|Additional remarks phenotype|
In the 'promoter-swap' mutant the promoter of DHHC2 replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes (the promoter of PBANKA_140060; clag; cytoadherence linked asexual protein). In addition, the mutant expresses the fusion protein GFP-Luciferase under control of the constitutive eef1a promoter.
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.
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 (see also mutant RMgm-1351 for a comparable promoter swap mutant).
The promoter-swap mutant produced normal numbers of female and male gametocytes and gametes. In addition, the mutant showed normal fertilisation rates and production of (diploid and tetraploid) zygotes. However zygotes failed to develop into mature ookinetes. No oocysts are formed.
Overnight ookinete cultures of mutant parasites stained with Hoechst showed signs of fertilisation. Image analyses of ploidy confirmed that fertilisation had not been affected with parasite DNA content of these forms being approximately 4N; the zygotes have thus completed meiotic DNA replication to tetraploidy. However, none of these zygotes developed into mature ookinetes and presented morphogenetic defects. In these cultures only clusters of round zygotes or zygotes with small, thin protrusions were present indicating initiated, but failed morphogenesis.
RMgm-1350: Unsuccessful attempts to disrupt the dhhc2 gene
RMgm-1351: A comparable promoter swap mutant as described here. The dhhc2 gene is tagged with GFP.
RMgm-1352, RMgm-1353: Mutants expressing a C-terminal GFP-tagged version of DHHC2