RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5005
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Details mutation: Deletion of (N-terminal) repeats of the central repeat sequence of CSP
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
PhenotypeNo phenotype has been described
Last modified: 27 May 2021, 11:56
  *RMgm-5005
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Not published (yet)
MR4 number
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 that expresses GFP under the control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherBalaban AE, Sinnis P
Name Group/DepartmentJohns Hopkins Malaria Research Institute and Department of Molecular Microbiology & Immunology
Name InstituteJohns Hopkins Bloomberg School of Public Health
CityBaltimore
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-5005
Principal nameΔRep1
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 expresses a mutated form of CSP with deletion of (N-terminal) repeats of the central repeat sequence of CSP (see below)

Published in bioRxiv preprint doi: https://doi.org/10.1101/2021.05.12.443759

Protein (function)
The CS protein is the major protein on the surface of sporozoites and is critical for development of sporozoites within the oocysts and is involved in motility and invasion of both the salivary gland of the mosquito and the liver cells. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. During substrate-dependent locomotion of sporozoites, CS is secreted at the sporozoite anterior pole, translocated along the sporozoite axis and released on the substrate at the sporozoite posterior pole. Following sporozoite invasion of hepatocytes, the CS is released in the host cell cytoplasm.

Phenotype
Normal numbers of salivary gland sporozoites.

To probe whether the length of the repeats was important for domain exposure, we generated three CSP repeat truncation mutants, ΔRep1, ΔRep2, and ΔRep3, in which different lengths or regions of the repeats were deleted (Interestingly, deletion of 25% of  the repeat region, whether it be in the N-terminal (ΔRep1) or the C-terminal (ΔRep3) portion of the repeats, does not significantly impact function. In contrast, deletion of 50% of the repeat region (ΔRep2) leads to the motility defects outlined above and increased TSR domain exposure. 
Additionally, to test whether the repetitive nature of the repeats contains important structural features we also generated a scrambled mutant, Scr, by randomly scrambling the amino acid sequence of the repeats. For this mutant, we avoided introduction of new secondary structures, as predicted by the Chou-Fasman method, but maintained amino acid content and length.

ΔRep1 and ΔRep3 had comparable salivary gland infections to controls. In contrast, ΔRep2 and Scr parasites were inhibited in their capacity to enter the salivary glands, with fewer salivary gland associated sporozoites. We confirmed that ΔRep2 and Scr salivary gland associated sporozoites were inside the salivary glands, as opposed to attached to the external surface of the glands by live confocal imaging of salivary glands

Additional information

WT CSP repeat sequence:
KLKQP PPPP NPNDPPPP NPNDPPPP NPNDPPPP NPNDPAPP NANDPAPP
NANDPAPP NANDPAPPNANDPAPP NANDPAPP NANDPAPP NANDPPPP
NPNDPAPP QGNNN PQPQ PRPQ PQPQ PQPQ PQPQ PQPQ PRPQ PQPQP

ΔRep1: lacking orange part
ΔRep2: lacking blue part
ΔRep3: lacking purple part

Scr Repeat Sequence

KLKQP PPPP PNNAPPRD GPPAAPPD PDQNPDPD PQPPQPQN PPNPPPNP
NPQDPNNP PQDQPANP NPPPPAQP PPDPDPPA QPNPPANP NNPPDPAP
NPNPPQPN QAPNPPQN QPNDPPPA NNDNAQPN PANRNPPP PPAQP

From the Abstract:
We found that sporozoite mutants with severely truncated or scrambled repeats have impaired motility due to altered adhesion site formation and dynamics, suggesting that the CSP repeats provide a cohesive environment in which adhesion sites can form. We show that the repeats are a stiff, linear spring with elastic properties, dependent upon length and lost when the repeats are scrambled. These data are the first evidence that the CSP repeat region serves a functional role during infection and motility, likely mediated through its biophysical properties.

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0403200
Gene Model P. falciparum ortholog PF3D7_0304600
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCSP
Details of the genetic modification
Short description of the mutationDeletion of (N-terminal) repeats of the central repeat sequence of CSP
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/construct(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
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationMutant csp repeat region oligonucleotides were commercially synthesized by Genscript (Piscataway, NJ) and provided in the pUC57 plasmid. Repeat sequences were digested from the pUC57 plasmid using HindIII-HF and SexAI and ligated into an intermediate CSP vector in Bluescript SK, containing the KpnI to PacI sequence from the csp locus which includes 500 bp of 5’utr and the full length csp sequence. The csp sequence in this vector was engineered to have HindIII-HF and SexAI restriction sites flanking the repeats. Once the mutant repeat sequences were ligated into the intermediate CSP vector, a KpnI and PacI digest was performed and this fragment was ligated into the previously described transfection vector, pCSRep, altered to have an additional 1 kb of 3’ UTR in order to minimize correction of the repeat mutations. Recombinant parasites were generated by double homologous recombination in which the native csp locus was replaced by a mutant copy of csp with its control elements and an upstream selection cassette. pCSRep was digested with XhoI and KasI
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(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
Selectable marker used to select the mutant parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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