Sporozoite | Reduced numbers of salivary gland sporozoites. Salivary gland sporozoites show wild-type morphology, motility and infectivity to hepatocytes in vitro. Light-microscopy analysis of oocysts between day 14 and 21 showed the presence of aberrant, enlarged, melanized sporozoites, either still inside or during release from oocysts.
On day 21 p.i. non-motile, enlarged sporozoites covered by melanin were found in the hemocoel, often in clusters or attached to salivary glands. In contrast, most CSP(mut) sporozoites inside salivary glands had a WT-like morphology, i.e. long and slender, without signs of melanization. |
Additional remarks phenotype | Mutant/mutation
The mutant expresses a mutated form of CSP with glutamine in region I of CSP replaced with alanine (Q92A). In addition, it expresses mCherry and luciferase under control of constitutive promoters. The mutated csp gene is introduced in the by GIMO transfection in the 'CSP-GIMO' line RMgm-4681.
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
Normal numbers of oocysts are produced. On day 10 after infection melanized oocysts are present in 26-48% of CSP(mut) infected mosquitoes (1-35 melanized oocysts/mosquito), while such oocysts were absent in WT-infected mosquitoes.
Reduced numbers of salivary gland sporozoites. Salivary gland sporozoites show wild-type morphology, motility and infectivity to hepatocytes in vitro. Light-microscopy analysis of oocysts between day 14 and 21 showed the presence of aberrant, enlarged, melanized sporozoites, either still inside or during release from oocysts.
On day 21 p.i. non-motile, enlarged sporozoites covered by melanin were found in the hemocoel, often in clusters or attached to salivary glands. In contrast, most CSP(mut) sporozoites inside salivary glands had a WT-like morphology, i.e. long and slender, without signs of melanization. Salivary gland sporozoites show wild-type morphology, motility and infectivity to hepatocytes in vitro. Wild type development of liver stages in vitro.
Additional information
From the paper:
'Circumsporozoite surface protein (CSP) is the most abundant protein on the sporozoite surface. We therefore hypothesized that CSP could be a prime target for immune recognition of QC-null parasites. We analyzed published proteomes for pGlu modification of CSP and identified multiple CSP peptides with pGlu at the N-terminus with the majority (>90%) having pGlu modification of the glutamine (Q) located in region 1 of CSP. This short, five amino acid region (KLKQP), contains a proteolytic cleavage site and the glutamine is conserved across different Plasmodium species. CSP cleavage is known to occur at the sporozoite surface and this processing step is essential for host cell invasion. To establish whether CSP is a main target for immune recognition of QC-null sporozoites, two independent P. berghei lines (csp(mut)1,2) were generated that express a mutated CSP with glutamine in region 1 replaced with alanine (Q92A), thereby preventing pGlu formation. Both csp(mut) mutants produced similar numbers of oocysts as WT parasites. No melanization of csp(mut) oocysts was observed until day 10,whereas at day 14-16 p.i., melanized oocysts were detected in 26-48% of csp(mut)-infected mosquitoes (1-35 melanized oocysts/mosquito), mainly rupturing oocysts. Clusters of melanized sporozoites were found in the hemocoel and csp(mut) sg-sporozoite numbers were reduced compared to WT, although not statistically significant. We confirmed CSP processing in both WT and csp(mut) sg-sporozoites by Western analysis and csp(mut) sg-sporozoites showed WT-like infectivity to cultured hepatocytes. These observations demonstrate that the absence of the single glutamine residue in CSP region 1 results in immune recognition of viable/infective sporozoites in the hemocoel, resulting in melanization'.
From the Abstract:
'We show that Plasmodium sporozoites of QC-null mutants are recognized by the mosquito immune system and melanized when they reach the hemocoel. Sporozoite numbers in salivary glands are also reduced in mosquitoes infected with QC-null or QC catalytically-dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito hemocytes or melanization immune responses. Mutation of a single QC-target glutamine of the major sporozoite surface protein (CSP) also results in immune recognition of sporozoites. These findings reveal QC-mediated post-translational modification of surface proteins as a major mechanism of mosquito immune evasion by Plasmodium sporozoites'.
Other mutants |