Lipoic Acid Biosynthesis and Protein Lipoylation Pathways in Gram-positive pathogens.

RUIZ, DM; MARTIN, N; DE MENDOZA, D; MANSILLA, MC

Mar del Plata

Congreso; VIII Congreso Argentino de Microbiología General; 2012

SAMIGE

Lipoic acid (LA) is a sulfur-containing cofactor required for the activity of several key enzyme complexes involved in oxidative and single carbon metabolism. The current model for LA synthesis is based on Escherichia coli, where octanoate from octanoyl-acyl carrier protein (ACP) is transferred to the lipoyl domains of the lipoate dependent proteins, by the octanoyltransferase LipB. Then, LipA, the lipoyl synthase, converts the octanoylated domains into the lipoyl derivatives. Exogenous free LA can also be scavenged by the lipoyl ligase, LplA. Recently, a new pathway of LA synthesis in the Gram-positive bacterium Bacillus subtilis was proposed. In this microorganism the octanoyl moieties are transferred from the ACP to the H protein of the glycine cleavage system (GcvH) by the octanoyl transferase LipM. The octanoyl moieties are then transferred by LipL, a novel GcvH:E2 amidotransferase, from GcvH to E2 subunits to form active dehydrogenase complexes. The LA synthase, LipA, inserts the sulfur groups on the protein bound octanoyl moites. In bacteria, the enzymes involved in protein lipoylation have gained increasing attention because of their implication in pathogenicity. Analysis of genome sequences revealed that LipL, LipM and GcvH homologues can be found in Gram-positive pathogens closely related to B. subtilis, such as Staphylococcus aureus and Bacillus anthracis. The aim of this work was to determine if the lipoylation mechanisms are conserved among Gram-positive microorganisms.To this end, B. subtilis LA auxotrophic mutants were complemented with S. aureus and B. anthracis genes and the growth and lipoylation pattern of the complemented strains were characterized.Although S. aureus encodes two lipoyl ligases homologs (lplJ1 and lplJ2), only LplJ1 was able to functionally replace B. subtilis LplJ. This suggests that LplJ1 is required for the attachment of free-LA to aproproteins while LplJ2 might be involved in the usage of lipopeptides from the host, as described for Listeria monocytogenes. In addition, S. aureus and B. anthracis LipM proteins were both able to restore growth of the respective B. subtilis mutants, suggesting the first step in LA biosynthesis is conserved. Besides, B. anthracis LipL was able to functionally replace its B. subtilis ortholog, but S. aureus LipL was not. Additionally when S. aureus lipL and gcvH genes were expressed in a B. subtilis ΔgcvH strain, prototrophy was not observed. Notably, the addition of branched-chain fatty acid precursors, the products of the one of the two LA-dependent enzymes was sufficient to restore growth in minimal media, indicating that there might be different substrate specificity in S. aureus lipoylation pathways. Due to the involvement of lipoic acid metabolic proteins in pathogenesis, multidrug resistance and intracellular growth of pathogens, the discovery of new enzymes in these important Gram positive pathogens should provide potential new targets for antimicrobial agents.