Mutant/mutation
The mutant expresses a GFP fusion with the CS protein (CSP; PBANKA_040320; under control of the eef1α promoter). The CS amino terminal region (amino acids 1-74), which includes the signal sequence and putative pexel motifs, was cloned in-frame at the N-terminus of GFP followed by the 3’ P.berghei dhfr UTR for proper expression. The fusion cs-gfp gene was introduced by transient transfection using circular plasmids. In this study three additional mutants are described. Since P.berghei show two putative pexel motifs, three mutants were made. The first contains a mutation in the pexel1 motif, the second is mutated in pexel2, and the third has mutation in both motifs simultaneously. For pexel motif mutagenesis, the conserved arginine and leucine were mutated to alanine using the Quick change XL-II site directed mutagenesis kit (Stratagene, USA).
The cs fusion gene is under the control of the constitutive eef1a promoter, resulting in expression of CS in blood stages.

Protein (function)
The CS protein is the major protein on the surface of sporozoites and is critical for development of sporozoites within the oocysts and is involved in motility and invasion of both the salivary gland of the mosquito and the liver cells. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. During substrate-dependent locomotion of sporozoites, CS is secreted at the sporozoite anterior pole, translocated along the sporozoite axis and released on the substrate at the sporozoite posterior pole. Following sporozoite invasion of hepatocytes, the CS is released in the host cell cytoplasm.

Phenotype
The analysis of blood stages by fluorescence microscopy showed that the fusion CS-GFP gene when expressed in blood stages entered the red blood cell cytoplasm. Mutation of the two putative pexel/VTS motifs in the CS gene resulted in abortion of transport of the fusion gene into the cytoplasm of the red blood cell. These results indicate that the putative pexel/VTS motifs are involved in transport of the CS protein. See also mutant RMgm-286 in which the endogeneous cs gene is replaced with a cs gene with two mutated pexel/VTS motifs. This mutant shows a defective transport of CS into the cytoplasm/nucleus of the hepatocyte.

Additional information

Other mutants
RMgm-286: A mutant in which the endogeneous cs gene is replaced with a cs gene with two mutated pexel/VTS motifs.