Summary

RMgm-4356
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PbANKA_0311600; Gene model (P.falciparum): PF3D7_0214800; Gene product: conserved Plasmodium membrane protein, unknown function
PhenotypeNo phenotype has been described
Last modified: 21 September 2017, 18:28
  *RMgm-4356
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification Unknown
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28902970
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherSayers CP, McFadden GI, Goodman CD
Name Group/DepartmentSchool of BioSciences
Name InstituteUniversity of Melbourne
CityParkville, Victoria
CountryAustralia

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PbANKA_0311600
Gene Model P. falciparum ortholog PF3D7_0214800
Gene productconserved Plasmodium membrane protein, unknown function
Gene product: Alternative name
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 usedYes
Name of PlasmoGEM construct/vector-
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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood stage growth.

The protein was identified in a bioinformatic screen for identifying apicoplast proteins.
27 candidate apicoplast membrane proteins were identified, 20 of which are annotated as putative membrane transporters.
Candidates were selected from P. falciparum putative membrane transporters belonging to known transport families and selected members of the predicted P. falciparum apicoplast proteome with multiple TMDs. Many P. falciparum membrane transporters have seven or more TMDs, whilst some have six or fewer. Without biological data to confirm membrane transport activity,  candidate proteins predicted to have at least six TMDs were selected. Although multiple TMDs is a minimum requirement of most membrane transporters, some of these proteins may be integral membrane proteins with other functions.

A genetic screen was performed in P. berghei to determine blood stage essentiality and subcellular localisation. Eight apparently essential blood stage genes were identified, three of which were apicoplast-localised: PbANKA_0614600 (DMT2), PbANKA_0401200 (ABCB4) and PbANKA_0505500. Nineteen candidates could be deleted at the blood stage, four of which were apicoplast-localised.Three apicoplast-localised candidates lack a canonical apicoplast targeting signal but do contain conserved N-terminal tyrosines with likely roles in targeting.

Sixteen candidate genes were HA-tagged for localisation experiments in P. berghei.
Immunofluorescence assays (IFAs) were performed for all tagged candidate genes. Five candidates co-localised with the apicoplast marker, ACP. PbANKA_0809500, PbANKA_0401200, PbANKA_0505500 were all predicted to have apicoplast targeting leader sequences whilst PbANKA_1103600, PbANKA_1304700  were not predicted to contain leader sequences. Another seven candidates localised to non-apicoplast structures within the parasite. Four candidates had no observable immunofluorescence signal at the blood stage so could not be localised in this life cycle stage: PbANKA_0942100, PbANKA_0417100, PbANKA_1446100 and PbANKA_0602400. Life cycle analysis of two candidates (PbANKA_1103600 and PbANKA_0942100) revealed that PbANKA_1103600 TAG was expressed and localised to the apicoplast in midgut oocysts, salivary gland sporozoites and in vitro liver cell stage parasites. No signal was observed for PbANKA_0942100 TAG parasites at any life cycle stage so its localisation remains unknown
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