RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1502
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0303900; Gene model (P.falciparum): PF3D7_0206200; Gene product: pantothenate transporter (PAT)
Name tag: GFP
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Sporozoite; Liver stage;
Last modified: 20 July 2016, 09:36
  *RMgm-1502
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) : 27427910
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/ResearcherKehrer J; Mair GR
Name Group/DepartmentIntegrative Parasitology, Center for Infectious Diseases
Name InstituteUniversity of Heidelberg Medical School
CityHeidelberg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-1502
Principal namepat::gfp
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteExpression in gametocytes (speckled appearance). Following activation of female gametocytes, PAT::GFP to distribute towards the plasma membrane (reminiscent of osmiophilic bodies).
Fertilization and ookineteExpression in ookinetes (speckled appearance)
OocystNot tested
SporozoiteExpression in sporozoites, indicative for micronemal (and surface) location. Co-localisation of PAT with the micronemal protein TRAP in sporozoites.
Liver stageLate liver stage forms did not produce any fluorescent cells
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of PAT.

Protein (function)
P. berghei PAT PBANKA_0303900 belongs to the major facilitator superfamily (MFS) of transporters and is a syntenic ortholog of the P. falciparum vitamin B5 transporter PAT PF3D7_0206200. The MFS protein family (Pfam:PF07690; Superfamily:SSF103473) is characterised by the presence of 10–12 transmembrane domains and is evolutionarily widely distributed from bacteria to plants and humans. Twelve such proteins are known in rodent and human malaria species. The PBANKA_030390 protein in P. berghei is 541 amino acids long and highly conserved between the various malaria species with 97.2% identity (98.9% similarity) between proteins from the rodent species P. berghei and P. yoelii, and 75.9% identity (85.6% similarity) between proteins from P. berghei and P. falciparum despite a 30 amino acid asparagine-rich insertion. Outside the genus Plasmodium the nearest neighbours are found in related apicomplexans including Toxoplasma gondii, Babesia and Eimeria. In global proteomic screens PAT has been identified in P. berghei sporozoites, and the P. falciparum ortholog PF3D7_0206200 was detected in stage V gametocytes as well as salivary gland sporozoites but not asexual stage parasites.
For P. falciparum evidence has been presented that the protein is a pantothenate transporter (PAT) and is essential for blood stage development. Studies in rodent malaria parasites demonstrate that this protein is not essential for blood stages but play a role dring transformation of gametocytes into ookinetes and for formation of oocysts and infective sporozoites (see below)

Phenotype
In the paper a number of mutants have been generated and analysed and provided evidence for the following phenotypes and  function of PAT:

A mutant lacking expression of PAT (Δpat; RMgm-1500);
PAT is not essential for blood stages; Δpat parasites did not form oocysts; male and female gametes fail to egress from their host erythrocytes after induction of gametogenesis. Females gametes fail to egress from the parasitophorous vacuole membane and erythrocyte membrane.

A mutant in which the endogeneous pat gene is replaced with the pat gene of P. falciparum 3D7: Δpat;pat(PF3D7)::mcherry; RMgm-1501)
P. falciparum PAT can complement P. berghei PAT

A mutant expressing a C-terminal GFP-tagged version of PAT (pat::gfp; RMgm-1502):
No expression in blood stages; expression in gametocytes (speckled appearance); expression in ookinetes (speckled appearance); expression in sporozoites, indicative for micronemal (and surface) location; no expression in late liver stages. Following activation of female gametocytes,  PAT::GFP to distribute towards the plasma membrane (reminiscent of osmiophilic bodies). Co-localisation of PAT with the micronemal protein TRAP in sporozoites.

A mutant expressing GFP-tagged PAT and mCherry-tagged G377 (pat::gfp;g377:mcherry; RMgm-1503):
Co-localisation of PAT with the osmiophilic body protein G377 (PBANKA_1463000). The presence of additional PAT+ but G377-negative (G377-) vesicles suggests that additional vesicles defined by PAT exist that are distinct from OBs containing G377.

A mutant expressing GFP-tagged PAT and mCherry-tagged PPLP2 (pplp2::mCherry;pat::gfp; RMgm-1504 :
Co-localisation of PAT with PPLP2 (PBANKA_1432400) suggesting that PPLP2 as well resides in vesicles defined by the membrane protein PAT.

A mutant lacking expression of PAT and expressing mCherry-tagged G377 (PBANKA_1463000) and a mutant lacking expression of PAT and expressing mCherry-tagged PPLP2 (PBANKA_1463000)
The absence of PAT did not influence the formation of G377- and PPLP-positive vesicles and trafficking of these vesicles to the membrane of females after induction of gametogenesis

A mutant expressing a GFP-tagged version of TRAP (ss::gfp::trap; RMgm-1505)

A 'promoter-swap' mutant expressing PAT under the promoter of the ccp1/lap2 promoter (PBANKA_1300700)(patccp.PP; RMgm-1506)
The ccp1/lap2 promoter is silent in asexual blood stages but is active in gametocytes and ookinetes. It is also silent in oocysts and sporozoites. This promoter-swap mutant produces normal number of oocysts. The large number of midgut sporozoites in the patccp.PP mutant did not result into highly infected salivary glands, which was not due to a failure of the mutant to exit the oocysts and enter the hemolymph. patccp.PP sporozoites are defective in adhesion and gliding (and fail to invade salivary glands). The failure to efficiently establish blood stage infections and the delay in prepatency after mosquito bite and i.v. infections of mice identifies a role for PAT in motility, which is required during salivary gland entry, skin passage, cell traversal and invasion of host hepatocytes.
Evidence is provived that in patccp.PP sporozoites micronemal secretion is affected

Additional information

Other mutants
See the link PF3D7_0206200 for other PAT mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0303900
Gene Model P. falciparum ortholog PF3D7_0206200
Gene productpantothenate transporter
Gene product: Alternative namePAT
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 modificationA 2417 bp product encompassing the entire ORF of pat (1626 bp) as well as 771 bp of the 5’UTR region was amplified from P. berghei genomic DNA with primers g1025 and g1027. The amplicon was cut with HincII and BamHI to release 1539 bp of the 3’ end of the ORF; this fragment was ligated into pLIS0010 (ΔNheI) containing the Toxoplasma dhfr/ts selection cassette to yield a C-terminal GFP fusion. The final plasmid pLIS0301 was digested with NheI, precipitated with 2.5 volumes ethanol and transfected into wildtype schizonts.
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