Summary

RMgm-4160
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1002100; Gene model (P.falciparum): PF3D7_0404400; Gene product: 6-cysteine protein (P36)
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
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 (P230p)
Phenotype Sporozoite; Liver stage;
Last modified: 28 May 2017, 14:30
  *RMgm-4160
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28506360
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 cl15cy1
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherManzoni G, Silvie O
Name Group/DepartmentCentre d'Immunologie et des Maladies Infectieuses
Name InstituteSorbonne Universités
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-4160
Principal namePbΔp36
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNormal sporozoite production. Sporozoites fail to establish an infection in hepatocytes. Analysis of the kinetics of PbΔp36 sporozoite invasion by FACS, in comparison to control PbGFP sporozoites, revealed that genetic ablation of p36 abrogates sporozoite productive invasion of HepG2 (SR-BI-dependent entry pathway) and Hepa1-6 cells (CD81-dependent entry pathway)(see below for more phenotype information).
Liver stageNormal sporozoite production. Sporozoites fail to establish an infection in hepatocytes. Analysis of the kinetics of PbΔp36 sporozoite invasion by FACS, in comparison to control PbGFP sporozoites, revealed that genetic ablation of p36 abrogates sporozoite productive invasion of HepG2 (SR-BI-dependent entry pathway) and Hepa1-6 cells (CD81-dependent entry pathway)(see below for more phenotype information).
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of P36 and expresses GFP under control of the constitutive hsp70 promoter. The mutant lacks a drug-selectable marker, which has been removed by negative selection using a ‘‘Gene Out Marker Out’’ strategy (see RMgm-1026).

Protein (function)
P52 (P36) and P36 are members of a small family of proteins, the 6-cysteine (cys) family of (surface) proteins (Thompson J. et al., Mol. Biochem. Parasitol. (2001)118, 147-54). The proteins are characterised by domains of roughly 120 amino acids in size that contain six positionally conserved cysteines (6-cys). Although some species of Plasmodium (may) contain unique members of the 6-cys family, ten members have been identified that are conserved both in structure as well as in genome organisation throughout the genus. Some of the conserved 6-cys proteins are encoded by genes that form paralogous gene-pairs which are closely linked in the genome separated by less then 2 kb of intergenic region. Most members have a GPI anchor and are predicted membrane surface proteins whereas others appear to be secreted and most members are expressed in a discrete stage-specific manner in gametocytes, sporozoites or merozoites.
Both genes have been shown to play a role in invasion/development of sporozoites in hepatocytes (see PF3D7_0404400 and PF3D7_0404500

Phenotype
Normal sporozoite production. Sporozoites fail to establish an infection in hepatocytes. Analysis of the kinetics of PbΔp36 sporozoite invasion by FACS, in comparison to control PbGFP sporozoites, revealed that genetic ablation of p36 abrogates sporozoite productive invasion of HepG2 (SR-BI-dependent entry pathway) and Hepa1-6 cells (CD81-dependent entry pathway)(see below for more phenotype information).

By combining inhibitory studies using antibodies against the hepatocyte proteins CD81 and SR-BI and experimental genetic approaches the following evidence is presented:

- SR-BI is a major entry factor for P. berghei sporozoites in CD81-null HepG2 cells.
- CD81 and SR-BI play redundant roles during productive invasion of hepatocytic cells by P. berghei sporozoites.
- CD81 but not SR-BI plays a central role during P. yoelii sporozoite invasion in HepG2/CD81 cells, similarly to P. falciparum in human hepatocytes.
- P52 and/or P36 are required for sporozoite productive invasion, in both P. berghei and P. yoelii, irrespective of the entry route.
- P. berghei P36 is a key determinant of CD81-independent entry via SR-BI. 

Additional information
From the paper:
'We further sought to investigate whether P52 and/or P36 proteins contribute to the selective usage of host cell receptors by different sporozoite species. We designed a trans-species genetic complementation strategy in which copies of P. berghei (Pb), P. yoelii (Py), P. falciparum (Pf) or P. vivax (Pv) P52 and P36 were introduced in the Δp52/p36 parasites.
For this purpose, we used centromeric plasmid constructs for stable expression of the transgenes from episomes. Complementing PbΔp52/p36 sporozoites with PbP52 and PbP36 restored sporozoite infectivity to both HepG2 and HepG2/CD81 cells, where the parasite formed UIS4-positive vacuoles, confirming that genetic complementation was efficient. Remarkably, complementation of PbΔp52/p36 with PyP52 and PyP36 restored infection in HepG2/CD81 but not in HepG2 cells, where only low numbers of UIS4-negative intranuclear EEFs were observed. Thus, the concomitant replacement of PbP52 and PbP36 by their P. yoelii counterparts reproduced a P. yoelii-like invasion phenotype in chimeric P. berghei sporozoites, indicating that P52 and/or P36 contribute to the selective usage of a CD81-independent entry pathway in P. berghei sporozoites.

Complementation of PbΔp52/p36 parasites with either P. falciparum or P. vivax P52 and P36 coding sequences did not restore infectivity of transgenic sporozoites, not only in HepG2 and HepG2/CD81 cells, but also in primary human hepatocytes, the most permissive cellular system for human malaria sporozoites in vitro. Hence it was not possible using this approach to assess the function of P. falciparum or P. vivax P52 and P36 in transgenic P. berghei sporozoites.

We next dissected the individual contribution of P52 and P36 by complementing PbΔp52/p36 parasites with mixed combinations of either PyP52 and PbP36 or PbP52 and 269 PyP36. This approach revealed that P36 determines the ability of P. berghei sporozoites to enter cells via a CD81-independent route. Indeed, PbΔp52/p36 complemented with PyP52 and PbP36 infected both HepG2/CD81 and HepG2 cells, forming UIS4-labeled PVs in both cell types. P52 therefore is not responsible for the phenotypic difference between P. berghei and P. yoelii. In sharp contrast, complementation of PbΔp52/p36 with PbP52 and PyP36 restored sporozoite infectivity to HepG2/CD81 but not HepG2 cells, thus reproducing a P. yoelii-like invasion phenotype.

In reciprocal experiments, we analysed whether expression of PbP36 would be sufficient to allow P. yoelii sporozoites to invade CD81-null cells. For this purpose, we performed genetic complementation experiments in PyΔp52/p36 parasites, using the same constructs employed with the P. berghei mutant. Strikingly, whilst HepG2 cells are normally refractory to P. yoelii productive invasion, complementation with P. berghei P52 and P36 protein was sufficient to confer chimeric P. yoelii mutants the capacity to infect HepG2 cells. Most importantly, PyΔp52/p36 parasites complemented with PyP52 and PbP36 283 infected both HepG2 and HepG2/CD81 cells. In particular, PyΔp52/p36 parasites expressing PbP36 became capable of forming UIS4-positive PVs in HepG2 cells. Thus, the transgenic expression of PbP36 appears to be sufficient to recapitulate a P. berghei-like invasion phenotype in P. yoelii sporozoites. In contrast, complementation of PyΔp52/p36 parasites with PbP52 and PyP36 restored sporozoite infectivity in HepG2/CD81 cells only, but not in HepG2 cells. This confirms that P52, although essential for sporozoite entry, is not directly associated with host receptor usage. Finally, invasion of PbP36-expressing PyΔp52/p36 sporozoites was abrogated by anti-SR-BI antibodies in HepG2 cells, demonstrating that P. berghei P36 is a key determinant of CD81-independent entry via SR-BI. 

From the abstract: ' Here we show that the two main species causing malaria in humans, P. falciparum and P. vivax, rely on two distinct host cell surface proteins, CD81 and the Scavenger Receptor BI (SR-BI), respectively, to infect hepatocytes. By contrast, CD81 and SR-BI fulfil redundant functions during infection by the rodent parasite P. berghei. Genetic analysis of sporozoite factors reveals the 6-cysteine domain protein P36 as a major parasite determinant of host cell receptor usage'

Other mutants
see PF3D7_0404400 and PF3D7_0404500
 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1002100
Gene Model P. falciparum ortholog PF3D7_0404400
Gene product6-cysteine protein
Gene product: Alternative nameP36
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationPbΔp52p36 and PbΔp36 mutant parasites were generated using a ‘‘Gene Out Marker Out’’ strategy (see RMgm-1026).
Wild type P. berghei ANKA blood stage parasites were transfected with pbp52p36 and pbp36 targeting constructs using standard transfection methods. GFP-expressing parasite mutants were isolated by flow cytometry after positive and negative selection rounds (see RMgm-1026)
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
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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationPbΔp52p36 and PbΔp36 mutant parasites were generated using a ‘‘Gene Out Marker Out’’ strategy (see RMgm-1026).
Wild type P. berghei ANKA blood stage parasites were transfected with pbp52p36 and pbp36 targeting constructs using standard transfection methods. GFP-expressing parasite mutants were isolated by flow cytometry after positive and negative selection rounds (see RMgm-1026)
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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 nameP230p
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