Summary

RMgm-4414
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1323600; Gene model (P.falciparum): PF3D7_1460000; Gene product: biotin--protein ligase 2 (HCS2)
PhenotypeNo phenotype has been described
Last modified: 3 March 2018, 12:27
  *RMgm-4414
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 29483266
MR4 number
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
The mutant parasite was generated by
Name PI/ResearcherDellibovi-Ragheb TA, Sinnis P, Prigge ST
Name Group/DepartmentW. Harry Feinstone Department of Molecular Microbiology and Immunology
Name InstituteJohns Hopkins Bloomberg School of Public Health
CityBaltimore
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-4414
Principal nameΔHCS2
Alternative name
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 ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks HCS2

Protein (function)
Acetyl-CoA carboxylase (ACC) is a biotin-dependent enzyme. Malaria parasites contain a plant-like ACC, and this is the only protein predicted to be biotinylated in the parasite. We found that ACC is expressed in the apicoplast organelle in liver- and blood-stage malaria parasites; however, it is activated through biotinylation only in the liver stages. Biotin is an essential metabolite in all known free-living organisms. It functions as a CO2 carrier in carboxylation and decarboxylation reactions catalyzed by a small family of enzymes, the biotin carboxylases. Biotin carboxylases participate in central metabolic processes such as gluconeogenesis, lipogenesis, amino acid metabolism, and energy transduction. Bacteria, plants, and some fungi can synthesize biotin de novo from a pimeloyl-CoA precursor, but other organisms acquire biotin from their diet or environment. Regardless of how biotin is acquired, a dedicated ATP-dependent biotin ligase, or holocarboxylase synthetase (HCS), is required to catalyze the covalent attachment of biotin to a specific lysine in the target carboxylase. Perhaps the most unusual feature of biotin metabolism in malaria parasites is the presence of two HCS paralogs (HCS1, HCS2) encoded in the genomes of Plasmodium species.

In the paper evidence is presented that: 
- PfHCS1 and PfHCS2 Are Active Biotin Ligases
- Biotin Is Not Essential for the Survival of Blood-Stage Parasites
- Biotinylated Proteins Cannot Be Detected in Blood-Stage Parasites
- Neither PbHCS1 Nor PbHCS2 Is Required for Blood-Stage Replication or Mosquito-Stage Development
- Loss of PbHCS1 Affects Early Liver-Stage Development
- Deletion of PbHCS1, but Not PbHCS2, Results in the Loss of Liver Stage Protein Biotinylation
- Parasites Lacking PbHCS1 Show Delayed Progression Through Liver Stages in Vivo. Deletion of PbHCS1 Affects Late Liver-Stage Development and Reduces Merosome Production.
- Biotin depletion increases the severity of the developmental defects, demonstrating that parasite and host biotin metabolism are required for normal liver-stage

Phenotype
Normal progression throughout the complete life cycle including normal liver stage development.

Additional information
See also mutant RMgm-4413 lacking HCS1 which shows the follwing liver stage phenotype:
Reduced growth of liver stages (At 24 hpi, ΔHCS1 parasites were 3 μm2 smaller, at 48h 53 μm2, at 56h 73 μm2 smaller than  WT controls). Reduced merozoite/merosome formation.
A delay of 2.1 days of the prepatent period after infecting mice with 5.000 sporozoites. Reduced infectivity of merozoites/merosomes produces as measured by blood infections in mice infected with merozoites/merosomes

Other mutants
See also mutant RMgm-4413 lacking HCS1


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1323600
Gene Model P. falciparum ortholog PF3D7_1460000
Gene productbiotin--protein ligase 2
Gene product: Alternative nameHCS2
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 parasitehdhfr
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