Substrate Analogues Entering the Lipoic Acid Salvage Pathway via Lipoate-Protein Ligase 2 Interfere with Staphylococcus aureus Virulence

Abstract

Lipoic acid (LA) is an essential cofactor in prokaryotic and eukaryotic organisms, required for the function of several multienzyme complexes such as oxoacid dehydrogenases. Prokaryotes either synthesize LA or salvage it from the environment. The salvage pathway in Staphylococcus aureus includes two lipoate-protein ligases, LplA1 and LplA2, as well as the amidotransferase LipL. In this study, we intended to hijack the salvage pathway by LA analogues that are transferred via LplA2 and LipL to the E2 subunits of various dehydrogenases, thereby resulting in nonfunctional enzymes that eventually impair viability of the bacterium. Initially, a virtual screening campaign was carried out to identify potential LA analogues that bind to LplA2. Three selected compounds affected S. aureus USA300 growth in minimal medium at concentrations ranging from 2.5 to 10 μg/mL. Further analysis of the most potent compound (Lpl-004) revealed its transfer to E2 subunits of dehydrogenase complexes and a negative impact on its functionality. Growth impairment caused by Lpl-004 treatment was restored by adding products of the lipoate-dependent enzyme complexes. In addition, Caenorhabditis elegans infected with LpL-004-treated USA300 demonstrated a significantly expanded lifespan compared to worms infected with untreated bacteria. Our results provide evidence that LA analogues exploiting the LA salvage pathway represent an innovative strategy for the development of novel antimicrobial substances.

Keywords: LplA2; Staphylococcus aureus; infection model; lipoic acid; salvage pathway; virtual screening.

Figure 1

Lipoyl-relay pathways of protein lipoylation in S. aureus. LA is produced de novo by a biosynthetic pathway involving three key enzymes (LipM, LipA, and LipL). During LA salvage, S. aureus utilizes two lipoate-protein ligases (LplA1 and LplA2). Hijacking of LA salvage enzymes by substrate analogues (gray dotted arrow) will result in ligation of these compounds to E2 domains of various oxidoreductases and thereby negatively impact their function and eventually impair viability.

Figure 2

Chemical structures of lipoic acid and virtual screening hits Lpl-004, Lpl-008, and Lpl-023 selected for experimental validation.

Scheme 1. Synthesis of Amine Building Blocks 4, 8, and 10

Scheme 2. Synthesis of Experimentally Validated Virtual Screening Hits Lpl-004, Lpl-008, and Lpl-023

Figure 3

Impact of Lpl-004, Lpl-008, and Lpl-023 on the growth of Gram-positive bacteria. S. aureus USA300 and B. subtilis JH642 were inoculated in RPMI or in SMM medium, containing 0.05% vitamin-free CAA, in the presence of different compound concentrations. Cultures were incubated for 12 h at 37 °C. Each bar represents the mean ± SD of three independent experiments. *p < 0.1, **p < 0.01, ***p < 0.001; ns, no significant difference between the treated and control condition (paired t test).

Figure 4

Predicted binding modes of the most potent compounds Lpl-004 (A) and Lpl-023 (B) in the active site of S. aureus LplA2 determined with the molecular docking program GOLD. Polar interactions are indicated as yellow dotted lines.

Figure 5

Compound Lpl-004 binds to lipoate-dependent complexes impairing their activity. (A) Growth of S. aureus USA300 in RPMI containing 0.05% vitamin-free CAA and different concentrations of Lpl-004 in presence and absence of acetate and BCFAP and (B) in presence and absence of 25 nM LA. Each bar represents the mean ± SD of three independent experiments. *p < 0.1, **p < 0.01; ns, no significant difference between the cultures with and without additives (paired t test). (C, D) Modified proteins recognized by antibodies against LA. (C) S. aureus USA300 or (D) S. aureus NE264 (ΔlipA) extracts of cells grown in the absence or presence of 20 μg/mL Lpl-004 or 30 μg/mL selenolipoate were analyzed by Western blot. Strains were grown in RPMI supplemented with vitamin-free CAA, BCFAP, and acetate for 6 h. The blot was probed with an antibody against RpoB to serve as loading control.

Figure 6

Lipoic acid salvage pathway is targeted by Lpl-004. (A) Growth of S. aureus USA300, NE1257 (ΔlplA1), NE266 (ΔlplA2), and FA-S912 (ΔlplA1 ΔlplA2) in RPMI medium containing 0.05% vitamin-free CAA, with or without Lpl-004 (20 μg/mL). Cultures were incubated for 12 h at 37 °C. Each bar represents the mean ± SD from three independent experiments. ns, no significant difference between growth in the absence and in the presence of Lpl-004; *p < 0.1, **p < 0.01, ***p < 0.001 (paired t test). (B) Cell extracts from S. aureus strains USA300 and FA-S1178 (ΔlipA ΔlplA1 ΔlplA2) grown in RPMI supplemented with vitamin-free CAA, BCFAP, acetate, and with or without Lpl-004 (20 μg/mL) were analyzed by Western blot with antibodies against LA. The blot was probed with an antibody against RpoB to serve as loading control.

Figure 7

LA analog selenolipoate produces lipoate-protein ligase-dependent growth inhibition in S. aureus. S. aureus USA300, NE1257 (ΔlplA1), NE266 (ΔlplA2), and FA-S912 (ΔlplA1 ΔlplA2) were cultured in RPMI medium containing 0.05% vitamin-free CAA with or without selenolipoate (30 μg/mL). Cultures were incubated for 12 h at 37 °C. Each bar on the graph represents the mean ± SD from three independent experiments; ns indicates no significant difference between growth in the presence and absence of selenolipoate; *p < 0.1, **p < 0.01, and ***p < 0.001 signifies statistical significance based on paired t test results.

Figure 8

Treatment of USA300 with Lpl-004 increases worm lifespan. (A) Kaplan–Meier survival plot of C. elegans TJ1060 L4 worms fed with S. aureus USA300 treated (black line) or untreated (gray line) with Lpl-004, or fed with NE264 (ΔlipA, gray line with closed circles); (B) worms colonized with the double mutant ΔlplA1 ΔlplA2, FA-S912 treated (black line) or untreated (gray line) with Lpl-004. Viability was scored every 24 h and is represented as the percentage of surviving worms.