Bissett, Ryan Eugene
Lipoic acid metabolism in Leishmania major.
PhD thesis, University of Glasgow.
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Protozoan parasites of the genus Leishmania are the causative agents of a complex of diseases referred to as leishmaniasis. Leishmania have a digenetic life cycle that involves a sand fly vector (promastigote stage) and a mammalian host (amastigote stage). The parasites reside within very different environmental niches in the two different hosts, and therefore must be able to adapt their energy metabolism to the available carbon and nitrogen sources.
Lipoic acid (LA) is a multifaceted molecule, and plays an important role as a water- and fat-soluble antioxidant. LA is also an essential cofactor of the alpha-ketoacid dehydrogenase complexes (alpha-KADHs) and of the glycine cleavage complex (GCC). The alpha-KADHs include the pyruvate dehydrogenase (PDH), branched-chain alpha-ketoacid dehydrogenase (BCKDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH), each of which is integral to cellular energy metabolism. In some organisms, LA can be acquired through salvage and biosynthesis pathways, and yet others only encode enzymes that permit one of the two pathways. Lipoylation of the PDH has been demonstrated in a parasite related to Leishmania called Trypanosoma brucei; however there have not been any investigations into the enzymes involved in LA metabolism in either Leishmania or Trypanosoma brucei.
In silico analyses identified genes encoding for proteins involved in both LA biosynthesis and salvage (lipoic acid synthase (LIPA), octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LIPB) and lipoate protein ligase (LPLA), respectively), and it was predicted that all three proteins possess mitochondrial targeting peptides. Targeting of these proteins to the mitochondrion was verified by a green fluorescence protein (GFP) reporter system, and by subcellular pre-fractionation using digitonin followed by western blotting. Functionality of L. major putative LIPA, LIPB and LPLA genes was determined by showing that the genes complemented the no-growth phenotype of bacteria deficient in either lipA or lipB genes on minimal medium.
Bioinformatics analyses also showed that L. major possesses genes encoding all of the subunits comprising the different alpha-KADHs and the GCC, and the subunits were predicted to possess mitochondrial targeting peptides. Western blotting of promastigote protein with an antibody recognising protein-bound LA (alpha-LA antibody) identified four proteins, which based upon predicted molecular sizes, correspond to the lipoylated transacylase subunits of the three alpha-KADHs and the H-protein of the GCC. Interestingly, the lipoylation pattern changes throughout promastigote growth in vitro, with alpha-KGDH being lipoylated throughout promastigote life while PDH and BCKDH are not lipoylated and presumably not active in metacyclic promastigotes. These findings indicate that modification of alpha-KADHs and the GCC by lipoylation is a dynamic process, possibly reflecting adaptations in the parasite’s energy metabolism during their developmental cycle.
Three approaches were taken to study the relative importance of the LA biosynthesis and salvage pathways in L. major promastigotes. First, LA analogues 8’ bromooctanoic acid (8-BOA) and octanoic acid (OA) were tested for their effects on growth in L. major maintained in lipid-depleted medium. The IC50 for 8-BOA was relatively high when compared to that determined in other organisms, suggesting that LA biosynthesis can compensate for a decrease in LA salvage in medium deficient in LA. Second, attempts to replace either LIPA or LPLA genes with selectable markers were unsuccessful. LPLA could however, be knocked-out when an extra copy of the gene was introduced into the parasite’s genome. These data suggest that both LA acquisition pathways might be essential for promastigote growth and development. Third, overexpression of C-terminal His-tagged versions of LIPB (LIPB-His), LPLA (LPLA-His) and a LPLA active site mutant, LPLAH118A (LPLAH118A-His), resulted in slow-growth phenotypes. Overexpression of LIPB-His and LPLAH118A-His resulted in lipoylation of the PDH and BCKDH in metacyclic promastigotes, which is not observed in wild-type metacyclic promastigotes. It is hypothesised that LA biosynthesis and salvage enzymes could have differential substrate-specificities in L. major.
A number of avenues require further investigation, including the mechanism that permits a relatively rapid turnover of lipoylated protein, and whether lipoylation patterns differ depending upon the carbon sources that are provided in the growth medium. Also, it will be interesting to determine whether LIPB and LPLA have intrinsic substrate-specificities, and whether this is sufficient to explain the fact that both LIPA and LPLA are essential in the promastigote stage in vitro.
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