Summary

RMgm-1473
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0512000; Gene model (P.falciparum): PF3D7_1027900; Gene product: palmitoyltransferase DHHC10, putative (DHHC10)
Name tag: GFP
TaggedGene model (rodent): PBANKA_0204500; Gene model (P.falciparum): PF3D7_0109100; Gene product: LCCL domain-containing protein (LAP3; LCCL/lectin adhesive-like protein 2; CCp5)
Name tag: mCherry
Phenotype Fertilization and ookinete;
Last modified: 1 July 2016, 12:52
  *RMgm-1473
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Gene tagging
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 P. berghei ANKA cl15cy1
Other information parent lineP. berghei ANKA 820cl1m1cl1 (RMgm-164) is a reference ANKA mutant line which expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 19438517).
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-1473
Principal name2492
Alternative namedhhc10::gfp;lap3::mCherry
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteAnalyses of the dhhc10::gfp;lap3::mCherry showed co-localisation of DHHC10::GFP and LAP3::mCherry in ookinetes. LAP3 has previously been localized to the ookinete crystalloids by both live fluorescence imaging and immunoelectron microscopy. The co-localisation confirms the crystalloid location of DHHC10
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant  expresses a C-terminal GFP-tagged version of DHHC10 and, in addition, it expresses a C-terminal mCherry-tagged version of LAP3 (PBANKA_0204500). The mCherry-tagged lap3 gene is introduced as an additional copy on a circular plasmid in the mutant that expresses a GFP-tagged version of DHHC10 (RMgm-1472).  The dhhc10::gfp;lap3::mCherry parasites are selected by WR99210 treatment.

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

This mutant showed normal progression throughout the life cycle, including ookinete, oocyst, and sporozoite production and transmission to mice via mosquito bites, indicating that GFP-tagging of DHHC10 did not affect protein function or parasite viability.

Analyses of the dhhc10::gfp;lap3::mCherry mutant showed co-localisation of DHHC10::GFP and LAP3::mCherry in ookinetes. LAP3 has previously been localized to the ookinete crystalloids by both live fluorescence imaging and immunoelectron microscopy. The co-localisation confirms the crystalloid location 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-1474).

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


  Tagged: Mutant parasite with a tagged 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
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
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 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_0204500
Gene Model P. falciparum ortholog PF3D7_0109100
Gene productLCCL domain-containing protein
Gene product: Alternative nameLAP3; LCCL/lectin adhesive-like protein 2; CCp5
Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagginglap3 mCherry tagging construct pLP-PbLAP3/mCherry was obtained by cloning mCherry into the published plasmid pLP-PbLAP3/EGFP (Saeed et al., Mol Biochem Parasitol 2010 170(1): p. 49-53).
Commercial source of tag-antibodies
Type of plasmid/constructCircular plasmid
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 modificationlap3 mCherry tagging construct pLP-PbLAP3/mCherry was obtained by cloning mCherry into the published plasmid pLP-PbLAP3/EGFP (Saeed et al., Mol Biochem Parasitol 2010 170(1): p. 49-53).
The coding sequence of mCherry plus 3' UTR of pbdhfr/ts were PCR-amplified from plasmid pDNR-mCherry with primers mCherryswap-F and mCherryswap-R and introduced into ApaI-digested pLP-PbLAP3/EGFP by In-Fusion® cloning system (Clontech® Laboratories, Inc.) to generate pLP-PbLAP3/mCherry. This construct contains the entire lap3 coding sequence plus 0.6 kb of its upstream sequence as well as the human dhfr/ts selectable marker. Circular plasmid was transfected into dhhc10::gfp parasites (RMgm-1472).
Additional remarks selection procedureThe mutant expresses a C-terminal GFP-tagged version of DHHC10 and in addition it expresses a C-terminal mCherry-tagged version of LAP3 (). The mCherry-tagged lap3 gene is introduced as an additional copy on a circular plasmid in the mutant that expresses a GFP-tagged version of DHHC10 (RMgm-1472). The dhhc10::gfp;lap3::mCherry parasites are selected by WR99210 treatment.
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