Dephospho‐CoA kinase, a nuclear‐encoded apicoplast protein, remains active and essential after Plasmodium falciparum apicoplast disruption

Abstract

Malaria parasites contain an essential organelle called the apicoplast that houses metabolic pathways for fatty acid, heme, isoprenoid, and iron–sulfur cluster synthesis. Surprisingly, malaria parasites can survive without the apicoplast as long as the isoprenoid precursor isopentenyl pyrophosphate (IPP) is supplemented in the growth medium, making it appear that isoprenoid synthesis is the only essential function of the organelle in blood‐stage parasites. In the work described here, we localized an enzyme responsible for coenzyme A synthesis, DPCK, to the apicoplast, but we were unable to delete DPCK, even in the presence of IPP. However, once the endogenous DPCK was complemented with the E. coli DPCK (EcDPCK), we were successful in deleting it. We were then able to show that DPCK activity is required for parasite survival through knockdown of the complemented EcDPCK. Additionally, we showed that DPCK enzyme activity remains functional and essential within the vesicles present after apicoplast disruption. These results demonstrate that while the apicoplast of blood‐stage P. falciparum parasites can be disrupted, the resulting vesicles remain biochemically active and are capable of fulfilling essential functions.

Roles of Ferredoxin-Dependent Proteins in the Apicoplast of Plasmodium falciparum Parasites

“…Alternatively, genes were targeted for deletion using the pRSng ( 32 ) or pRSng (BSD) plasmid in combination with the pCasG plasmid ( 31 ). To generate the pRSng (BSD) plasmid, harmonized blasticidin- S -deaminase (hBSD) was cut with BamHI and HindIII from a synthetic plasmid ( 79 ) and ligated into the same sites in the pRSng plasmid to replace the sequence encoding human dihydrofolate reductase (hDHFR).…”

The mitochondrion of Plasmodium falciparum generates essential acetyl-CoA for protein acetylation

“…Plasmid pCasG-LacZ was digested with Bsa I for insertion of double-stranded DNA adaptamers (generated from primers listed in supplementary file 1 ) using LIC or standard ligation; positive colonies were selected using blue/white colony screening. To generate double knockout lines, the gene encoding human Dihydrofolate Reductase ( dhfr ) was excised from plasmid pRSng using Bam HI/ Hin dIII and replaced using LIC (primers pRsBSD.F and pRsBSD.R) with a sequence encoding Aspergillus terreus Blasticidin-S Deaminase codon harmonized for expression in P. falciparum 79 to generate the pRSngB plasmid.…”

Pantothenate and CoA biosynthesis in Apicomplexa and their promise as antiparasitic drug targets

“…Experimental localization data (in black) were obtained from Oppenheim and colleagues [ 34 ], Lunghi and colleagues [ 16 ], and Barylyuk and colleagues (only PBAL is identified in this study) [ 17 ] for T . gondii , Tjhin and colleagues [ 35 ] and Swift and colleagues [ 36 ] for P . falciparum .…”

“…This reaction is followed by the sequential formation of 4′-phosphopantothenoylcysteine by phosphopantothenoylcysteine synthetase (PPCS), 4′-phosphopantetheine by phosphopantothenoylcysteine decarboxylase (PPCDC), dephospho-CoA by phosphopantetheine adenylyltransferase (PPAT), and, finally, CoA by dephospho-CoA kinase (DPCK). Although this essential CoA pathway is highly conserved, there are differences between phyla and even between apicomplexan parasites ( Fig 2 ) [ 20 – 22 , 36 , 50 , 51 ]. Most bacteria and Saccharomyces cerevisiae contain a single PanK [ 52 ].…”

“…While all these CoA synthesis enzymes, including Tg DPCK, are likely cytoplasmic, the last step performed by Pf DPCK has recently been localized to the apicoplast. This enzyme is essential for intra-erythrocytic parasite survival and remains active in the vesicles that accumulate after apicoplast disruption [ 36 ].…”