Malaria parasiteP. yoelii
MutatedGene model (rodent): PY17X_1337400; Gene model (P.falciparum): Not available; Gene product: duffy receptor, beta form precursor (erythrocyte binding like protein, EBL; PyEBL)
Details mutation: The second Cys position in Region 6 of the ebl gene is substituted with Arg.
Phenotype Asexual bloodstage;
Last modified: 18 June 2009, 17:26
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) : 19346470
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17X
Name parent line/clone Not applicable
Other information parent line17X is a non-lethal strain of P. yoelii
The mutant parasite was generated by
Name PI/ResearcherH. Otsuki; M. Torii
Name Group/DepartmentDepartment of Molecular Parasitology
Name InstituteEhime University Graduate School of Medicine
CityToon, Ehime
Name of the mutant parasite
RMgm numberRMgm-276
Principal name17X-CtoR
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageMerozoites of the mutant 17X-CtoR invaded erythrocytes of various ages (reticulocytes and normocytes) whereas the parent 17X line (non-lethal) of P. yoelii mainly invades reticulocytes. Mice infected with blood stages of mutant 17X-CtoR developed higher parasitemias compared to mice infected with the parent line 17X (non-lethal) but did not die at day 7 like mice infected with the 17XL (lethal) line (see also 'Additional information').
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a mutated form of PY04764 (erythrocyte binding like protein, EBL; pyEBL). The endogenous Pyebl is replaced by the mutated form. In the mutated gene the second Cys position in Region 6 is substituted with Arg (as occurs in the lethal strain 17XL; see 'Protein (function)' and 'Additional information').

Protein (function)
PY04764 (Pyebl) is a gene encoding a type I integral transmembrane protein encoded by the ebl (erythrocyte-binding-like) gene family. Upon release from the micronemes, EBL proteins recognize erythrocyte receptors and initiate the formation of the tight junction. Plasmodium vivax uses an EBL orthologue, PvDBP (Pv110810), to recognize the Duffy antigen on the erythrocyte surface. P. falciparum has an expanded family of EBL proteins, for example EBA175 (PF07_0128), EBA181/JESEBL (PFA0125c), EBA140/BAEBL (MAL13P1.60), EBL1 (PFD1145c).

EBL proteins possess 2 Cys-rich regions conserved among EBL orthologues. The N-terminal Cys-rich region named the DBL (Duffy-binding-like) domain or region 2 recognizes a specific erythrocyte surface receptor. The C-terminal Cys-rich region named the C-cys domain or region 6 is located adjacent to the transmembrane domain, and the number and location of Cys residues are well conserved among  Plasmodium species. Region 6 exhibits structural similarity to the KIX-binding domain of the coactivator CREB-binding protein and has been proposed to be a protein trafficking signal for transportation to the micronemes.

Only one 1 nonsynonymous nucleotide substitution was found in region 6 of Pyebl between the nonlethal 17X and lethal 17XL lines. The nonlethal 17X line possesses 8 conserved Cys residues that form 4 disulfide bridges, whereas the lethal 17XL line possesses an Arg instead of Cys at the second Cys position.
All Plasmodium EBL orthologues for which protein expression was validated possess 8 conserved Cys residues in this region,  indicating that the Cys residues play an important role. The observed substitution from Cys to Arg is likely to abolish the native conformation of region 6.

The phenotype analyses indicate that mutation of Pyebl results in a changed erythrocyte-type preference of the parasite. Expression of the mutated PyEBL with the second Cys position in Region 6 substituted with Arg changes the reticulocyte-restricted invasion of the nonlethal P. yoelii 17X line to invasion of erythrocytes of various ages and results in the development of higher parasitemias in mice.

Additional information
In the 17X line, PyEBL localized to the apical end of each merozoite in both the segmented schizont-stage parasite and individual merozoites, where it colocalized with AMA1, a known microneme protein. In the 17XL line PyEBL did not colocalize with AMA1  and showed a more diffused but granular distribution. Immunoelectron microscopy revealed that PyEBL localized in micronemes in the 17X line whereas in the 17XL line, PyEBL localized not in the microneme but in another microorganelle—the dense granules.
To evaluate whether the Arg substitution at the second Cys position is responsible for the altered trafficking of PyEBL, the Cys and Arg in the 17X and 17XL lines were mutated (17X-CtoR and 17XL-RtoC (see RMgm-277 for the 17XL-RtoC mutant).

In the 17X line, replacement of Cys with Arg (mutant 17X-CtoR) altered the PyEBL localization from an apical pattern to a nonapical diffused pattern, and PyEBL did not colocalize with AMA1. Furthermore, the replacement of Arg with Cys in the 17XL line (17XL-RtoC; see RMgm-277) altered the PyEBL localization from a nonapical diffused pattern to an apical pattern. These results indicate that the observed substitution from Cys to Arg is responsible for the altered localization of PyEBL from micronemes to dense granules in the 17XL line.

To determine whether altered localization of PyEBL affects erythrocyte-type invasion preference, infected erythrocytes were examined by microscopy, and a selectivity index (SI) was obtained by calculating multiple parasite infection of single erythrocytes for each parasite line on postinfection day 3 in mice. We found that 17XL-RtoC (RMgm-277) predominantly invaded reticulocytes in the same way as the nonlethal 17X line. The SI of the 17XL line (2.38) was increased in 17XL-RtoC (≈35; P < 0.001). On the other hand, 17X-CtoR was able to invade a variety of ages of erythrocytes, including mature erythrocytes, comparable to the lethal 17XL line, with the SI of the 17X line (16.78) reduced in 17X-CtoR (≈4; P < 0.001). These results indicate that the localization of PyEBL is responsible for the erythrocyte-type preference of the parasite.

Because erythrocyte-type preference frequently correlates with virulence in malaria parasites, the mutamt parasites were analysed for differences in the course of infection and survival of parasite-infected mice. Mice infected with the 17XL-RtoC mutant (RMgm-277) developed significantly lower parasitemias compared with the parental 17XL with 100% survival whereas all mice infected with 17XL  line died by day 7. The pattern observed for the 17XL-RtoC line was identical to that observed for the nonlethal 17X line. These results indicate that trafficking of PyEBL to the micronemes causes the virulence of the 17XL line to be reduced to the same level as the nonlethal 17X line, suggesting that PyEBL is a critical virulence determinant in the 17XL line. The parasitemia of mice infected with 17X-CtoR increased significantly compared with those infected with parental 17X  line during the acute phase of infection on days 4 to 5 (P < 0.001). However, the parasitemia did not reach the level observed for the lethal 17XL line, and it reduced to the same level observed for the 17X line by day 9. No parasites were detectable by microscopy at day 17. This suggests that the 17X-CtoR line is able to invade a greater repertoire of erythrocyte types than 17X but is unable to invade as many types as the 17XL line. This reduced capacity to invade multiple erythrocyte types compared with the 17XL line results in a nonlethal infection, in which all mice survive. Thus, displacement of the EBL from microneme was not sufficient to make this line fully lethal, suggesting the existence of other determinant(s).

Other mutants
RMgm-275: Unsuccessful attempts to disrupt  PY04764 (Pyebl).
RMgm-277: A P. yoelii 17XL (lethal) mutant expressing a mutated version of pyebl (first Arg in Region 6 substituted with Cys)

  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1337400
Gene Model P. falciparum ortholog Not available
Gene productduffy receptor, beta form precursor
Gene product: Alternative nameerythrocyte binding like protein, EBL; PyEBL
Details of the genetic modification
Short description of the mutationThe second Cys position in Region 6 of the ebl gene is substituted with Arg.
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid XhoI
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationSee Otsuki et. al., PNAS 2009 106:7167-72 for detailed information about generation of the replacement constructs used to introduce the mutated PY04764 gene containing the amino acid substitution.
The endogenous Pyebl is replaced by the mutated form. In the mutated gene the second Cys position in Region 6 is substituted with Arg (as occurs in the lethal strain 17XL; see 'Additional information')
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