RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-405
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1468100; Gene model (P.falciparum): Not available; Gene product: rhoptry protein (235EBP-1; Py235EBP-1; erythrocyte binding protein 1)
PhenotypeNo phenotype has been described
Last modified: 1 December 2013, 10:39
  *RMgm-405
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 21379566
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii YM
Name parent line/clone Not applicable
Other information parent lineP. yoelii YM is a virulent strain of P. yoelii
The mutant parasite was generated by
Name PI/ResearcherS.A. Ogun; A.A. Holder
Name Group/DepartmentDivision of Parasitology
Name InstituteMRC National Institute for Medical Research
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-405
Principal namePY01365-KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of Py235EBP-1 (PY01365), one member of the py235 gene family.
(The gene model of P. yoelii X (XNL) is shown; however, the gene has been deleted in P. yoelii YM; sequence differences exists between members of the py235 (reticulocyte binding protein) gene family between these two laboratory strains)

Protein (function)
Of the Plasmodium erythrocyte adhesion ligand families identified to date, one of the most studied is the erythrocyte binding ligand family (EBL), which includes P. falciparum erythrocyte binding antigen (EBA)-175 and the Duffy binding protein (DBP) of P. vivax and P. knowlesi, located in the apical organelles of the merozoite.

A second group of high molecular mass adhesion proteins, which was first described in the rodent malaria parasite P. yoelii as Py235 is the reticulocyte binding-like (RBL) super family, so named because of sequence homology with the reticulocyte binding protein RBP-1 and RBP-2 of P. vivax. In P. vivax, these proteins are thought to be involved in erythrocyte selection as they bind to reticulocytes but not mature erythrocytes thereby restricting P. vivax to the invasion of reticulocytes. P. falciparum contains a small group of genes coding for proteins with similarities to Py235 and PvRBP, the PfRH family. In contrast to the PvRBP and PfRH gene families, which are small, the Py235 multigene family contains at least 11 members. Analysis of the sequences on fifteen contigs identified in the P. yoelii genome database, which represent members of the Py235 gene family (and some of which are incomplete), show they have overall conserved structural elements.
The Py235 proteins have been implicated in the selection, recognition and invasion of erythrocytes. Populations of P. yoelii asexual blood stage parasites express multiple members of the py235 gene family. The member Py235EBP-1 (encoded by the single copy gene PY01365) is recognized by mAb 25.77.

Phenotype
Host cell preference (reticulocytes, normocytes), growth/multiplication rate of asexual blood stages was not different from wild type parasites.
Evidence is presented that there was no compensatory significant upregulation of expression of any of the other Py235 genes in the mutant parasite.
It is suggested that that the protein PY01185 (designated PY235EBP-2) is able to fulfill the role of Py235EBP-1 by serving as an invasion ligand (see additional information).

The pehenotype of an independent mutant lacking expression of  PY01365 (RMgm-615) has been reported. The phenotype of this mutant is different from the phenotype reported in this study (see 'Additional Information' below).

Additional information
The mAb 25.77 had previously been used to identify Py235EBP-1, the product of the PY01365 gene. By IFA, this mAb gives a punctuate pattern of fluorescence in the WT parasite line. A similar pattern was also observed for the PY01365-KO parasite line, even though Py235ebp-1 is no longer being expressed. Evidence is presented that mAb 25.77 also recognizes two other members of the Py235 family: gene PY01185 and gene PY05995/PY03534. The PY01365, PY01185, and PY05995/PY03534 sequences form a discrete subset of the family with a degree of similarity in pairwise alignment of greater than 80% at the amino acid sequence level. Evidence is presented that the protein encoded by  PY01185 binds to erythrocytes and has been designated Py235 erythrocyte binding protein-2 (Py235EBP-2), as the second known erythrocyte binding protein from this family.
The similar growth and invasion phenotype of mutant and wild type asexual blood stages may suggest that that the PY01185 gene product (PY235EBP-2) is able to fulfill the role of Py235EBP-1.

The Py235 family of proteins belongs to the reticulocyte binding-like (RBL) super family, with homology with the reticulocyte binding protein RBP-1 (PVX_098585) and RBP-2 (PVX_121920) of P. vivax. P. falciparum contains a small group of genes coding for proteins with similarities to Py235 and PvRBP, the PfRH family (for example see the reticulocyte binding protein 2 homologue a (PF13_0198) and the reticulocyte binding protein 2 homologue b (MAL13P1.176)).

In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another. Based on these results it is proposed that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional redundancy at the protein level gives the parasite phenotypic plasticity in the absence of differences in gene expression.

In an independent study the phenotype has been analysed from an independent P. yoelii mutant lacking expression of PY01365 (see RMgm-615). This mutant showed a different (delayed) course of parasitemia in BALB/c mice and BALB/c mice showed a delayed death from infection. Moreover data is presented indicating that disruption of PY01365 leads to a change in the overall transcription pattern of different members of Py235.

Other mutants
RMgm-615: An independent mutant lacking expression of PY01365


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1468100
Gene Model P. falciparum ortholog Not available
Gene productrhoptry protein
Gene product: Alternative name235EBP-1; Py235EBP-1; erythrocyte binding protein 1
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
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gtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctgggtaccgggccccccctcgaggtcgacggtatcgataagcttgcatgcctgcaggtcaacaataaataataaataaatattgtggaaataaaataacatataattatttttaatacattgatttcccttttatttttttaaatttcattgatataaaaatatataataataacatatatgatttcaaattaatcttttcaaaaatggtgtttatttttgtatgtttgtgtatgaattaatcacataacacatctattaaattgagttggtaatatagacacaaataaatatatatatttttatagcttaaaagtgtgttatgaatattttaagcatattttctttttctttggattgtgtaaaatgaactcatataatgcgtttttttgtttttgttattttgtcattttgttattttgctattttatggattaatttttgtttataaaatgggaaataattttaacatatttaaataaatggagaaaaaatataaaataattataaaaaaaagttaatacacattttttcctgttatagaccttatatttatttatccatatatatatatatatatatatatatatatatatacataccaagtgaattaagaggaaagctaatttattattcagaataatatatgaactatatataatttttattattttggtgtatattaatctgtctatatgcatacatgcaataatttatcgacttatatatcaaataacataaaatagaagtgttttaaattatggatatatgctcaatattcattttttttaataagttagctatatttaaattatacattttatatatggtctcttttttttttaaatattatttaagtgatcatgaaaatataaataatttttttttatttaatatccttttgcttgcatgtggtaaatggaaatttggatgtgttttgaargttcggatatagttgtatggacatataatatattttgtgaaaaattggttttatgtttatacttatgccaatactttttgagtaaaacaaagcaagtgcttataaataattaaagccaattttataatatatatttttttatttaatttgaatttagtagtataattttttatggtaagtgctcaaagagagttgcttataaagtatggtttgtttctttttcgccattttgaattacacattaaaaatatatagatacatatattataatatgaaatcattaataatttagggaaattctacaaatttaaaaacgaataaaataattgtttttcatcatgccataacacaatattgatatatacatgtacaaacattttttttatttggaaaatataaattatataaaaaaaaatgtatagtatacaaaatgagcatattcacacggggtggacgttcattttttcatttttcccctgttttttatgagtatatgataaaattttatgaacatttacacaaaatgaaaatggatatataggaaaaatggagcggtatttcatttatctttgattgtcatttggatattatattaccytgggtaggcaattaaaaatgttaaataacaatttaaggaaattatattttatatattaaaattaacactgtattatatgattcgcttataaaagccactctttccccatgcaaagctgtttaatatcaattttaacaaattacacacatgttaatatatttatatatataatttatatatttataatttatatatttatatttttattatttatatatttattatttattgtgtgtgtcaattcgggtaggatatacctcttttttattgtttaaagcgatttgtattctaaaatataaagratttgaaaaagagaaagatagaatatgatcccatcatatatagccctataatttttatttagcagcgaattaatttttctattaagtttatgtgtaattaaaataacggaatatatataatacaataaaaaagtgcataaattaaaattttttcaattaaatttttttttttaaggggttatataatattaaatatataaaatacgattatatatttttgctacaattttttatattaagatataaatagtaaataaatggtattatatggcatgtaatatataaattttttccaatttttattttatatacacttttcctttttttgtcataaaacttaaacaatttacacattcattttaaaaattgactatttgtttcaacattttttgagtttccgttttataatagtattttcatttgtatattgcttatatatataaatacacacctaaatgttacaaaggatcaatgcataaaccggtgtgtctggtcgtcgcgatgacccccaagaggggcatcggcatcaacaacggcctcccgtggccccacttgaccacagatttcaaacacttttctcgtgtgacaaaaacgacgcccgaagaagccagtcgcctgaacgggtggcttcccaggaaatttgcaaagacgggcgactctggacttccctctccatcagtcggcaagagattcaacgccgttgtcatgggacggaaaacctgggaaagcatgcctcgaaagtttagacccctcgtggacagattgaacatcgtcgtttcctcttccctcaaagaagaagacattgcggcggagaagcctcaagctgaaggccagcagcgcgtccgagtctgtgcttcactcccagcagctctcagccttctggaggaagagtacaaggattctgtcgaccagatttttgtcgtgggaggagcgggactgtacgaggcagcgctgtctctgggcgttgcctctcacctgtacatcacgcgtgtagcccgcgagtttccgtgcgacgttttcttccctgcgttccccggagatgacattctttcaaacaaatcaactgctgcgcaggctgcagctcctgccgagtctgtgttcgttcccttttgtccggagctcggaagagagaaggacaatgaagcgacgtatcgacccatcttcatttccaagaccttctcagacaacggggttccctacgactttgtggttctcgagaagagaaggaagactgacgacgcagccactgcggaaccgagcaacgcaatgagctccttgacgtccacgagggagacaactcccgtgcacgggttgcaggctccttcttcggccgcagccattgccccggtgttggcgtggatggacgaagaagaccggaaaaaacgcgagcaaaaggaactgattcgggccgttccgcatgttcactttagaggccatgaagagttccagtaccttgatctcattgccgacattattaacaatggaaggacaatggatgaccgaacgggcgttggtgtcatctccaaattcggctgcactatgcgctactcgctggatcaggcctttccacttctcaccacaaagcgtgtgttctggaaaggggtcctcgaagagttgctgtggttcattcgcggcgacacgaacgcaaaccatctttctgagaagggcgtgaagatctgggacaagaatgtgacacgcgagttcctcgattcgcgcaatctcccccaccgagaggtcggagacatcggcccgggctacggcttccagtggagacacttcggcgcggcatacaaagacatgcacacagactacacagggcagggcgtcgaccagctgaagaatgtgatccagatgctgagaacgaatccaacagatcgtcgcatgctcatgactgcctggaatcctgcagcgctggacgaaatggcgctgccgccttgtcacttgttgtgccagttctacgtgaacgaccagaaggagctgtcgtgcatcatgtatcagcggtcgtgcgatgtcggcctcggcgtccccttcaacatcgcttcctattcgcttttgacgctcatggttgcacacgtctgcaacctaaaacctaaggagttcattcacttcatggggaacacgcatgtctacacgaaccatgtcgaggctttaaaagagcagctgcggagagaaccgagaccgttccccattgtgaacatcctcaacaaggaacgcatcaaggaaatcgacgatttcaccgccgaggattttgaggtcgtgggctacgtcccgcacggacgaatccagatggagatggctgtctagcggaaatacagaagctagctttgatcccgtttttcttacttatatatttataccaattgattgtatttataactgtaaaaatgtgtatgttgtgtgcatatttttttttgtgcatgcacatgcatgtaaatagctaaaattatgaacattttattttttgttcagaaaaaaaaaactttacacacataaaatggctagtatgaatagccatattttatataaattaaatcctatgaatttatgaccatattaaaaatttagatatttatggaacataatatgtttgaaacaataagacaaaattattattattattattatttttactgttataattatgttgtctcttcaatgattcataaatagttggacttgatttttaaaatgtttataatatgattagcatagttaaataaaaaaagttgaaaaattaaaaaaaaacatataaacacaaatgatgttttttccttcaatttcgatatcgaattcctgcagcccgggggatccactagttctagagcggccgccaccgcggtggagctccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttag
Restriction sites to linearize plasmid ApaI, XbaI
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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 1gccgggggcccACTATAACACTAATTATTTATTATAAAACG
Additional information primer 15'UTR forward (ApaI)
Sequence Primer 2gccggaagcttATGTATGTATCTATGTATGCATGCATG
Additional information primer 25'UTR reverse (HindIII)
Sequence Primer 3gccgggaattcACGAACTCACTCGAATACAAAGTCGTTTAG
Additional information primer 33'UTR forward (EcoRI)
Sequence Primer 4ggcggtctagaATAATTTTTATATTTTGCATCATCATTATTATTATGG
Additional information primer 43'UTR reverse (XbaI)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6