RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-876
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1319500; Gene model (P.falciparum): PF3D7_1455800; Gene product: LCCL domain-containing protein (CCp2; LAP4)
Name tag: EGFP
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 30 December 2018, 21:00
  *RMgm-876
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) : 23684590
Reference 2 (PMID number) : 30367865
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/ResearcherSaeed, S; Dessens, T
Name Group/DepartmentDepartment of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases
Name InstituteLondon School of Hygiene & Tropical Medicine
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-876
Principal namePbLAP4/GFP
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 ookineteGFP expression in ookinetes. Ookinetes exhibited GFP-based fluorescence that typically distributed to 2–3 regions visibly associated with clusters of malariapigment, characteristic of crystalloid targeting.
However, crystalloid biogenisis is abnormal
OocystReduced growth of oocysts and premature sporogony
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of LAP4 (CCp2). The tagged gene is under the control of the lap4 promoter region and the pbdhfr 3'UTR.

Protein (function)
The lap4 (ccp2) gene is a member of a small conserved gene family (6 genes), encoding proteins with multiple adhesive domains, for example a Lgl1 (LCCL)-lectin adhesive domain. In P. falciparum the LCCL domain-containing proteins are termed PfCCp's and in P. berghei PbLAP's

Phenotype
GFP expression in ookinetes. Ookinetes exhibited GFP-based fluorescence that typically distributed to 2–3 regions visibly associated with clusters of malaria-pigment, characteristic of crystalloid targeting.
No GFP expression in gametocytes and mature oocysts. mRNA is present in mature gametocytes indicating translational repression in gametocytes.

In reference PMID 30367865 (Saeed et al., 2018) a more detailed analysis of this mutants is provided. It is shown that: 'the LAP4 mutant that has abnormal crystalloid biogenesis and produces oocysts that display reduced growth and premature sporogony'.

Additional information
Evidence has been presented in different studies for protein expression of LAP1, LAP2, LAP3 in gametocytes and ookinetes whereas protein expression of LAP4, LAP5 and LAP6 is absent in gametocytes. All proteins are associated with crystalloid bodies in ookinetes. See also mutants with different lap genes deleted: all these gene deletion studies show a defect during oocyst/sporozoite development.

From the second paper PMID 30367865 (Saeed et al., 2018):

'In confocal microscopic examination, ookinetes and young oocysts of LAP4/GFP parasites possessed discrete fluorescent areas that co-localised strongly with pigment clusters, which is typical of crystalloid proteins. Electron microscopic examination of LAP4/GFP ookinetes revealed that the cells did not possess normal crystalloids, but instead had abnormal crystalloid-like structures that are absent in wild type and LAP null mutant ookinetes. To assess the effects of the crystalloid defect identified in the LAP4/GFP parasite on oocyst and sporozoite development, Anopheles stephensi vector mosquitoes were infected and parasite development was monitored in comparison with parasites that form normal crystalloids and display wild type sporogony (parasite line LAP3/GFP expressing LAP3::GFP)and with parasites that fail to form crystalloids and lack sporozoite formation (parasite line LAP3-KO, a LAP3 null mutant).
LAP4/GFP parasites produced oocysts in numbers comparable to the other lines, indicating that the crystalloid defect had not adversely affected ookinete fitness or infectivity. LAP4/GFP oocysts were significantly smaller than control oocysts (P < 0.0001) at 14 days p.i., and already lagged significantly in size at 8 days p.i. (P < 0.01). Thus, LAP4/GFP parasites display premature oocyst growth arrest. The large majority of LAP4/GFP oocysts underwent cytokinesis and produced sporozoites of normal morphology and size. Despite the ability of LAP4/GFP oocysts to produce sporozoites, salivary gland sporozoite numbers at 21 days p.i. were highly reduced (>10-fold) compared with LAP3/GFP control parasites. Moreover, we were repeatedly unable to  transmit LAP4/GFP sporozoites to naive mice by mosquito bite (five unsuccessful transmissions in five attempts), while control LAP3/GFP parasites were readily transmitted (two successful transmissions in two attempts). '

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1319500
Gene Model P. falciparum ortholog PF3D7_1455800
Gene productLCCL domain-containing protein
Gene product: Alternative nameCCp2; LAP4
Details of the genetic modification
Name of the tagEGFP
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 parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationA ca. 3 kb fragment of pblap4 corresponding to the 3'-part of the coding sequence was PCR amplified from genomic DNA withprimers P3 (ACGAAGTTATCAGTCGACAAGATGTCGAAAATATTTGTGCAT) and P4 (ATGAGGGCCCCTAAGCTTTCACATTCTGATATACACTGATTTATCA) and introduced into SalI/HindIII-digested pLP-PbLAP6/EGFP (see RMgm-878) to give pLP-PbLAP4/EGFP
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 1ACGAAGTTATCAGTCGACAAGATGTCGAAAATATTTGTGCAT
Additional information primer 1P3
Sequence Primer 2ATGAGGGCCCCTAAGCTTTCACATTCTGATATACACTGATTTATCA
Additional information primer 2P4
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