RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1137
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0506500; Gene model (P.falciparum): PF3D7_1022300; Gene product: ZIP domain-containing protein, putative (ZIP domain-containing protein, ZIPCO)
Phenotype Liver stage;
Last modified: 26 October 2014, 13:26
  *RMgm-1137
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) : 25257508
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).

An independent mutant lacking ZIPCO was generated in parasites of the NK65 strain (NK65 Paris or New York)
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The mutant parasite was generated by
Name PI/ResearcherSahu T; Menard R; Baldacci P
Name Group/DepartmentUnité de Biologie et Génétique du Paludisme
Name InstituteInstitut Pasteur
CityParis
CountryFrance
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Name of the mutant parasite
RMgm numberRMgm-1137
Principal nameZIPCO-F (ANKA); ZIPCO (NK-65)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot different from wild type
Liver stageNormal sporozoite production. Sporozoites had normal motility, cell traversal capacity and infectivity to hepatocytes in vitro.

Equal numbers of WT-F or ZIPCO-F salivary gland sporozoites were injected into mice intradermally (ID) or intravenously (IV), and pre-patent periods of infection were assessed by Giemsa-stained blood smears. ZIPCO-F blood-stage parasites emerged with a 3–4 day delay compared to WT-F, independently of the sporozoite injection route. Since the multiplication in the blood stages is normal, these results indicated a approximately 10(3) loss in infectivity for the mutant during pre-erythrocytic development. A similar delay was observed with ZIPCO sporozoites after IV injection.

Although numbers of WT-F and ZIPCO-F EEFs were counted in vitro by fluorescence microscopy at 24 hpi, significantly fewer ZIPCO-F than WT-F EEFs were observed at 48 hpi (43%) and 65 hpi (30%). Most notably, the average size of ZIPCO-F EEFs was significantly smaller at all time points, being approximately 65, 80 and 78% that of WT-F EEFs at 24, 48 and 65 hpi, respectively.
EEF development was also examined by intra-vital fluorescence microscopy at 24, 48 and 65 after IV of sporozoites. Like in vitro, ZIPCO-F EEFs were significantly smaller than the WT-F, displaying a approximately 90% reduction in size at 48 hpi. At 65 hpi, WT-F EEFs were no longer observed, having liberated merozoites, while a few ZIPCO-F EEFs were still detected. These data indicated that mutant EEFs were affected both in growth and viability, resulting in reduced and delayed release of merozoites into the blood. At 66 hpi, at the peak of merosome release in the WT-F EEFs, approximately 1,900 times fewer ZIPCO-F than control merozoites were counted.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of ZIPCO (zinc transporter, putative; ZIP domain-containing protein) and expresses GFP under the control of the constitutive eef1a promoter.

An independent mutant lacking ZIPCO was generated in parasites of the NK65 strain (NK65 Paris or New York).

Protein (function)
PBANKA_050650 is predicted to code for a protein with seven trans-membrane (TM) domains, including a putative N-terminal signal peptide, and has been annotated as a putative zinc transporter due to the presence of a conserved ZIP domain (Pfam02535). The ZIP family of proteins have a conserved signature of 15 amino acids, located either completely or partially within the fourth TM region, and 13 of them are present in PBANKA_050650. Sequence comparison using the forty sequences that constitute the seed of the Pfam02535 profile found that the well-conserved amino acids of this profile are present in PBANKA_050650. The genomes of P. falciparum, P. chabaudi, P. knowlesi, P. vivax, Saccharomyces cerevisiae and Arabidopsis thaliana were screened with the Pfam02535 profile, and a phylogenetic tree was generated that indicated the presence of two paralogs of the ZIP family in the screened Plasmodium species. In P. berghei, the paralog of PBANKA_050650 is PBANKA_010770, annotated as a putative permease. PBANKA_050650 thus encodes a protein of the ZIP family, which was named ZIP domain-containing protein, ZIPCO.

Phenotype
Phenotype analyses indicate that mutant  ZIPCO-F parasites undergo a delayed and inefficient schizogonic process in the hepatocyte, resulting in reduced formation of merozoites.
Evidence is presented that iron or zinc supplementation to the medium rescues the growth defect of liver stages suggesting that ZIPCO is required for parasite utilization of iron and possibly zinc, consistent with its predicted function as a metal transporter

Additional information
Quantitative RT-PCR analysis of various P. berghei developmental stages indicated that PBANKA_050650 transcripts were sevenfold higher in sporozoites than mixed RBC or exo-erythrocytic forms (EEFs) developing inside HepG2 hepatoma cells.
Analysis of a transgenic mutant expressing a HA-tagged version of ZIPCO (ZIPCO-HA; the endogenous zipco gene is tagged; see RMgm-1138) showed peak expression of ZIPCO in late liver stages and evidence is presented for localisation of ZIPCO in the parasite plasma membrane.

Evidence is presented that iron or zinc supplementation to the medium rescues the growth defect of liver stages suggesting that ZIPCO is required for parasite utilization of iron and possibly zinc, consistent with its predicted function as a metal transporter.

Other mutants
A transgenic mutant expressing a HA-tagged version of ZIPCO (ZIPCO-HA; the endogenous zipco gene is tagged; see RMgm-1138)

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0506500
Gene Model P. falciparum ortholog PF3D7_1022300
Gene productZIP domain-containing protein, putative
Gene product: Alternative nameZIP domain-containing protein, ZIPCO
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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 genePartial
Additional remarks partial/complete disruption A 540 bp-long internal fragment of ZIPCO encoding the fourth TM region, including the ZIP signature sequence and part of the fifth TM region of the protein, was deleted and replaced by the hDHFR selectable cassette.
Selectable marker used to select the mutant parasitehdhfr
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 15’-GGGCCCGAAGTGTTATAATATATTTAATAG-3’
Additional information primer 1
Sequence Primer 25’-TCCCCCGGGTTTAATAAAATATATAATATTC-3’
Additional information primer 2
Sequence Primer 35’-ATAAGAATGCGGCCGCAGTCATCCCCATTTCATTCAAATG-3’
Additional information primer 3
Sequence Primer 45’-GGCGCGCCTTGCAATTCTTCTTAAGATACACTT-3’
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren