Mutant/mutation
Seven different mutants are described that express mutated forms of the P.yoelii EBL protein. For localization analysis a GFP tag was fused at the C-terminus of the EBL protein. It was tried to independently substitute all eight Cys residues in PyEBL region 6 to Ala residues. Parasite lines were obtained substituting seven Cys with the exception of the 6th Cys at position 754; all transgenic lines showed a GFP signal. 

The 7 mutants are:
Cys1st   (717) C717A 
Cys2nd (726) C726A 
Cys3rd  (738) C738A 
Cys4th  (747) C747A 
Cys5th  (748) C748A 
Cys7th  (768) C768A 
Cys8th  (780) C780A

Protein (function)
Erythrocyte-binding-like (EBL) proteins secreted from micronemes are involved in tight junction formation; and a lack of the erythrocyte receptor or the disruption of the gene locus coding for EBL for Plasmodium knowlesi or Plasmodium yoelii, respectively, resulted in failed cell invasion without forming a tight junction. This is explained by the experimental demonstration that P. vivax EBL, termed the Duffy binding protein (PvDBP), recognizes the Duffy antigen and P. vivax cannot invade erythrocytes lacking the Duffy antigen. 
EBL is a type I transmembrane protein with distinct cysteine-rich domains at the N-terminal and C-terminal sides of the extracellular region. The N- terminal Cys-rich domain binds erythrocyte surface receptor molecules and is called region 2. This region has been extensively studied in vaccine development for P. vivax PvDBP and the P. falciparum ortholog EBA-175. Region 2 has also been studied for its role in erythrocyte invasion, such as conferring the binding ability to recognize erythrocyte surface receptor molecules. In contrast, studies on the C-terminal cysteine- rich domain, termed region 6, are relatively limited. 
Unlike human and simian malaria parasites, EBL molecules in rodent malaria parasites are not represented by a multigene family. Therefore, the rodent malaria model, such as P. yoelii, provides a platform in which EBL function can be analyzed without interference from paralogous molecules.
EBL region 6 has eight Cys residues which are conserved among known functional EBL molecules of Plasmodium spp. The P. yoelii 17X strain shows low parasitemia and a preference to invade immature erythrocytes. The 17XL strain was derived from the 17X strain following passage in mice and in contrast, it shows significantly higher parasitemia, invasive ability into all erythrocytes, and lethality in mice. The P. yoelii 17X non-lethal strain contains eight Cys residues in PyEBL region 6; whereas the 2nd Cys within the domain is substituted to Arg in the 17XL lethal strain.
The PyEBL protein is located on the dense granules in the 17XL strain, in contrast to a micronemal localization for all known EBLs possessing eight intact Cys residues in region 6. The Cys to Arg substitution in the 17XL strain was shown to be responsible for altering the parasite infection course, virulence, and PyEBL localization. 
It has been reported that P. falciparum EBA-175 region 6 is responsible for its trafficking to the micronemes. The crystal structure of EBA-175 region 6 suggested the disulfide bonding pattern of eight Cys residues. However, it remains unclear if other Cys residues in PyEBL region 6 are also responsible for correct PyEBL localization in the micronemes, and if different PyEBL localization patterns are associated with parasite virulence. Region 6 amino acids are highly conserved among Plasmodium parasites and this suggests a functional importance for this domain. The eight cysteines likely form four disulfide bonds which underpin the domain stability and conformation. Functional analysis via comprehensive modification of the disulfide bonds within the domain, and the impact of the modifications on intracellular localization, may cast light on the importance and uniqueness of this vaccine candidate molecule. 

Phenotype
It was tried to independently substitute all eight Cys residues in PyEBL region 6 to Ala residues. Parasite lines were obtained substituting seven Cys with the exception of the 6th Cys at position 754; all transgenic lines showed a GFP signal (for localization analysis a GFP tag was fused at the C-terminus of the mutated EBL's).

The 7 mutants are:
Cys1st   (717)  C717A 
Cys2nd (726)  C726A 
Cys3rd  (738)  C738A 
Cys4th  (747)  C747A 
Cys5th  (748)  C748A 
Cys7th  (768)  C768A 
Cys8th  (780)  C780A

The P. yoelii 17X parasite shows a non-lethal phenotype with low parasitemia and a preference to invade reticulocytes, whilst the 17XL lethal strain shows a higher parasitemia of more than 80% and no preference for a target erythrocyte type. Our previously reported transgenic 17X parasite in which the 2nd Cys in PyEBL region 6 was substituted to Arg also showed a higher parasitemia, almost as high as the lethal 17XL strain. 
The infection course and parasitemia of the 7 mutants on day 6 can be roughly classified into two groups. One group contains the C717A, C726A, C747A, C748A, and C768A lines and showed higher parasitemias than the 17X strain, a similar pattern to 17XL. The other group consists of the C738A and C780A lines, with them growing slightly slower and leading to lower parasitemia on day 6 than the parental 17X strain, although the difference was not significant. 

Infection with the parental 17X strain did not kill any mice, whereas the lethal 17XL strain parasite killed all the infected mice by day 9. 
The survival of mice infected with the seven mutants was classified into two distinct groups. One is the sublethal group, including the C726A, C747A, and C748A lines, which killed more than two mice but not all. These lines showed similar levels of parasitemia as the lethal 17XL strain and were significantly higher than the other transgenic lines. The second group did not kill any mice by day 9; specifically, the C717A and C768A lines with higher parasitemia and the C738A and C780A lines.

To investigate whether the altered infection courses observed in the transgenic lines were caused by a difference of erythrocyte preference in the invasion process, a selectivity index (SI) was calculated by multiple parasite infections of single erythrocytes for each parasite line on day 4 post infection. A higher SI value indicates that the parasites invaded into a limited population of erythrocytes, and lower SI means that the  parasites invaded into a broader population of erythrocytes.
We found that five lines with higher parasitemia (C717A, C726A, C747A, C748A, and C768A) showed low SI (0.5~7.3), which is similar to the lethal 17XL strain (SI = 6.8) and lower than the parental 17X strain (SI = 14.6, no significance). This suggests that they invaded a wider range of erythrocyte populations than the 17X strain. In contrast, the C738A and C780A lines showed significantly higher SI (46.8 and 37.3) than the parental 17X strain (p < 0.01) suggesting that they preferred to invade into a more limited population of erythrocytes than the non-lethal 17X strain, which is consistent with the slower growth of these two transgenic lines than the 17X strain.

In our previous study, the parasite infectivity and erythrocyte preference of P. yoelii 17X lineages were related to the localization of PyEBL; specifically, the 17X-type showed microneme localization whereas the 17XL-type showed dense granule localization.
To investigate the relationship between PyEBL localization and parasite infectivity, IFA was carried out on mature merozoites in schizonts for each parasite line. PyAMA-1 was used as a microneme marker at the merozoite apical end. The C738A and C780A lines with lower parasitemia showed a clear punctate pattern that largely overlapped PyAMA-1. These two Cys residues are reported to form a disulfide bond in the EBA-175 region 6 structure. Thus, these results suggest that disruption of one of the four disulfide bonds in PyEBL region 6 does not affect the PyEBL localization in the micronemes. In contrast, the other five lines with high parasitemia showed a somewhat dispersed pattern and were not well colocalized with PyAMA-1, suggesting that the location of PyEBL and PyAMA-1 are different. 

Additional information
Immunoelectron microscopy was carried out to further confirm the localization of PyEBL in the merozoite. In merozoites obtained from the C738A and C780A lines, which showed lower parasitemia, gold particle deposition indicating EBL localization was observed on the micronemes. In the other five transgenic parasite lines that showed higher parasitemia, no deposition of gold particles was detected in either the organelles or cytoplasm of merozoites. This is in contrast to our previous study, in which the lethal 17XL strain parasite showed PyEBL localization on dense granules after staining with mouse anti-PyEBL R1-6 antibodies.

Other mutants
Other mutants with mutated (Py) EBL proteins