Summary

RMgm-609
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CS, CSP)
Details mutation: deletion of entire N terminus excluding the signal sequence (including region I)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 5 March 2011, 11:40
  *RMgm-609
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 21262960
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/ResearcherA. Coppi; P. Sinnis
Name Group/DepartmentDepartment of Medical Parasitology
Name InstituteNew York University School of Medicine
CityNew York
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-609
Principal name∆Nfull
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
OocystMutant oocysts produced between 50 and 100% more sporozoites per oocyst compared with wild type oocysts.
SporozoiteMutant oocysts produced between 50 and 100% more sporozoites per oocyst compared with wild type oocysts. However, salivary glands contained 10-fold lower numbers of salivary gland sporozoites compared to wild type.
Mutant salivary gland sporozoites showed a higher invasion efficiency of hepatocytes in vitro compared with wild type. Mutant salivary gland sporozoites showed a higher infectivity in vivo after intravenous inoculation. After intradermal inoculation, most sporozoites did not reach the liver but were trapped in the skin.
Liver stageMutant salivary gland sporozoites showed a higher invasion efficiency of hepatocytes in vitro compared with wild type. Mutant salivary gland sporozoites showed a higher infectivity in vivo after intravenous inoculation. After intradermal inoculation, most sporozoites did not reach the liver but were trapped in the skin.
Additional remarks phenotype

Mutant/mutation
In the mutant the wild type csp is replaced with a mutated csp containing a specific deletion of entire N terminus, (including region I) and  excluding the signal sequence (amino acids NKSIQAQRNLNELCYNEGNDNKLYHVLNSKNGKIYNRNTVNRLLADAPEGKKNEKKNEKIERNNKLKQP)

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
It has been shown that proteolytic processing of CSP occurs by a cysteine protease during invasion of hepatocytes. Processing occurs on the sporozoite surface (Coppi et al., 2005, J. Exp. Med. 201, 27-33).
Phenotype analyses of a mutant lacking region 1, ∆RI, (see figure below and RMgm-308) suggest that the CSP cleavage site lies within region I and demonstrate that region I is required for complete processing of CSP. The phenotype of ΔRI sporozoites  establishes the link between CSP cleavage and hepatocyte invasion and demonstrates that efficient cleavage requires region I. Moreover, evidence is presented that proteolytic processing of CSP is required for TSR exposure during hepatocyte invasion, but not for its exposure during sporozoite development in the mosquito. The TSR domain is a known cell-adhesive motif C-terminal to the repeats termed the type I thrombospondin repeat (TSR).
In the ∆Nfull sporozoites only 'cleaved' CSP is expressed. Mutant  sporozoites expressed normal amounts of CSP∆Nfull and was exported in normal amounts to the sporozoite surface. Evidence is presented that the TSR-domain is exposed on the surface of ∆Nfull sporozoites in contrast to wild type sporozoites where the TSR-domain is only exposed after proteolytic processing. Despite increased numbers of sporozoites per oocyst, there was a 10-fold reduction in the number of ∆Nfull sporozoites that reached the salivary glands.These observations suggest that exposure of the TSR on hemolymph sporozoites leads to their nonspecific adhesion throughout the mosquito, explaining the low numbers that reach the salivary glands.
The large differences in infectivity between mutant salivary gland sporozoites that are imoculated via the intradermal or the inrtravenous route and the observations of the migration of mutant sporozoites in the skin suggest that the exposure of the TSR- domain on the ∆Nfull sporozoites enhances sporozoite invasion of hepatocytes but inhibits migration of the sporozoites out of the skin (see also figure below).
 
Additional information

 
Figure: Mutated csp genes from the different P. berghei mutant parasites as described in Coppi et al., 2011, J. Exp. Med. 208, 341-56. (RCon: wild type csp; ∆RI: see RMgm-608)

Figure: A model for the role of CSP in the sporozoite's journey (from: Coppi et al., 2011, J. Exp. Med. 208, 341-56).

Other mutants
RMgm-608: A mutant in which the wild type csp gene is replaced with a mutated csp gene, lacking region I (region I is a 5-aa sequence at the N terminus of the repeats).


  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 nameCS, CSP
Details of the genetic modification
Short description of the mutationdeletion of entire N terminus excluding the signal sequence (including region I)
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructPlasmid 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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn the mutant the wild type csp is replaced with a mutated csp containing a specific deletion of entire N terminus, (including region I) and excluding the signal sequence (amino acids NKSIQAQRNLNELCYNEGNDNKLYHVLNSKNGKIYNRNTVNRLLADAPEGKKNEKKNEKIERNNKLKQP.

The mutant CSP gene was generated using a PCR-based approach. Two gene fragments flanking the region to be deleted and including engineered or endogenous restriction sites were amplified and cut to yield fragments that when ligated make a CSP mutant containing the desired deletion.

For the ∆Nfull mutant, the amino acids NKSIQAQRNLNELCYNEGNDNKLYHVLNSKNGKIYNRNTVNRLLADAPEGKKNEKKNEKIERNNKLKQP, which encompass the entire N terminus excluding the signal sequence, were deleted as follows: a 5’ 722-bp fragment was amplified using forward primer P5 (5’-AAAAAAGGTACCAAATATTATATGC-3’; existing KpnI site underlined) and reverse primer P6 (5’-AGAGCAGCTCGCCATATCCTGGAAGTAGAG-3’; introduced PvuII site underlined). Next, a 776-bp 3’ CSP fragment was amplified using forward primer P3 (5’- AGCGTAATAATAAATTGAAACAAAGGCCTCCACCACCAAACCC- 3’; introduced StuI site underlined) and reverse primer P4 (5’- TTATTTAATTAAAGAATACTAATAC-3’; existing PacI site underlined). Both PCR products were gel purified, digested SspI and StuI (resp.) to remove region 1, and ligated. The correct PmlI-PacI ligation product was determined by pcr and subsequently sub-cloned, sequenced and cloned into the pCSComp transfection plasmid.

To generate the final pCSRep targeting construct that would replace the endogenous CSP locus, a CSP 5’UTR targeting region was cloned upstream of the selection cassette in pCSComp. The 5’UTR targeting region was obtained from p9.5∆E by PCR using p9.5∆E specific primers (see article).
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 1AAAAAAGGTACCAAATATTATATGC
Additional information primer 1P5 (KpnI); 5'CSP forward
Sequence Primer 2AGAGCAGCTCGCCATATCCTGGAAGTAGAG
Additional information primer 2P6 (PvuII); 5'CSP reverse
Sequence Primer 3GAGCGTAATAATAAATTGAAACAAAGGCCTCCACCACCAAACCC
Additional information primer 3P3 (StuI); 3'CSP ORF forward
Sequence Primer 4GTTTATTTAATTAAAGAATACTAATAC
Additional information primer 4P4 (PacI); 3'CSP ORF reverse
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6