RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-224
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1349100; Gene model (P.falciparum): PF3D7_1335100; Gene product: merozoite surface protein 7 (MSP7)
Phenotype Asexual bloodstage;
Last modified: 16 December 2011, 19:10
  *RMgm-224
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) : 15339842
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/ResearcherR. Tewari, A.A. Holder
Name Group/DepartmentDivision of Parasitology
Name InstituteNational Institute for Medical Research
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-224
Principal nameMSP7-ko
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageMutants show a minor growth defect resulting in a lower growth rate in Balb/c mice during the initial phase of infection. When reticulocytes were prevalent (after day 9 after infection), the growth rate was similar to that of wild type parasites
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of MSP7 (merozoite surface protein 7). Rodent malaria parasites have three MSP7-related genes with an adjacent chromosomal location. The exact orthology with the 6 family members of MSP7 (PF13_0197) and the five MSP7-related genes of P. falciparum (MSRP1-5) is not clear. The gene disrupted in this study is in the middle of the cluster of the 3 P. berghei genes (see 'Additional Information'). No information is however given for the sequence of the disrupted gene and no information is available for the primers used to amplify the target regions for disruption of the msp7 gene by homologous recombination.

Protein (function)
In P. falciparum MSP7 ((PF13_0197) is a protein of the surface coat of merozoites. The surface coat of the merozoite is composed of a number of proteins, the most abundant of which is a complex of  MSP1, MSP6, and MSP7. Glycosylphosphatidyl inositol (GPI) membrane-anchored MSP1 is processed during maturation of merozoites and erythrocyte invasion. Following invasion, only a C-terminal 19-kDa fragment (MSP119) is retained on the parasite membrane within the newly invaded erythrocyte. The rest of the MSP1 protein is shed as a protein complex comprising four polypeptides of MSP1 and associated with 36- and 22-kDa polypeptides derived from MSP6 and MSP7, respectively.
MSP7 is synthesized as a 48-kDa precursor and binds full-length MSP1 in a pre-Golgi compartment. Prior to its translocation to the membrane, MSP7 undergoes a first proteolytic cleavage, releasing an N-terminal 20-kDa fragment (MSP720), while the C-terminal 33-kDa fragment (MSP733) remains in association with the MSP1 precursor (31). The second proteolytic processing of MSP733 yields a 22-kDa C-terminal fragment (MSP722) and occurs along with the primary processing of MSP1. Upon erythrocyte invasion by the merozoite, MSP7 can be detected as a 22- or 19-kDa component of the shed MSP1 complex.

Phenotype
Phenotype analyses indicate that MSP7 is not essential for blood stage parasite growth. However, parasites lacking expression of MSP7 showed a minor growth defect resulting in a lower growth rate in mice during the initial phase of infection. When reticulocytes were prevalent, the growth rate was similar to that of wild type parasites, suggesting that the mutant parasites prefer to invade/growth in reticulocytes. The mutant lacked expression of MSP7 as shown by Northern analysis and IFA using recombinant P. yoelii MSP7 antibodies (no details or reference is provided for these antibodies). No data is provided on the expression of the other two MSP7-like proteins, MSRP1 and MSRP2 of P. berghei (see 'Additional information').

Additional information
In all Plasmodium genomes examined so far, msp7 is a member of a small family of related genes located at the same locus. For example, P. falciparum has MSP7 (PF13_0197) and five MSP7-related genes (MSRP1-5), and in rodent parasites, there are three genes in the family. In both rodent and human parasites these genes have an adjacent chromosomal location. In P. falciparum there is evidence that some or all of these genes are expressed, and therefore, it is possible that one or more MSRPs may substitute for MSP7.
P. berghei and P. yoelii have 3 MSP7-like genes, MSP7, MSRP1 and MSRP2 (see Figure 1 and 2) and in P. yoelii evidence has been presented for expression of both MSP7 and MSRP1. This might indicate  that functional redundancy exists in the rodent parasites between the three family members. For the mutant described here no data is provided on the expression of the other two MSP7-like proteins of P. berghei.

Figure 1
Syntenic mapping of P.yoelii and P.berghei MSRP genes. Three P.yoelii MSRP genes were mapped as described earlier (ref. 1). Based on this analysis, PY02147 is MSRP2, PY02148 is MSP7 and PY02149 is MSRP1. Corresponding P.berghei MRSP genes were mapped to each of the P.yoelii genes. The gene PY02149 was truncated in the PlasmoDb annotation compared with that found in earlier analysis (ref. 1) and therefore this more complete annotation was used instead. Manual re-annotation of PB107618.00.0 revealed an ORF to the 3` of the gene, which was highly similar to the PY02140 (MRSP1) gene. These two ORFs were conceptually joined for mapping analysis, but indicate that PB107618 is most likely a pseudogene.
Figure 2
Phylognetic analysis. After re-annotation, the three and P.berghei gene models were subjected to multiple alignment using BioEdit (version 7.0.9.0) with default alignment settings. The evolutionary history was inferred using the Maximum Parsimony method conducted in MEGA4 (ref. 2). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. This showed that the following genes could be matched pair-wise: PY02147 and PB000441.03.0, PY02148 and PB000442.03.0, PY02149 and PB107618.00.0 and that all genes were located in the same order on the P.yoelii and P.berghei contigs.
References
1. Mello,K., Daly,T.M., Morrisey,J., Vaidya,A.B., Long,C.A. and Bergman,L.W. (2002) A multigene family that interacts with the amino terminus of plasmodium MSP-1 identified using the yeast two-hybrid system. Eukaryot. Cell, 1, 915-925.
2. Tamura,K., Dudley,J., Nei,M. and Kumar,S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol., 24, 1596-1599.
(Data of Figure 1 and 2 are obtained from Dr. J. Fonager).



Other mutants
RMgm-671 and RMgm-673: Independent mutants lacking expression of MSP7 (merozoite surface protein 7)


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1349100
Gene Model P. falciparum ortholog PF3D7_1335100
Gene productmerozoite surface protein 7
Gene product: Alternative nameMSP7
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
Restriction sites to linearize plasmid
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption No sequence information of the target sequences is provided. No information is provided for the primers used to amplify the target regions for disruption of the msp7 gene homologous recombination.
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationNo sequence information of the target sequences is provided. No information is provided for the primers used to amplify the target regions for disruption of the msp7 gene homologous recombination.
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