RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4129
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1202000; Gene model (P.falciparum): PF3D7_1003600; Gene product: inner membrane complex protein 1c, putative (IMC1c; ALV2)
Details mutation: Three carboxy-terminal cysteines substituted and tagged with mCherry
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 24 February 2017, 17:59
  *RMgm-4129
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) : 28223095
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 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherTremp AZ; Dessens JT
Name Group/DepartmentDepartment of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases
Name InstituteLondon School of Hygiene and Tropical Medicine
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4129
Principal nameIMC1c/mCherry-Mutant 1
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNormal growth; mCherry expression in asexual blood stages
Gametocyte/GametemCherry expression in macrogametocytes
Fertilization and ookineteNormal ookinete production. Normal ookinete motility. Evidence is presented that mutant ookinetes possess reduced tensile strength compared to wild type ookinetes as determined in hypo-osmotic exposure experiments
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 C-terminal mCherry tagged version of IMC1c in which the three carboxy-terminal cysteines have been substituted (wild type IMC1c: ... AKPVGCCTGTCR; mutant  IMC1c: ... AKPVGLETGTWR)

Protein (function)
The three motile and invasive stages (zoites) of Plasmodium species (i.e. ookinetes, sporozoites and merozoites), as well as zoites of other apicomplexan parasites, possess a similar cortical structure termed the pellicle. The pellicle is essentially made up of the plasma membrane and an underlying double membrane structure termed the inner membrane complex (IMC). Closely associated with the IMC on its cytoplasmic side is a network of intermediate filaments termed the subpellicular network (SPN), which supports the pellicular membranes and provides mechanical strength to the cell. Several members of an Apicomplexa-specific family of proteins termed IMC1 proteins have been identified as components of the SPN. Structurally related proteins from ciliates and dinoflagellate algae have since been added to this protein family renamed ‘alveolins’, which now defines the Alveolata infrakingdom. In the genus Plasmodium, the number of members of the alveolin family has risen to 12, which are encoded by conserved and syntenic genes. The alveolin family members display differential expression between the three zoite stages of the parasite, with the largest repertoires present in the ookinete and sporozoite according to proteomic studies. It has been shown in the rodent malaria species Plasmodium berghei that the disruption of individual alveolin family members expressed in sporozoites (PbIMC1a), in ookinetes (PbIMC1b) or in both these zoites (PbIMC1h) results in morphological abnormalities that are accompanied by reduced tensile strength of the zoite stages in which they are expressed. Besides roles in morphogenesis and mechanical strength, the Plasmodium alveolins are also involved in gliding motility in both ookinetes and sporozoites, most likely through interactions with components of the glideosome that are situated within the pellicular cytoplasm.

PbIMC1c is composed of 278 amino acids encoded by a single exon. PbIMC1c and its orthologous. proteins share a highly conserved amino-terminal domain related to the IMCp domain superfamily (Pfam12314) that defines the IMC1 proteins/alveolins.
PbIMC1c and its Plasmodium orthologues have a single conserved cysteine motif at their carboxy-terminus that is predicted to act as a palmitoylation site. In P. falciparum, the protein was detected in the palmitoylated proteome of asexual blood stages indicating that it is indeed palmitoylated. PbIMC1c is expressed in all three zoites where it displays a cortical localisation consistent with that of the SPN, and is essential for asexual blood stage parasite development (see RMgm-1120 for unsuccessful attempts to disrupt Pbimc1c)

Phenotype
See RMgm-1122 for a 'wild type' mutant expression mCherry-tagged IMC1c. This mutant showed mCherry expression in merozoites, ookinetes and sporozoites.

IMC1c/mCherry-Mutant 1 showed normal asexual blood stage growth, production of ookinetes, oocysts and infective sporozoites demonstrating that the carboxy-terminal cysteine motif of PbIMC1c is dispensable for parasite development in the mouse and  mosquito.

Evidence is presented that mutant ookinetes possess reduced tensile strength compared to wild type ookinetes as determined in hypo-osmotic exposure experiments

Additional information
Evidence is presented that IMC1c is palmitoylated on its carboxy-terminal cysteine motif.
From the summary: 'These findings support the hypothesis that alveolin (IMC1c) palmitoylation enhances cytoskeletal function by strengthening the connection between the subpellicular network (SPN) and the adjoining inner membrane complex via lipid anchoring'

Other mutants
See IMC1c


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1202000
Gene Model P. falciparum ortholog PF3D7_1003600
Gene productinner membrane complex protein 1c, putative
Gene product: Alternative nameIMC1c; ALV2
Details of the genetic modification
Short description of the mutationThree carboxy-terminal cysteines substituted and tagged with mCherry
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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPlasmid pLP-IMC1c/mCherry (RMgm-1122) was PCR-amplified with primers IMC1C-mut1-F (CTCGAGACAGGTACATGGAGAAGCTTAGGGGCCCTC) and IMC1C-Mut1-R (CCATGTACCTGTCTCGAGTCCAACTGGTTTAGCTTCTTCA) and circularised by in-fusion to give plasmid pLP-IMC1c/mCherry-Mutant 1. The site-directed mutagenesis was designed to substitute the three conserved cysteine residues at the carboxy terminus, and at the same time to introduce a diagnostic XhoI restriction site at the site of the mutation. The resulting targeting vector was digested with KpnI and SacII to remove the plasmid backbone prior to transfection of purified P. berghei schizonts as described. Pyrimethamine-resistant parasites were selected and dilution cloned to give parasite line IMC1c/mCherry-Mutant 1.
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