Summary

RMgm-903
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0932100; Gene model (P.falciparum): PF3D7_1115900; Gene product: palmitoyltransferase DHHC9 (DHHC9)
Name tag: triple-HA
Phenotype Asexual bloodstage; Oocyst; Sporozoite; Liver stage;
Last modified: 22 April 2016, 18:44
  *RMgm-903
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 23638681
Reference 2 (PMID number) : 27084458
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 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherK. Frénal, J.Rayner, O. Billker, E. Bushell, D. Soldati-Favre
Name Group/DepartmentMalaria Programme
Name InstituteWellcome Trust Sanger Insitute
CityCambridge Hinxton
CountryUK
Name of the mutant parasite
RMgm numberRMgm-903
Principal namePbDHHC9 _3xHA_TAG
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageSchizont expression confirmed by IFA and Western Blotting. IFA co-localises the tagged protein with PbMSP1, in late, but not early schizonts, suggesting an IMC localisation.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystDHHC3-HA, it was detectable in oocyst sporozoites, but not in salivary gland sporozoites.
Generally the level of DHHC9-HA expression detectable by immunofluorescence was low in sporozoites.
SporozoiteDHHC3-HA, it was detectable in oocyst sporozoites, but not in salivary gland sporozoites.
Generally the level of DHHC9-HA expression detectable by immunofluorescence was low in sporozoites.
Liver stageIn liver stages, expression was not observed until 48 hours after invasion of the mammalian host cell, where it appeared to localize with dividing hepatic merozoites, a staining pattern that persisted into mature, 65 hour liver stage parasites.
At 48 hours, DHHC9-HA was also localized to a discrete streak in the periphery of the parasitophorous vacuole, which showed partial overlap with staining for uis4 (up-regulated in infective sporozoites gene 4). This distinct DHHC9-HA streak was observed in all infected hepatocytes at this time point in liver stage development, but was absent from parasites at 65 hours after infection.
Additional remarks phenotype

Mutant/mutation/phenotype

Reference PMID 23638681
The mutant expresses a C-terminal triple-HA-tagged version of the protein. The endogenous gene has been tagged with the triple-HA tag using a PlasmoGEM construct.

Schizont expression confirmed by IFA and Western Blotting. IFA co-localises the tagged protein with PbMSP1, in late, but not early schizonts, suggesting an IMC localisation.

This protein was selected in a study aimed at determining the repertoire of  DHHC-CRD S-acyl transferase protein family as putative candidates for protein S-acyl transferases (PATs). The genes encoding these proteins were targeted for deletion to analyse their essentiality for blood stages In addition, mutants were generated that express a HA-tagged version of the protein to identify its cellular location

The transgenic parasite has not been cloned and the phenotype of the gene deletion mutant has not been not analysed in detail

Reference PMID 27084458
In this study the mutant has been cloned and analysed in more detail during mosquito and liver stage development. See above for phenotype descriptions with expression observed in sporozoites and maturing liver stages.

Background:
Protein palmitoylation in particular is  a fundamental, dynamic, and widespread posttranslational mechanism that controls transport, properties and activity of proteins across eukaryotes. Unlike other irreversible lipid modifications such as myristoylation and prenylation, the addition of a 16-carbon saturated palmitate group to the sulfhydryl group of a cysteine to form a hydroxylamine-sensitive thioester linkage, is a reversible modification. This constitutes a fast and dynamic mechanism to spatiotemporally control protein function by impacting reversibly on protein trafficking, stability and clustering. While palmitoylation frequently facilitates membrane association of a soluble protein by the addition of a hydrophobic anchor, this modification also occurs on transmembrane proteins, involving other effects such as structural conformation changes, protein-protein interactions or the clustering to specific lipid domains leading, for example, to assembly of signalling complexes.
Palmitoylation can control the affinity of a protein for lipid membranes, which allows it to impact protein trafficking, stability, folding, signalling and interactions. The publication of the palmitome of the schizont stage of P. falciparum implicated a role for palmitoylation in host cell invasion, protein export and organelle biogenesis.

The enzymes mediating transfer of palmitate from palmitoyl-CoA to a protein substrate were first identified in Saccharomyces cerevisiae and subsequently in mammals. Protein S-acyl transferases (PATs) belong to the DHHC family of proteins that exhibit a catalytic Asp-His-His-Cys conserved motif located within a cysteine-rich domain (CRD) and frequently between two transmembrane regions, facing the cytosol. Substrate recognition and catalysis occur after the protein substrates have associated with membrane via another lipidation.

Other mutants
See the link DHHC for a number of other related mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0932100
Gene Model P. falciparum ortholog PF3D7_1115900
Gene productpalmitoyltransferase DHHC9
Gene product: Alternative nameDHHC9
Details of the genetic modification
Name of the tagtriple-HA
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vectorPbGEM-121234
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) 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