Mutant/mutation
The mutant expresses a GFP-tagged version of CSP. The GFP-tagged csp gene is under control of the uis4 (PBANKA_0501200) promoter (and with a shortened 3'UTR of csp) and is introduced in a locus on chromosome 12 between the genes PBANKA_122210 and PBANKA_122220.

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
High numbers of salivary gland-derived fluorescent sporozoites are produced, but their fluorescence was weak and variable.

To investigate CSP function by live fluorescence microscopy, we recently generated a series of P. berghei parasites expressing internally tagged CSP-GFP fusion proteins (Singer & Frischknecht, 2021; see mutants RMgm-4901). A replacement of CSP with a CSP-GFP fusion where the GFP was inserted between the signal peptide and the N-terminus allowed sporozoite formation. However, sporozoites degenerated within the cysts, and the CSP-GFP fusion was not detectable at the plasma membrane but within internal membranes suggesting processing of the protein shortly after the fusion protein was made. In contrast, two other internally tagged proteins allowed CSP-GFP expression and correct plasma membrane localization, albeit they were only expressed as additional copies from the csp locus. In these parasite lines, the GFP was inserted between either the repeats and the TSR (R-GFP-CSP) or the TSR and the GPI anchor (TSR-GFP-CSP). This allowed full sporozoite formation and targeted the protein to the plasma membrane. Yet, these parasites failed to egress from oocysts (Singer & Frischknecht, 2021; see mutants RMgm-4901).

Here, we sought to explore different strategies to overcome this block in sporozoite egress from oocysts. First, we generated a parasite line, where the N-terminus and the repeats were replaced by GFP. This parasite line did not form sporozoites. Second, we generated a parasite line with decreased expression of an internally tagged CSP-GFP. This allowed sporozoite formation but the sporozoites did not egress. Third, we crossed egress-deficient CSP-GFP parasite lines with wild-type parasites. This allowed sporozoite egress and yielded low numbers of fluorescent salivary gland sporozoites. Lastly, we explored a strategy of expressing an additional copy of CSP-GFP from a silent locus and two promoters that are only active upon salivary gland entry, uis4 (PBANKA_0501200) and spect2 (PBANKA_1006300). This gave high numbers of salivary gland-derived fluorescent sporozoites, but their fluorescence was weak.

Additional information

Other mutants