct pLIS0209 contains the entire dhhc2 ORF (amplified with primers 3010 and 0644) and promoter sequence from the cytoadherence linked asexual (protein) gene (clag) PBANKA_140060 (amplified with primers 3024 and 3025). The human dhfr selection marker is flanked by a second upstream targeting region (amplified with primers 0739 and 0740) to allow for double cross-over homologous recombination following digestion with KpnI and BglI. Transfection of line 676m1cl1 with pLIS0209 was followed by WR99210 selection resulting in the parasite line 2593 (clag::dhhc2).
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 nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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