|Additional remarks phenotype|
The mutant expresses a C-terminal GFP-tagged version of DHHC2.
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 mutant RMgm-1349, RMgm-1351).
Analyses of the mutant expressing a C-terminal GFP-tagged version of DHHC2 indicates expression in blood stages, gametocytes, ookinetes, (maturing) oocysts and sporozoites.
See also mutant RMgm-1353 that also expresses a C-terminal GFP-tagged version of DHHC2 (using the 3'UTR of the dhhc2 gene) and shows a similar phenotype.
The mutant showed normal development throughout the complete life cycle indicating that the GFP tag did not affect the function of the DHHC2 gene
Transcriptional patterns were analysed by semi-quantitative RT-PCR using cDNA prepared from blood and mosquito stage parasites; compared to the control transcripts of hsp70 and 18S ribosomal RNA, dhhc2 was found to be highly transcribed in asexual blood stage parasites and in gametocytes, while mRNA levels were absent in mature ookinetes; in oocysts/midgut sporozoites the gene was transcribed anew but absent in mature salivary gland sporozoites. An RNAseq analysis of different P. berghei life cycle stages31 also found high transcript levels in schizonts and gametocytes compared to ring forms and trophozoites.
RMgm-1350: Unsuccessful attempts to disrupt the dhhc2 gene
RMgm-1349, RMgm-1351: 'Promoter swap mutants' in which the promoter of DHHC2 is replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes.
RMgm-1353: Another mutant expressing a C-terminal GFP-tagged version of DHHC2 (using the 3'UTR of the dhhc2 gene)