RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-972
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1107100; Gene model (P.falciparum): PF3D7_0507500; Gene product: subtilisin-like protease 1 (SUB1)
Details mutation: The mutant contains a FRTed ORF of the sub1 locus
Details conditional mutagenesis: The ORF of sub1 is removed in the sporozoite stage by the Flp/FRT system.
Transgene
Transgene not Plasmodium: FlpL recombinase of yeast (FlpL)
Promoter: Gene model: PBANKA_0501200; Gene model (P.falciparum): Not available; Gene product: early transcribed membrane protein up-regulated in infective sporozoites
3'UTR: Gene model: PBANKA_1349800; Gene product: sporozoite surface protein 2 thrombospondin-related anonymous protein (sporozoite surface protein 2; SSP2; SSP-2)
Insertion locus: Gene model: PBANKA_0501200; Gene product: early transcribed membrane protein up-regulated in infective sporozoites
Phenotype Liver stage;
Last modified: 22 December 2013, 21:26
  *RMgm-972
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) Reference 1 (PMID number) : 24348254
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone RMgm-278
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherC. Suarez; O. Billker; M.J. Blackman
Name Group/DepartmentDivision of Parasitology
Name InstituteMedical Research Council National Institute for Medical Research, Mill Hill
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-972
Principal namecondSUB1(short); condSUB1
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 stageGFP expression in mutant oocysts/sporozoites showed high levels (>90%) of SSR (site-specific recombination) in salivary gland sporozoites, which resulted in the removal of the FRTed ORF of SUB1.
The mutant sporozoites showed an infectivity to HepG2 cells that was comparable to wild type parasites and produced liver stages at 28 hour after incubation of mutant sporozoites with HepG2 cells that were comparable in number and size to that of wild type sporozoites. However, 48 hour mutant liver stages were significantly smaller than wild type liver stages. The majority of the PbSUB1-deficient parasites completely lacked a detectable MSP1 signal. Multiple nuclei were observed in the early PbSUB1-deficient schizonts, indicating normal nuclear replication, but in later stages many of the schizont nuclei appeared condensed and abnormal. Mutant liver stages were deficient in merosome formation. SUB1-deficient liver stages are unable to establish a blood stage infection.
Additional remarks phenotype

Mutant/mutation
The mutant is a 'FlpL/FRT conditional knock-out mutant' of SUB1 (subtilisin-like protease 1). The mutant expresses the yeast FlpL recombinase under the control of the uis4 promoter  and contains a FRTed ORF (open reading frame) of the sub1 locus.  This mutant has been generated by replacement of the endogenous sub1 gene by an sub1 gene with a FRTed ORF in the mutant RMgm-278 that expresses FlpL.
The sub1 ORF is removed from the genome by using the Flp/FRT site-specific recombination (SSR) system (see RMgm-269). Removal of the FRTed ORF of sub1  has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase in sporozoites that resulted in the excision of the ORF of sub1 which was flanked by FRT sequences. In addition, excision of the ORF of sub1 results in placing the orf of GFP under the control of the constitutive hsp70 promoter. Thus sporozoites and liver stages from which the ORF of sub1 is excised express GFP.

Protein (function)
The subtilisin-like protease SUB1, a bacterial-like serine protease, is a Plasmodium merozoite product essential for the parasite erythocytic cycle. In P. falciparum, SUB1 was shown to have a dual function. It is crucial for merozoite egress from erythrocytes and renders released merozoites competent for a new round of erythrocyte invasion. In the process of P. falciparum merozoite egress from erythrocytes, SUB1 carries out the maturation of the family of papain-like proteases called serine repeat antigens (SERAs). It remains unknown whether the SERAs have a direct role in membrane disruption, or whether they in turn maturate other effectors, but it is established that their SUB1-dependent maturation is essential for the egress of merozoite. The reported role of SUB1 in P. falciparum merozoite invasion is to undertake the processing of merozoite surface proteins (MSPs), including MSP1. After SUB1 maturation, the membrane-anchored C-terminal 42-kDa fragment of MSP1, remains associated with the other MSP1 fragments and additional partners, including MSP6 and MSP7.

Phenotype
The Flp/FRT site-specific recombination (SSR) system of yeast has been used to silence SUB1 expression specifically in liver stages. Removal of the FRTed ORF of sub1  has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase in sporozoites that resulted in the excision of the FRTed ORF of sub1. In addition, excision of the ORF of sub1 results in placing the orf of GFP under the control of the constitutive hsp70 promoter. Thus sporozoites and liver stages from which the ORF of sub1 is excised express GFP.

GFP expression in mutant oocysts/sporozoites showed high levels (>90%) of SSR (site-specific recombination) in salivary gland sporozoites, which resulted in the removal of the FRTed ORF of SUB1.

The mutant sporozoites showed an infectivity to HepG2 cells that was comparable to wild type parasites and produced liver stages at 28 hour after incubation of mutant sporozoites with HepG2 cells that were comparable in number and size to that of wild type sporozoites. However, 48 hour mutant liver stages were significantly smaller than wild type liver stages. The majority of the PbSUB1-deficient parasites completely lacked a detectable MSP1 signal. Multiple nuclei were observed in the early PbSUB1-deficient schizonts, indicating normal nuclear replication, but in later stages many of the schizont nuclei appeared condensed and abnormal. Mutant liver stages were deficient in merosome formation. SUB1-deficient liver stages are unable to establish a blood stage infection.

Additional information
See also RMgm-952 for an independent mutant containing a FRTed ORF of the sub1 locus. Analyses of this mutant provided evidence for  normal development of liver merozoites but a reduced egress of liver merozoites from the mature liver schizonts. Evidence is presented that the reduced egress is due to lack of rupture of the PVM indicating a role of SUB1 in PVM rupture. in addition, evidence is presented for a role of SUB1 in processing of SERA3 and MSP1. SUB1-deficient liver stage merozoites are unable to establish a blood stage infection.

See RMgm-973 for a mutant mutant expressing a HA-tagged version of SUB1. Staining of PbSUB1-HA schizonts with the anti-HA mAb again produced a clear punctate signal. The foci were associated with but distinct from individual merozoite nuclei, and similar to the exoneme-specific signal previously observed in P. falciparum blood stage schizonts. No PbSUB1-HA IFA signal was detected in salivary gland sporozoites or early liver stage schizonts.

Other mutants
RMgm-952: An independent mutant containing a FRTed ORF of the sub1 locus
RMgm-973: A mutant expressing a HA-tagged version of SUB1.


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1107100
Gene Model P. falciparum ortholog PF3D7_0507500
Gene productsubtilisin-like protease 1
Gene product: Alternative nameSUB1
Details of the genetic modification
Short description of the mutationThe mutant contains a FRTed ORF of the sub1 locus
Inducable system usedFlp/FRT
Short description of the conditional mutagenesisThe ORF of sub1 is removed in the sporozoite stage by the Flp/FRT system.
Additional remarks inducable system
Type of plasmid/constructPCR construct 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/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationWorking from a P. berghei genomic DNA library clone from the PlasmoGEM resource (http://plasmogem.sanger.ac.uk/) containing 9.6 kb of the pbsub1 locus and neighbouring genes, we constructed allelic exchange vectors designed to flank the entire pbsub1 coding sequence with FRT sites, while at the same time inserting a C-terminal HA epitope tag into pbsub1. The 59 FRT site was introduced into one of two alternative positions in the large upstream intergenic region together with a constitutive promoter sequence of the P. berghei hsp70 gene. A promoterless GFP coding sequence was positioned immediately downstream of the second FRT site. Precise placement of the FRT sites and other exogenous sequence both upstream and downstream of pbsub1 was achieved in 4 steps by recombinase mediated genetic engineering in E. coli, using both the improved Red/ET recombinase system of lambda phage and transient expression of Flp. The constructs were designed such that, following integration by ends-out homologous recombination into the parasite genome, correct recombinase-mediated excision of the sequence lying between the FRT sites (which included the epitope-tagged pbsub1 gene) would reposition the GFP reporter adjacent to the hsp70 promoter, driving constitutive GFP expression only in those parasites in which deletion of the pbsub1 gene had occurred. To prevent the FRT site from interfering with initiation of translation, the start codon for GFP expression was placed just upstream of the 59 FRT site, such that after excision the FRT sequence would be translated into a 12 residue N-terminal extension of GFP. The final constructs, called pJazz-FRTed-pbsub1 and pJazz-FRTed-pbsub1short (which differed only in the placement of the 59 FRT site at either ,2.3 kb or ,1.8 kb respectively upstream of the start ATG of the pbsub1 gene) contained,7 kb and 700 bp regions of homology respectively at their 59 and 39 ends for homologous integration into the P. berghei genome.
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 nameFlpL recombinase of yeast (FlpL)
Details of the genetic modification
Inducable system usedFlp/FRT
Additional remarks inducable system The mutant expresses the yeast FlpL recombinase under the control of the promoter of uis4. The mutant does not contain a selectable marker. This marker (the hdhfr gene) has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system. Removal of the hdhfr gene has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase that resulted in the excision of the hdhfr gene that was flanked by FRT sequences.
Type of plasmid/constructPlasmid 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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe flpl gene was cloned between 1.5 kb of 5′ regulatory sequence of uis4 and 0.6 kb of 3′ regulatory sequence of trap. The plasmid was linearized in the 5′ uis4 regulatory sequence to direct gap repair and plasmid integration by single crossover recombination at the homologous chromosomal locus, leaving the endogenous uis4 gene intact. The plasmid contained the hdhfr selectable marker that is flanked with the 34bp FRT sequences. The mutant expresses the yeast Flp recombinase under the control of the promoter of uis4. The mutant does not contain the hdhfr selectable marker that has been used to introduce the flp gene. This marker has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system. Removal of the hdhfr gene has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase that resulted in the excision of the hdhfr gene that was flanked by FRT sequences.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0501200
Gene Model P. falciparum ortholog Not available
Gene productearly transcribed membrane protein up-regulated in infective sporozoites
Gene product: Alternative name
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 15'-GCGCCCCGGGTGTGATAGTGTAGATTTTTTTGTTTGACCATTG-3'
Additional information primer 15'- GCGCCCATGGTTTATTCAGACGTAATAATTATGTGCTGAAAGG-3'
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_1349800
Gene productsporozoite surface protein 2 thrombospondin-related anonymous protein
Gene product: Alternative namesporozoite surface protein 2; SSP2; SSP-2
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 / InsertionInsertion locus
Gene Model of Parasite PBANKA_0501200
Gene productearly transcribed membrane protein up-regulated in infective sporozoites
Gene product: Alternative name
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