Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of QC and ECP1 and expresses mCherry and luciferase under control of constitutive promoters.
The ECP1 gene has been deleted in mutant RMgm-5057 (line 3172cl1(2nd)) that lacks expression of QC and expresses luciferase under the control of the eef1α (PBANKA_1133300) promoter and, in addition, mCherry under the control of the hsp70 (PBANKA_0711900) promoter. Mutant RMgm-5057 does not contain a drug-selectable marker (SM) that has been removed by negative selection from mutant RMgm-5056 (line 2930cl1).
Protein (function)
QC: N-terminal modification of glutamine or glutamic acid residues to pyroglutamic acid (pGlu; 5-oxo-L-46 proline) is a posttranslational modification (PTM), catalyzed by glutaminyl cyclases (QCs) found in eukaryotes and prokaryotes. Two evolutionary unrelated classes exist; mammalian QCs and QCs of bacteria, plants and parasites, which share no sequence homology, supporting a different evolutionary origin. Mammalian cells can express two forms, the secreted glutaminyl‐peptide cyclotransferase (QPCT) or its iso-enzyme (QPCTL), localized in the Golgi complex. pGlu is implicated in maturation and stabilization of mammalian proteins such has neuropeptides and cytokines. QC activity has been associated in humans with pathological processes such as amyloidotic diseases and QPCTL is critical for pGLu formation on CD47, facilitating myeloid immune evasion. A single gene encoding a glutaminyl cyclase (QC), named glutaminyl-peptide cyclotransferase, has been identified by electronic annotation in all sequenced Plasmodium genomes. Plasmodium QCs share 70-76% sequence similarity and 50-54% identity and contain a transmembrane domain. QC of the human malaria parasite P. falciparum (PfQC) shows 21-27% identity to QCs of various 64 bacteria and the plant Carica papaya (CpQC). All 9 amino acids of the catalytic site of bacterial and plant QCs are conserved in Plasmodium QC.
CRM4:
The Cysteine Repeat Modular Proteins (CRMP1–4) of Plasmodium, are encoded by a small gene family that is conserved in malaria and other Apicomplexan parasites. They are very large, predicted surface proteins with multipass transmembrane domains containing motifs that are conserved within families of cysteine-rich, predicted surface proteins in a range of unicellular eukaryotes, and a unique combination of protein-binding motifs, including a > 100 kDa cysteine-rich modular region, an epidermal growth factor-like domain and a Kringle domain.
CRM4 is involved in egress of mature, viable sporozoites from the mature oocysts (RMgm-585).
Phenotype
Normal numbers of oocysts are produced. Sporozoite formation inside oocysts. No sporozoite egress from oocysts. No salivary gland sporozoites. No melanized oocysts (or sporozoites).
In mosquitoes infected with QC 'single knockout' mutants (RMgm-5056, RMgm-5057, RMgm-5058) melanized oocysts are present on day ten after infection in 55-65% of QC-null infected mosquitoes (1-85 melanized oocysts/mosquito), while such oocysts were absent in WT-infected mosquitoes. In addition, reduced numbers of salivary gland sporozoites are present. 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.
From the paper about ‘double knock-out’ QC-null mutants:
'The absence of melanized QC-null oocysts before day 10 p.i. and absence of distinct deposition of melanin on the oocyst capsule may suggest that QC-null sporozoites, either still inside rupturing oocysts or after release into the hemocoel, are specifically recognized by the immune system. If this is the case, abolishing sporozoite formation inside QC-null oocysts or blocking egress of OC-null sporozoites would prevent oocyst melanization. To test this hypothesis we deleted genes encoding proteins involved in sporozoite formation (CSP, ROM3) or in sporozoite egress (ECP1, CRMP4) in P. berghei QC-null parasites. These ‘double knock-out’ QC-null mutants (RMgm-5062, RMgm-5063, RMgm-5064, RMgm-5065) produced WT numbers of oocysts; however, melanized oocysts were completely absent. In contrast, melanized oocysts/sporozoites were observed in 79% of mosquitoes infected with a ‘double knock-out’ QC-null mutant (RMgm-5066) lacking the trap gene that releases sporozoites into the hemocoel that cannot invade the salivary glands (1-60 melanized oocysts/mosquito). These observations confirm that melanization only occurs when QC-null oocysts rupture and sporozoites are released into the hemocoel'.
Additional information
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
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