|Additional remarks phenotype|
The mutant lacks expression of DHHC10 and expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter. In addition it expresses a C-terminal GFP-tagged version of LAP2. Lap2-tagging was performed in the background parasite line RMgm-1471 that lacks expression of DHHC10 and expresses the transgenes GFP and mCherry in males and females respectively.
Many proteins are post-translationally modified by the addition of lipids. Palmitoylation results in the addition of a C-16 fatty acid to a cysteine residue within a given protein. Palmitoylation is reversible and thus can dynamically regulate a protein’s subcellular localization, gene expression and activity.
Blocking palmitoylation in P. falciparum with 2-bromopalmitate (2-BMP) results in a complete failure to develop merozoites during the blood stage of the life cycle. Preventing palmitoylation of proteins through targeted mutagenesis of cysteine residues within the modification target results in the mis-localization of proteins found in the inner membrane complex (IMC).
The palmitoylation reaction is catalysed by TM-spanning enzymes called palmitoyl-S-acyl-transferases (PAT). One family of PATs is characterised by the presence of a conserved DH(H/Y)C motif, and certain apicomplexan organisms express more than 10 individual S-acyltransferases. They differ in localisation and timing of expression, and therefore are likely to modify distinct protein populations and biological functions.
The global extent of palmitoylation in asexual blood stages of P. falciparum comprises several hundred proteins; they include factors involved in gliding motility, invasion, adhesion, IMC function, signalling, protein transport and proteolytic activity. Of 11 PATs known from rodent malaria parasites five have been detected in blood stage parasites of P. berghei using an HA-tagging approach: they are DHHC3 (IMC), DHHC5 (ER), DHHC7 (rhoptry), DHHC8 (punctate_not_Golgi), and DHHC9 (IMC). Seven DHHC-PATs were found to be redundant for P. berghei blood stage development in a reverse genetic screen: they are DHHC 3, 5, 6, 7, 9, 10 and 11.
Three PATs are under putative translational control in the female P. berghei gametocyte: dhhc2, dhhc3 and dhhc10.
Phentype analyses of a mutants lacking expression of DHHC10 (RMgm-1470, RMgm-1471) showed the following phenotype: Normal numbers of gametocytes are produced. Normal fertilisation rate and ookinete production. Ookinetes however lack crystalloids (crystalloid body; crystalloid organelle). Normal numbers of oocysts are produced. However, oocysts fail to produce sporozoites.
Attempts to disrupt the dhhc2 gene in P. berghei were unsuccessful (see RMgm-1350) indicating an essential role of DHCC2 for blood stage development/multiplication. Phenotype analyses of the promoter-swap mutant indicate that DHHC2 plays an important role in the development of zygotes into mature ookinetes (RMgm-1349; RMgm-1351). The promoter-swap mutant produced normal numbers of female and male gametocytes and gametes and normal fertilisation rates and production of zygotes. However zygotes failed to develop into mature ookinetes. No oocysts are formed.
Phentype analyses of mutants lacking expression of DHHC10 (RMgm-1470, RMgm-1471) showed the following phenotype: Normal numbers of gametocytes are produced. Normal fertilisation rate and ookinete production. Ookinetes however lack crystalloids (crystalloid body; crystalloid organelle). Normal numbers of oocysts are produced. However, oocysts fail to produce sporozoites.
Phenotype analyses of the mutant expressing GFP-tagged DHHC10 (RMgm-1472) showed GFP-fluorescence in distinct cytoplasmic foci in ookinetes known for members of the LAP/CCp protein family typical for the ookinete/oocyst-specific crystalloid organelle.
Analyses of the Δdhhc10;lap2::gfp ookinetes showed diffuse localisation of LAP2::GFP in the cytoplasm ookinetes. LAP2 has previously been localized to the ookinete crystalloids by both live fluorescence imaging and immunoelectron microscopy. The cytoplasmic location of LAP2::GFP and absence of 'focal' localisation in crystalloids confirms the absence of crystalloid formation in mutants lacking expression of DHHC10
dhhc10 transcripts are present in female gametocytes but protein is absent. Evidence is presented for translational repression in female gametocytes. The protein is produced after fertilisation in developing zygots/ookinetes.
Evidence is presented for a location of DHHC10 in the crystalloid organelles (see also RMgm-1472, RMgm-1473).
Crystalloids are formed by the microtubule (MT)-dependent transport and assembly of endoplasmic reticulum-derived vesicles. Concomitantly, LAP proteins such as LAP3 are shuttled to common assembly points and incorporated into mature crystalloids. DHHC10::GFP was detected as early as 3 h postfertilization, and both DHHC10 and LAP3 showed signs of protein concentration by 9 h postfertilization. At 12 h, more than 50% of the retorts displayed focal accumulation of both proteins, and by the end of ookinete development (24 h), GFP and mCherry signals colocalized in the crystalloid in more than 90% of mature ookinetes. Thus, no significant differences between the two proteins in either the timing of expression or subcellular localization were evident.