Apicoplast fatty acid synthesis is essential for organelle biogenesis and parasite survival inToxoplasma gondii

Abstract

Apicomplexan parasites are the cause of numerous important human diseases including malaria and AIDS-associated opportunistic infections. Drug treatment for these diseases is not satisfactory and is threatened by resistance. The discovery of the apicoplast, a chloroplast-like organelle, presents drug targets unique to these parasites. The apicoplast-localized fatty acid synthesis (FAS II) pathway, a metabolic process fundamentally divergent from the analogous FAS I pathway in humans, represents one such target. However, the specific biological roles of apicoplast FAS II remain elusive. Furthermore, the parasite genome encodes additional and potentially redundant pathways for the synthesis of fatty acids. We have constructed a conditional null mutant of acyl carrier protein, a central component of the FAS II pathway in Toxoplasma gondii. Loss of FAS II severely compromises parasite growth in culture. We show FAS II to be required for the activation of pyruvate dehydrogenase, an important source of the metabolic precursor acetyl-CoA. Interestingly, acyl carrier protein knockout also leads to defects in apicoplast biogenesis and a consequent loss of the organelle. Most importantly, in vivo knockdown of apicoplast FAS II in a mouse model results in cure from a lethal challenge infection. In conclusion, our study demonstrates a direct link between apicoplast FAS II functions and parasite survival and pathogenesis. Our genetic model also offers a platform to dissect the integration of the apicoplast into parasite metabolism, especially its postulated interaction with the mitochondrion. apicomplexa ͉ fatty acid biosynthesis ͉ lipoic acid ͉ Plasmodium ͉ plastid

Lipidomic Analysis of Toxoplasma gondii Reveals Unusual Polar Lipids

“…Since acyl lipid labeling is abolished by treatment with haloxyfop, a reported inhibitor of the apicoplast acetyl CoA carboxylase, Bisanz et al (13) concluded that an active FAS II was critical for the bulk of the acyl lipid synthesis. Combining conditional mutant analyses and metabolic labeling, Mazumdar et al (5) demonstrated that FAS II was critical for the biogenesis of the apicoplast itself, and subsequently for parasite survival, but was unlikely the source of the acyl moiety of the bulk of acyl lipids. Rather, most glycerolipids from free T. gondii cells appears to be produced using acyl chains generated by FAS I or FAEs, which are resistant to the FAS II inhibitor thiolactomycin, but sensitive to the general FAS inhibitor cerulenin (5).…”

“…Combining conditional mutant analyses and metabolic labeling, Mazumdar et al (5) demonstrated that FAS II was critical for the biogenesis of the apicoplast itself, and subsequently for parasite survival, but was unlikely the source of the acyl moiety of the bulk of acyl lipids. Rather, most glycerolipids from free T. gondii cells appears to be produced using acyl chains generated by FAS I or FAEs, which are resistant to the FAS II inhibitor thiolactomycin, but sensitive to the general FAS inhibitor cerulenin (5). Together, these analyses highlight the importance [1] of the parasite FAS for bulk acyl lipid syntheses in free stages and [2] of the FAS II activity for the apicoplast biogenesis.…”

“…Thus far, manipulation of fatty acid synthesis (1)(2)(3)(4)(5), phosphatidylcholine metabolism (6), and sphingolipid synthesis (7) have been shown to affect tachyzoite growth and survival. The origin of fatty acids for synthesis of acyl lipids has been intensely investigated by molecular and metabolic labeling approaches.…”

“…The apicoplast harbors an active fatty acid synthase of type II (FASII) (1,2,(8)(9)(10)(11)13). T. gondii also contains a cytosolic fatty acid synthase of type I (FAS I) and fatty acyl-elongases (FAEs) (1,5,12 …”

What new cell biology findings could bring to therapeutics: is it time for a phenome-project in Toxoplasma gondii?

“…The obvious advantage of this strategy is that hypothetical genes that might otherwise be neglected in targeted approaches will be identified with equal probability. The Tet-inducible transactivator system is currently the most robust method to generate conditional mutants for essential genes in a targeted approach and appears to be especially well suited for the characterisation of factors involved in invasion and apicoplast biology (Meissner et al 2002, Mital et al 2005, Huynh & Carruthers 2006, Mazumdar et al 2006, Kessler et al 2008, Plattner et al 2008) whereas employment of the system for other essential GOIs might result only in weak phenotypes (Fleige et al 2008) or might not be possible at all (unpublished observations). A major advantage of the Tet-inducible system is that virtually any GOI can be regulated whereas regulation at the protein level using ddFKBP might not work for proteins targeted to the secretory system (Herm-Gotz et al 2007, and unpublished observations).…”