Summary

RMgm-1474
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0512000; Gene model (P.falciparum): PF3D7_1027900; Gene product: palmitoyltransferase DHHC10, putative (DHHC10)
TaggedGene model (rodent): PBANKA_1300700; Gene model (P.falciparum): PF3D7_1475500; Gene product: LCCL domain-containing protein (LAP2; LCCL/lectin adhesive-like protein 2; CCp1)
Name tag: EGFP
Transgene
Transgene not Plasmodium: RFP
Promoter: Gene model: PBANKA_1319500; Gene model (P.falciparum): PF3D7_1455800; Gene product: LCCL domain-containing protein (LAP4; LCCL/lectin adhesive-like protein 4; CCp2)
3'UTR: Gene model: PBANKA_1359600; Gene product: transmission blocking target antigen precursor 6-cysteine protein (P48/45)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Transgene
Transgene not Plasmodium: GFP (gfp-mut3)
Promoter: Gene model: PBANKA_0416100; Gene model (P.falciparum): PF3D7_0905300; Gene product: dynein heavy chain, putative
3'UTR: Gene model: PBANKA_1010600; Gene product: calmodulin, putative (cam)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 5 July 2016, 14:29
  *RMgm-1474
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene tagging, Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 27303037
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-1471
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherSantos JM; Janse CJ; Mair GR
Name Group/DepartmentParasitology, Department of Infectious Diseases
Name InstituteUniversity of Heidelberg Medical School
CityHeidelberg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-1474
Principal name2433
Alternative nameΔdhhc10;lap2::gfp
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteΔdhhc10;lap2::gfp parasites showed LAP2::GFP-positive female gametocytes with protein dispersed throughout the cytoplasm, as described previously.
Fertilization and ookineteAnalyses of the Δdhhc10;lap2::gfp ookinetes showed diffuse localisation of LAP2::GFP in the cytoplasm ookinetes. LAP2 has previously been localized to the ookinete crystalloids by both live fluorescence imaging and immunoelectron microscopy. The cytoplasmic location of LAP2::GFP and absence of 'focal' localisation in crystalloids confirms the absence of crystalloid formation in mutants lacking expression of DHHC10
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of DHHC10 and expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter. In addition it expresses a C-terminal GFP-tagged version of LAP2. Lap2-tagging was performed in the background parasite line RMgm-1471 that lacks expression of DHHC10 and expresses the transgenes GFP and mCherry in males and females respectively.

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.

Phentype analyses of a mutants lacking expression of DHHC10 (RMgm-1470, RMgm-1471) showed the following phenotype: Normal numbers of gametocytes are produced. Normal fertilisation rate and ookinete production. Ookinetes however lack crystalloids (crystalloid body; crystalloid organelle). Normal numbers of oocysts are produced. However, oocysts fail to produce sporozoites.

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 (RMgm-1349; RMgm-1351). The promoter-swap mutant produced normal numbers of female and male gametocytes and gametes and normal fertilisation rates and production of zygotes. However zygotes failed to develop into mature ookinetes. No oocysts are formed.

Phenotype
Phentype analyses of mutants lacking expression of DHHC10 (RMgm-1470, RMgm-1471) showed the following phenotype: Normal numbers of gametocytes are produced. Normal fertilisation rate and ookinete production. Ookinetes however lack crystalloids (crystalloid body; crystalloid organelle). Normal numbers of oocysts are produced. However, oocysts fail to produce sporozoites.

Phenotype analyses of the mutant expressing GFP-tagged DHHC10 (RMgm-1472) showed GFP-fluorescence in distinct cytoplasmic foci in ookinetes known for members of the LAP/CCp protein family typical for the ookinete/oocyst-specific crystalloid organelle.

Analyses of the Δdhhc10;lap2::gfp ookinetes showed diffuse localisation of LAP2::GFP in the cytoplasm ookinetes. LAP2 has previously been localized to the ookinete crystalloids by both live fluorescence imaging and immunoelectron microscopy. The cytoplasmic location of LAP2::GFP and absence of 'focal' localisation in crystalloids confirms the absence of crystalloid formation in mutants lacking expression of DHHC10

Additional information
dhhc10 transcripts are present in female gametocytes but protein is absent. Evidence is presented for translational repression in female gametocytes. The protein is produced after fertilisation in developing zygots/ookinetes.

Evidence is presented for a location of DHHC10 in the crystalloid organelles (see also RMgm-1472, RMgm-1473).
Crystalloids are formed by the microtubule (MT)-dependent transport and assembly of endoplasmic reticulum-derived vesicles. Concomitantly, LAP proteins such as LAP3 are shuttled to common assembly points and incorporated into mature crystalloids. DHHC10::GFP was detected as early as 3 h postfertilization, and both DHHC10 and LAP3 showed signs of protein concentration by 9 h postfertilization. At 12 h, more than 50% of the retorts displayed focal accumulation of both proteins, and by the end of ookinete development (24 h), GFP and mCherry signals colocalized in the crystalloid in more than 90% of mature ookinetes. Thus, no significant differences between the two proteins in either the timing of expression or subcellular localization were evident.
 
Other mutants
See palmitoyltransferase
See DHHC10


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0512000
Gene Model P. falciparum ortholog PF3D7_1027900
Gene productpalmitoyltransferase DHHC10, putative
Gene product: Alternative nameDHHC10
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 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 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

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1300700
Gene Model P. falciparum ortholog PF3D7_1475500
Gene productLCCL domain-containing protein
Gene product: Alternative nameLAP2; LCCL/lectin adhesive-like protein 2; CCp1
Details of the genetic modification
Name of the tagEGFP
Details of taggingC-terminal
Additional remarks: taggingIn situ C-terminal GFP-tagging of lap2 was performed by single cross-over homologous recombination into the endogenous locus using a previously published construct (RMgm-355).
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single 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 markerpbdhfr
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationIn situ C-terminal GFP-tagging of lap2 was performed by single cross-over homologous recombination into the endogenous locus using a previously published construct (RMgm-355). The construct used contains the human dhfr/ts selectable marker. Linearised plasmid was transfected into Δdhhc10-a parasites (RMgm-1471) using standard methods.
Additional remarks selection procedureSelection of mutant parasites was performed with the drug WR99210, resulting in the following transgenic line: 2433 (Δdhhc10;lap2::gfp).
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 nameRFP
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 markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe mutant expressses GFP under control of the male gametocyte specific promoter of PB000791.03.0 (dynein heavy chain, putative) and RFP under the control of the 'female gametocyte specific' promoter PB000504.02.0 (LCCL domain-containing protein CCP2). The rfp and gfp genes are stably integrated into the genome in the 230p locus. The mutant does not contain a drug selectable marker which has been removed by the negative selection procedure using the negative selectable marker yfcu (yeast cytosine deaminase and uridyl-phosphoribosyltransferase) and the drug 5-fluorocytosine (5-FC).
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1319500
Gene Model P. falciparum ortholog PF3D7_1455800
Gene productLCCL domain-containing protein
Gene product: Alternative nameLAP4; LCCL/lectin adhesive-like protein 4; CCp2
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_1359600
Gene producttransmission blocking target antigen precursor 6-cysteine protein
Gene product: Alternative nameP48/45
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mut3)
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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe mutant expressses GFP under control of the male gametocyte specific promoter of PB000791.03.0 (dynein heavy chain, putative) and RFP under the control of the 'female gametocyte specific' promoter PB000504.02.0 (LCCL domain-containing protein CCP2). The rfp and gfp genes are stably integrated into the genome in the 230p locus. The mutant does not contain a drug selectable marker which has been removed by the negative selection procedure using the negative selectable marker yfcu (yeast cytosine deaminase and uridyl-phosphoribosyltransferase) and the drug 5-fluorocytosine (5-FC).
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0416100
Gene Model P. falciparum ortholog PF3D7_0905300
Gene productdynein heavy chain, putative
Gene product: Alternative name
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_1010600
Gene productcalmodulin, putative
Gene product: Alternative namecam
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