Development of a biological screening for detection of compounds interfering with Staphylococcus aureus protein lipoylation

JIRGENSONS, A; SCATTOLINI, A; MANSILLA, MC; NIKITJUKA, A

Congreso; Congreso Conjunto SAIB-SAMIGE 2020; 2020

Infections with methicillin resistant Staphylococcus aureus (MRSA) are a global problem. Besides hospital acquired MRSA, also community associated MRSA strains have emerged that cause skin and soft tissue infections but also life threatening endocarditis and pneumonia. The increasing appearance of multidrug resistant strains urgently requires novel therapeutic approaches in order to keep the drug discovery pipeline filled. We have recently validated the enzymes of the lipoic acid (LA) biosynthesis and salvage pathways as attractive targets for antibacterial compounds development. LA is an organosulfur compound distributed in all domains of life. Is an essential cofactor of six multienzyme complexes, which are involved in oxidative and one carbon metabolism. We have characterized LA metabolism in the model Gram-positive bacterium Bacillus subtilis, which has two redundant pathways for protein lipoylation: de novo synthesis and scavenging of exogenous lipoate. The synthesis pathway requires three proteins. First, an octanoyltransferase (LipM) transfers the octanoyl moiety from ACP to GcvH. Then, a lipoate synthase (LipA) inserts sulfur atoms into C6 and C8 of octanoyl-GcvH and, finally, an amidotransferase (LipL) transfers the lipoyl residue from GcvH to the E2 subunits. The scavenging pathway involves a lipoate ligase (LplJ) which attaches the exogenous lipoate to GcvH or the E2 subunit of oxoglutarate dehydrogenase, followed by transference of lipoyl moiety to E2s by LipL. The amidotransferase plays an essential role in lipoate metabolism due to its participation in both synthesis and uptake of LA. As LipL is highly conserved in Gram-positive bacteria and does not share sequence identity with eukaryotic enzymes, we use it as a target for novel therapeutic approaches. To identify new antimicrobial compounds interfering with LipL activity we developed a biological assay, taking advantage of the differences between Escherichia coli and B. subtilis LA biosynthesis pathways. We constructed a B. subtilis reporter strain carrying the octanoyltransferase (LipB) of E. coli under a xylose-inducible promoter. This strain cannot grow in minimal medium containing the non-functional LA analogous selenolipoate, since LplJ and LipL ligate it to the E2s subunits, becoming inactivated. In the presence of an inhibitor of LipL the reporter strain would be unable to synthesise LA but also would not ligate the added selenolipoate. In this condition, if xylose is added to the medium, the reporter strain would be able to grow since LipB can transfer octanoyl moieties directly from ACP to E2s. The performance of the method was validated using a lipL mutant that express LipB under a xylose-inducible promoter. This powerful tool would help us in the search of new bioactive molecules targeting amidotransferase activity for further drug development against S. aureus.