Additional remarks phenotype | Mutant/mutation
The mutant is a 'Flp/FRT conditional knock-out mutant' of SufS. The mutant expresses the yeast FlpL recombinase under the control of the trap promoter and contains a FRTed 3'-UTR of the SufS locus.
This mutant has been generated by replacement of the endogenous 3'-UTR of the SufS locus with a FRTed 3'-UTR in the mutant RMgm-4186 that expresses FlpL. This mutant expresses the yeast FlpL recombinase under the control of the promoter of trap and does not contain a selectable marker. The flpl gene is integrated into the silent 230p locus by double cross-over integration.
The 3'-UTR of SufS is removed from the genome by using the Flp/FRT site-specific recombination (SSR) system (see RMgm-269, RMgm-747). Removal of the FRTed 3'-UTR of SufS has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase in oocysts/sporozoites that resulted in the excision of the 3'-UTR of SufS which was flanked by FRT sequences.
Protein (function)
Iron-sulfur [Fe-S] clusters are inorganic cofactors that are found on proteins from a range of biological processes and are present in most organisms. The assembly of [Fe-S] clusters on apoproteins is not a spontaneous process but is mediated by distinct pathways that mobilise sulfur atoms from L-cysteines, and assemble them onto scaffold components. The scaffold also receives iron from iron donors and the assembled [Fe-S] clusters are transferred to the target apoprotein via carrier protein(s). In bacteria, three distinct sets of factors/enzymes encoded as operons assemble [Fe-S] clusters; the bacterial isc, suf and nif operons encode proteins that are components of the ISC (iron-sulfur cluster formation), SUF (sulfur mobilization) and NIF (nitrogen fixation) assembly systems. The ISC system is the major [Fe-S] biogenesis system with the SUF system being activated under iron starvation or oxidative stress.
In eukaryotes, [Fe-S] assembly systems are thought to have evolved from bacterial endosymbionts. The SUF pathway is found in plastid-containing protozoa and plants. The e apicoplast, the relict plastid of the malaria parasite, harbours a functional SUF pathway. Except apicoplast-encoded SufB in Plasmodium spp., all constituent proteins of the SUF pathway are nuclear-encoded and targeted to the apicoplast. These include the cysteine desulfurase SufS and its interacting partner SufE [18], SufC and SufD that are predicted to constitute the scaffold complex with SufB, and putative carrier proteins SufA and Nfu. Components of the SUF pathway in P. berghei have also been reported to be refractory to gene deletion. Apicoplast proteins that likely require [Fe-S] clusters include ferredoxin, two enzymes (IspG and IspH) of the DOXP pathway of isoprenoid biosynthesis, LipA (lipoic acid synthase), and MiaB (tRNA methylthiotransferase).
Phenotype
In the SufScKO line, the 3'-UTR of SufS is excised during development in the mosquito midgut. Evidence is presented for normal numbers of oocysts, reduced sporulation in oocysts, strong reduction of the number of salivary gland sporozoites and reduced 'infectivity' of sporozoites
Additional information
Since SufS is essential for blood stage develepment/multiplication a stage-specific knockout (cKO) mutant using the FlpL/FRT system was generated.
The mutant was used to determine the role of SufS in oocysts/sporozoites. Evidence is presented that SufS plays a role in the formation of (infectious) sporozoites (inside oocysts).
Other mutants
|