| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) |
Gene disruption
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| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 17335349
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| MR4 number |
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| Parent parasite used to introduce the genetic modification |
| Rodent Malaria Parasite | P. berghei |
| Parent strain/line | P. berghei ANKA |
| Name parent line/clone |
P. berghei ANKA 2.34
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| Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943). |
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| The mutant parasite was generated by |
| Name PI/Researcher | J.D. Raine, R.E. Sinden |
| Name Group/Department | Division of Cell and Molecular Biology |
| Name Institute | Imperial College |
| City | London |
| Country | United Kingdom |
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| Name of the mutant parasite |
| RMgm number | RMgm-119 |
| Principal name | ∆pblap4 |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype |
| Asexual blood stage | Not different from wild type |
| Gametocyte/Gamete | Not different from wild type |
| Fertilization and ookinete | Not different from wild type |
| Oocyst | Normal numbers of oocysts are produced. Sporozoite formation within the oocysts is blocked. The diameters of oocysts were significantly larger than that of wild type on days 14 and 21 of infection. Light microscopy revealed the presence of two distinct populations of oocysts: those that displayed a phenotype reminiscent of immature wild type oocysts (i.e., non-sporulated), and those that appeared vacuolated/degenerate compared to wild type. Transmission electron microscopy analysis of oocysts further confirmed these findings and revealed that oocysts of these parasites possessed an endoplasmic reticulum that was highly vacuolated compared to that of wild type parasites. On day 13 p.i., the nuclear organization of oocysts appeared “immature” as indicated by the presence of few but large nuclei. By comparison, wild type oocysts of the same age had formed sporozoites, each with their own (haploid) nucleus. |
| Sporozoite | No midgut sporozoites were observed on day 10/11 p.i. By day 18 p.i., reduced numbers (typically 0%–12%) of sporozoites were observed in dissected midguts. The number of sporozoites in salivary gland preparations infections was consistently reduced to <1% of wild type. The expression and targeting of the circumsporozoite protein in midgut sporozoites was indistinguishable from that in wild type.
To test if the observed sporozoites were infectious to mice, infected mosquitoes were allowed to feed on mice on days 21 and 28 p.i. Blood stage parasites were observed in all mice bitten by wild type-infected mosquitoes when screened on day 4/5 post-bite. In contrast, mice bitten by mutant-infected mosquitoes remained uninfected. |
| Liver stage | Not tested |
| Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of LAP4 (LCCL/lectin adhesive-like protein 4; LCCL domain containing protein CCp2)
Protein (function)
The lap4 (ccp2) gene is a member of a small conserved gene family, encoding proteins with multiple adhesive domains, for example a Lgl1 (LCCL)-lectin adhesive domain. In P. falciparum the LCCL domain-containing proteins are termed PfCCp's.
Phenotype
Phenotype analyses indicate a role during oocyst development and sporozoite formation/production (see also additional information).
Additional information
Crossings of the mutant females with wild type males did not rescue the formation/production of sporozoites. Crossing of the mutant males with wild type females resulted in wild type production of sporozoites that were infectious to C57BL/6 mice. The lack of rescue of the ∆pblap4 mutant phenotype by crossing of mutant females with wild type males is suggestive of a role of LAP4 within a few hours after fertilisation.
Other members of the LAP/CCp family of proteins have been analysed by targeted gene disruption. A number of mutants lacking expression of these proteins show a comparable defect in development of oocysts and formation of sporozoites (RMgm-113, RMgm-114, RMgm-115: LAP1/PbSLAP/PbSR/CCp3; RMgm-118: LAP2/CCp1; RMgm-98: LAP5/FNPA; RMgm-120: LAP6/CCp4).
A P. falciparum mutant has been generated that lacks expression of CCp2 (LAP4). This mutant showed 'normal' sporozoite formation (sporulation) within oocysts in Anopheles freeborni. Howver, no hemocoel or salivary gland sporozoites were detected (Pradel G. et al., 2004, J. Exp. Med 199, 1533-1544).
Other mutants |
*RMgm-119
