RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4826
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1119200; Gene model (P.falciparum): PF3D7_0620000; Gene product: PIMMS43 protein (secreted ookinete protein 25 (PSOP25), ookinete surface-associated protein 8 (PSOP8))
Details mutation: P. berghei pimms43 gene replaced by the pimms43 gene of P. falciparum
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Sporozoite;
Last modified: 20 June 2020, 20:23
  *RMgm-4826
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) : 32165544
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 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherUkegbu CV, Vlachou D
Name Group/DepartmentDepartment of Life Sciences
Name InstituteImperial College London
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4826
Principal namePb(Pfc34)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GametePfPIMMS43 prominently localizes on the surface of female gametes or early stage zygotes found in the A. coluzzii blood bolus , as well as on the surface of ookinetes traversing the mosquito midgut epithelia and sporozoites found in the mosquito salivary gland lumen.
Fertilization and ookinetePfPIMMS43 prominently localizes on the surface of female gametes or early stage zygotes found in the A. coluzzii blood bolus , as well as on the surface of ookinetes traversing the mosquito midgut epithelia and sporozoites found in the mosquito salivary gland lumen.
OocystNot different from wild type
SporozoitePfPIMMS43 prominently localizes on the surface of female gametes or early stage zygotes found in the A. coluzzii blood bolus , as well as on the surface of ookinetes traversing the mosquito midgut epithelia and sporozoites found in the mosquito salivary gland lumen.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
In the mutant the P. berghei pimms43 gene has been replaced by the pimms43 gene of P. falciparum

Protein (function)
PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43).
PIMMS43 encode deduced proteins of 505 and 350 amino acids, respectively. N-terminal signal peptides (amino acids 1 to 25 for PfPIMMS43 and 1 to 22 for PbPIMMS43) and C-terminal transmembrane domains (amino acids 482 to 504 for PfPIMMS43 and 327 to 350 for PbPIMMS43) are predicted for both proteins. The transmembrane domains are predicted by PredGPI to also contain signals for attachment of a GPI lipid anchor with 99% probability.

Our transcriptomic profiling of field P. falciparum isolates from Burkina Faso in the midgut of sympatric Anopheles coluzzii (previously Anopheles gambiae M form) and Anopheles arabiensis mosquitoes and a laboratory P. berghei strain in the midgut of A. coluzzii identified hundreds of genes exhibiting conserved and differential expression during gametocyte-to-oocyst development. Several of them encoding putatively secreted or membraneassociated proteins were made part of a screen to identify genes that function during parasite infection of the mosquito midgut. These genes were given a candidate gene number preceded by the acronym PIMMS, for “Plasmodium Infection of the Mosquito Midgut Screen.”
Here, we report the characterization of P. falciparum and P. berghei PIMMS43 that encodes a membrane-bound protein found on the surface of ookinetes and sporozoites. The gene was first reported in P. berghei to be a target of the transcription factor AP2-O, has a role in mosquito midgut invasion and oocyst development, and was named POS8. A later study by another group reported the gene as being important for ookinete maturation, designating it as PSOP25. Evidence is presented that PIMMS43 has no detectable function in ookinete maturation or mosquito midgut invasion but plays a key role in ookinete evasion of the mosquito complement-like response.

Phenotype
Analysis of mutants lacking PIMMS43 (RMgm-4824, RMgm-4825) showed the following:
Normal blood stage development; normal production of gametocytes, gametes and ookinetes. In Anopheles coluzzi no oocyst formation; no sporozoite formation (in A stephensi low numbers of oocysts were observed, but no sporozoites).

We also raised a rabbit polyclonal antibody against a codonoptimized coding fragment of P. falciparum PIMMS43 (amino acids 25 to 481) expressed in E. coli cells and lacking the predicted signal peptide and C-terminal transmembrane domain (α-Pfc43opt). We examined the affinity and specificity of this antibody by generating and using a P. berghei c507 transgenic line (PbPfc43), where PbPIMMS43 was replaced by PfPIMMS43. PCR genotypic analysis confirmed successful modification of the endogenous PbPIMMS43 genomic locus, and RT-PCR analysis confirmed that PfPIMMS43 is transcribed in in vitro-cultured P. berghei ookinetes. Western blot analysis of total protein extracts prepared from purified in vitro-cultured PbPfc43 ookinetes using the α-Pfc43opt antibody revealed a strong band of ∼60 kDa, corresponding to the predicted molecular weight of the deduced PfPIMMS43 protein. PfPIMMS43 prominently localizes on the surface of female gametes or early stage zygotes found in the A. coluzzii blood bolus , as well as on the surface of ookinetes traversing the mosquito midgut epithelia and sporozoites found in the mosquito salivary gland lumen.

Next, we investigated whether PfPIMMS43 could complement the function of its P. berghei ortholog by infecting naïve A. coluzzii mosquitoes with the PbPfc43 parasite line and counting the number of oocysts detected in the mosquito midguts. Infections with c507 and Δc43 parasites served as positive and negative controls, respectively. The results showed that the PbPfc43 line exhibited an intermediate phenotype compared to c507 and Δc43 both in terms of both infection prevalence and intensity. Oocysts were morphologically variable and smaller in size compared to c507 oocysts and contained a small number of sporozoites that could not be transmitted to a new mouse host, resembling the phenotype obtained with Δc43 infections following silencing of the mosquito complement-like system. We examined whether this partial complementation phenotype could be affected upon LRIM1 silencing. Indeed, a significant increase in both the infection prevalence and oocyst numbers was observed , yet oocysts remained small and morphologically variable and produced few sporozoites. These results suggest that PfPIMMS43 can only partly complement the function of its PbPIMMS43 ortholog and corroborate the dual function of PIMMS43 in ookinete to oocyst transition and in oocyst maturation and sporozoite development, respectively.

Additional information
Evidence is presented that:
- Ookinetes Lacking PIMMS43 Are Killed by the Mosquito Complement-Like Response upon Midgut Traversal.
- PIMMS43 Is Additionally Required for Oocyst Maturation and Sporozoite Development
- PIMMS43 KO Leads to Compromised Ookinete Fitness and Attack by the Complement-Like Response

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1119200
Gene Model P. falciparum ortholog PF3D7_0620000
Gene productPIMMS43 protein
Gene product: Alternative namesecreted ookinete protein 25 (PSOP25), ookinete surface-associated protein 8 (PSOP8)
Details of the genetic modification
Short description of the mutationP. berghei pimms43 gene replaced by the pimms43 gene of P. falciparum
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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo express P. falciparum c43 in P. berghei, the transgenic parasite Pb(Pfc43) was created in the c507 line. The Pfc43 replacement construct was generated using the plasmid pL0035 which carries the hDHFR selection cassette. A 1.7 kb HindIII/ApaI fragment upstream of the PbPIMMS43 coding DNA sequence was amplified using primer pair P12/P13 and cloned upstream of the pL0035 selectable marker cassette. The 1.5 kb Pfc43 CDS was amplified from cDNA using the ApaI and SacII primer pair P14/P15 and cloned downstream of the 1.7kb fragment. A 518 bp region corresponding to the 3’UTR, downstream of the PbPIMMS43 stop codon, was amplified as a SacII fragment using primers P16/P17 and cloned downstream of the 1.5 kb Pfc43 CDS. Downstream of the pL0035 selectable marker, a 700 bp region corresponding to part of the PbPIMMS43 coding region and part of the 3’UTR was amplified using primers P18/P19 and cloned as a XhoI and SmaI fragment. This replacement vector was linearized by HindIII and SmaI digestion.
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 nameGFP (gfp-mu3)
Details of the genetic modification
Inducable system usedNo
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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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 / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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