Abstract
Eukaryotic pathogens of the phylum Apicomplexa contain a non-photosynthetic plastid, termed apicoplast. Within this organelle distinct iron-sulfur [Fe-S] cluster proteins are likely central to biosynthesis pathways, including generation of isoprenoids and lipoic acid. Here, we targeted a nuclear-encoded component of the apicoplast [Fe-S] cluster biosynthesis pathway by experimental genetics in the murine malaria parasite Plasmodium berghei. We show that ablation of the gene encoding a nitrogen fixation factor U (NifU)-like domain containing protein (NFUapi) resulted in parasites that were able to complete the entire life cycle indicating redundant or non-essential functions. nfu – parasites displayed reduced merosome formation in vitro, suggesting that apicoplast NFUapi plays an auxiliary role in establishing a blood stage infection. NFUapi fused to a combined fluorescent protein-epitope tag delineates the Plasmodium apicoplast and was tested to revisit inhibition of liver stage development by azithromycin and fosmidomycin. We show that the branched apicoplast signal is entirely abolished by azithromycin treatment, while fosmidomycin had no effect on apicoplast morphology. In conclusion, our experimental genetics analysis supports specialized and/or redundant role(s) for NFUapi in the [Fe-S] cluster biosynthesis pathway in the apicoplast of a malarial parasite.
Highlights
Iron-sulfur [Fe-S] clusters are inorganic cofactors that constitute one of the most ancient and ubiquitous prosthetic groups
Malaria parasite genomes encode three nitrogen fixation factor U (NifU)-like domain containing proteins, related to the bacterial nitrogen fixation (NIF) system. Two of these were predicted to target to the mitochondrion, one ISU/ IscU ortholog (PBANKA_131820) and one NFU1/NfuA ortholog (PBANKA_083170)
Phylogenetic analyses of the NifU-like domains confirmed the separation of Plasmodium NifU-like domain containing proteins into three groups
Summary
Iron-sulfur [Fe-S] clusters are inorganic cofactors that constitute one of the most ancient and ubiquitous prosthetic groups. Different biogenesis machineries have been identified that assemble [Fe-S] clusters in various cellular compartments, namely the cytoplasmic iron-sulfur protein assembly (CIA) [5], the mitochondrial ironsulfur cluster (ISC) [6], and the plastid-localized sulfur utilization factor (SUF) [7,8] systems. No phenotypical analyses of any experimentally modified apicomplexan parasite are available that indicate either essential, distinct stage-specific, or dispensable roles for any component of the [Fe-S] cluster biogenesis pathway in the apicoplast. We present an experimental genetics analysis of the NifUlike domain containing protein (NFU) in the [Fe-S] biosynthesis pathway of the Plasmodium berghei apicoplast. BPutative targeting of the P. falciparum SUF pathway proteins to the apicoplast or mitochondrion was predicted using four different algorithms. Note that no analysis was done for SUFB as the gene is encoded on the apicoplast genome and needs no targeting sequences. doi:10.1371/journal.pone.0067269.t001
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