Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of lipoate-protein ligase B (LipB) and expresses GFP-Luciferase under the constitutive eef1a promoter (a second independent mutant has been generated in wild type P. berghei ANKA background)
Protein (function)
The FAS-II pathway generates an eight-carbon chain precursor (octanoic acid) that is used for the synthesis of lipoic acid (6,8-thioctic acid), a cyclic disulphide-containing derivative of octanoic acid that is an essential cofactor for a number of multienzyme complexes found in almost all eukaryotic cells.
In addition to being synthesized de novo, lipoic acid can also be absorbed from dietary sources. Cells maintain active systems to scavenge non-protein bound lipoic acid from their environment.
In Plasmodium, covalently attached lipoic acid regulates the function of three a-ketoacid dehydrogenases, namely pyruvate dehydrogenase (PDH), a-ketoglutarate dehydrogenase (KGDH) and branched-chain α-ketoacid dehydrogenase (BCDH). These multi-enzyme complexes contribute to amino acid and energy metabolism and consist of multiple copies of a substrate-specific a-ketoacid decarboxylase (the E1 subunit), an acyltransferase (the E2 subunit) and a dihydrolipoamide dehydrogenase (the E3 subunit). These a-ketoacid dehydrogenases generally convert an a-ketoacid, NAD+ and coenzyme A (CoA) to CO2, NADH and acyl-CoA.
PDH, comprised of the lipoylated subunit E2 as well as subunits E1 and E3, is located in the parasite apicoplast.
In the apicoplast, PDH is thought to catalyze the oxidative decarboxylation of pyruvate to generate acetyl-CoA. In contrast to PDH-E2, the other lipoylated proteins (KGDH-E2, BCDH-E2 and the H-protein) have been localized to the mitochondria.
Plasmodium parasites synthesize lipoic acid within the apicoplast, where the FAS-II pathway produces octanoic acid attached to acyl-carrier protein (ACP) as one of its products. The derivation of lipoic acid from octanoyl-ACP requires two apicoplast-targeted enzymes: octanoyl-ACP transferase (LipB) and lipoate synthase (LipA). LipB transfers the octanoyl group to a lipoyl-accepting domain, whereas LipA is responsible for catalyzing the introduction of two sulphurs at positions 6 and 8, forming the lipoyl group. LipA is capable of generating lipoic acid from the octanoyl-ACP precursor before or after its transfer by LipB, although it is thought that LipA prefers the E2 protein bound substrate. In the apicoplast, these enzymes mediate the attachment of lipoic acid to PDH-E2 . In P. falciparum, LipB was earlier shown to be important for lipoylating PDH-E2 but was itself nonessential to blood stage replication. LipB-deficient (PfLipB) parasites were reported to have an increased growth rate during the asexual blood stages, a reduced level of lipoylated PDH-E2 protein, and a reduction in the total lipoic acid content in the parasite. The lipoate protein ligase LplA2, which is apparently targeted to both the apicoplast and the mitochondria, was hypothesized to compensate for the loss of PfLipB function and to account for the residual PDH-E2 lipoylation that was observed in those knockout (KO) parasites
In addition to the lipoic acid synthesis pathway, Plasmodium parasites have an active scavenging pathway that appears to be essential for both blood- and liver stage development and that has been shown to lead to lipoylation of KGDH-E2, BCDH-E2 and the H-protein in the mitochondrion. Host lipoic acid may be imported via the parasite pantothenate transporter. Scavenged radiolabeled lipoic acid was found to sequester solely in the mitochondrion and appeared to be attached to these proteins by the action of lipoic acid protein ligase LplA1.
The synthesis and scavenging pathways have generally been considered to operate separately, with no lipoic acid exchange occurring between the mitochondrion and apicoplast organelles
Phenotype
Normal blood stage development and development of mosquito stages. Normal production of salivary gland sporozoites (see also Additional information).
Reduced infectivity of sporozoites. Mice infected with sporozoites show a 4 day delay in prepatent period. Mutant liver stages have major defects during late liver stage development and do not produce detached cells during in vitro culture indicating an important role of LipB for formation of fully mature liver schizonts and viable merozoites..
Additional information
Apicoplast of mutant blood stages had a similar morphology to that of wild type parasites as shown by immunofluorescence analysis using antibodies against the apicoplast-specific protein ACP (PBANKA_030560; acyl carier protein).
Evidence is presented for markedly reduced levels of lipoylated PDH-E2 and in the LipB-deficient parasites compared to the WT control. KGDH-E2 lipoylation levels appeared unaffected by the loss of LipB. This is consistent with the proposal that LipB lipoylates PDH-E2 within the apicoplast. We note that PbLipB contains an apicoplast-targeting motif as predicted by PlasmoAP and PDH-E2 is the only protein within the apicoplast that is known to require lipoylation. The other three proteins known to be lipoylated in Plasmodium are BCDH-E2, KGDH-E2, and the H-protein, which appear to localize to the mitochondria based on GFP-fusion studies in P. falciparum. These proteins were predicted to be lipoylated by the mitochondrial ligases LplA1 or LplA2.
By analysis of a mutant (RMgm-852) expressing a GFP-tagged version of BCDH-E2 evidence is presented that BCDH-E2 is NOT located in the apicoplast. These results suggest that LipB might be responsible for the lipoylation of the mitochondrial protein BCDH-E2, and evoke the possible movement of lipoic acid from the apicoplast to the mitochondria. An alternative explanation would be that LipB, which has a N-terminal bipartite peptide that is predicted to traffic this protein to the apicoplast, could in part also traffic to the mitochondria.
Evidence is presented that the mutant has a reduced growth of asexual blood stages when mice has reduced serum lipid concentrations (induced by treatment of mice with clofibrate).
Apicoplast of mutant liver stages (after 24h) show an aberrant morphology (smaller and more constricted) compared to that of wild type parasites as shown by immunofluorescence analysis using antibodies against the apicoplast-specific protein ACP (PBANKA_030560; acyl carier protein).
Other mutants
A mutant (RMgm-852) expressing a GFP-tagged version of BCDH-E2 |