Summary

RMgm-1351
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)
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 5 April 2016, 09:52
  *RMgm-1351
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
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 RMgm-1353
Other information parent lineThe parent line 202cl1m1 expresses a C-terminal GFP-tagged version of the DHHC2 protein
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-1351
Principal name209cl2
Alternative nameclag::dhhc2::gfp::3'utr
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). This promoter swap modification is made in a mutant parent line (RMgm-1353) that expresses a C-terminal GFP-tagged version of DHHC2.

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-1349 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-1349: 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 202cl1m1 with pLIS0209 was followed by WR99210 selection on four consecutive days; a final clonal line (209cl2) was established by limiting dilution.
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