Mutant/mutation
A series of mutants are described that express an additional copy of CSP that is tagged with GFP at different locations in the protein.  

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 following five parasite lines were generated:
- GFP-GPI, parasites expressing a protein consisting of the signal peptide of CSP, GFP and the C-terminal 22 amino acids of CSP corresponding to the GPI-anchor sequence. This line was obtained through insertion at a silent locus in chromosome 12 of P. berghei strain ANKA.
- SP-GFP-CSP_add, parasites expressing a CSP-GFP fusion protein with the GFP placed between the signal peptide (SP) and the N-terminus of CSP. This line, expressed SP-GFP-CSP in addition to the endogenous CSP and was obtained by linear insertion of the sp-gfp-csp plasmid into the csp locus on chromosome 4.
- SP-GFP-CSP_rep, where the endogenous csp was replaced by the sp-gfp-csp gene.
- R-GFP-CSP, where the GFP was placed between the repeat region and TSR of CSP
- TSR-GFP-CSP; where the GFP was placed between the TSR and the GPI-anchor. Exact positioning of the GFP insertion sites for R-GFP-CSP and TSR-GFP-CSP was assisted by the crystal structure of the TSR domain. In these lines, the fusion proteins were also expressed in addition to the endogenous CSP as obtained by linear insertion of the respective plasmids into the csp locus on chromosome 4. Note that for SP-GFP-CSP_add, the DNA is inserted such that the resulting modified locus features an endogenous csp with a truncated 3’UTR and the fluorescent copy a truncated 5’UTR, while this is inversed for R-GFP-CSP and TSR-GFP-CSP. For SP-GFP-CSP_rep, the gene is flanked by the complete 5’UTR and 3’UTR.

See the paper for a detailed Figure of the modifications/tagging of CSP and explanation about processing events

Normal numbers of oocysts are produced in all 5 lines. 'Normal' sporozoite formation inside oocysts.
Investigating the GFP-CSP localization revealed that all but the two SP-GFP-CSP lines showed the expected surface localization of the fusion proteins. Close inspection also revealed that the GPI-GFP expressing parasite often showed additional small vesicular localization in the proximity of the plasma membrane at the estimated nuclear exit site or Golgi localization.
R-GFP-CSP and TSR-GFP-CSP but not SP-GFP-CSP localize to the plasma membrane.

Strongly reduced numbers of salivary gland sporozoites. Evidence is presented that the expression of tagged proteins inhibited sporozoite egress from oocysts.

Additional information

Other mutants