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. 2024 May 9;10(6):2172–2182. doi: 10.1021/acsinfecdis.4c00148

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

Albertina Scattolini †,‡, Konstantinos Grammatoglou §, Anna Nikitjuka §, Aigars Jirgensons §, María Cecilia Mansilla †,‡,*, Björn Windshügel ∥,⊥,*
PMCID: PMC11184557  PMID: 38724014

Abstract

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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: lipoic acid, Staphylococcus aureus, LplA2, salvage pathway, virtual screening, infection model

The Gram-positive pathogen Staphylococcus aureus is responsible for a broad range of diseases, including bacteremia, pneumonia, and infections of skin and bones.1 Over 60 years ago, methicillin-resistant S. aureus (MRSA) strains emerged and have become a major problem in hospitals in many countries.2 The adaptability of S. aureus as a pathogen is not solely dependent on its ability to release virulence factors that compromise the host immune defense; it also relies on its capacity to adapt to host nutritional restrictions by scavenging essential nutrients.3−6S. aureus has evolved various mechanisms to evade the host organism phagocytic leukocytes, which are able to produce virulence factors against pathogens/bacteria.3 Treatment of S. aureus infections typically involves the use of antibiotics, but the efficacy of this approach is increasingly limited due to the emergence of antibiotic-resistant strains.7 Therefore, alternative approaches that aim to target bacterial virulence rather than viability have been suggested.8−10

Lipoic acid (LA) is a sulfur-containing cofactor that is essential in prokaryotic and eukaryotic organisms for the function of several multienzyme complexes: pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase (ODH), branched-chain 2-ketoacid dehydrogenase (BKDH), the glycine cleavage system (GCS), acetoin dehydrogenase, and 2-oxoadipate dehydrogenase.11−14 Dehydrogenase complexes are made up of several copies of three subunits (E1, E2, and E3). The central core is composed of multiple E2 subunits with a conserved lysine residue, to which LA is covalently linked. The subunits of the GCS do not form a stable complex; instead, they function as independent proteins that are loosely associated with each other. The H subunit (GcvH) is the one found to be lipoylated.

In bacteria, protein lipoylation can be accomplished using two independent pathways: de novo LA biosynthesis or its salvage from the environment. In presence of two possible pathways, the choice between them is determined by the nutritional limitations of the environment (host tissue), which can influence the modulation of metabolic gene regulatory programs for the biosynthesis or salvage of cofactors.15S. aureus modifies its proteins with LA according to the lipoyl-relay paradigm (Figure 1), described for the Gram-positive model bacterium Bacillus subtilis.16,17 LA biosynthesis comprises three sequential steps. During the first step catalyzed by the octanoyl transferase LipM, the octanoyl moiety from octanoyl-acyl carrier protein conjugate (octanoyl-ACP, resulting from fatty acid synthesis) is transferred to a conserved lysine residue of GcvH.18 In the following step, the lipoate synthase LipA catalyzes the formation of LA by replacing two hydrogen atoms of octanoic acid with sulfur atoms,19 which results in formation of a lipoyl-GcvH conjugate. Finally, the amidotransferase LipL transfers the lipoyl moiety from lipoyl-GcvH to the E2 subunits of dehydrogenase complexes.16

Figure 1.

Figure 1

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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.

S. aureus also requires a lipoyl-relay for LA salvage. It possesses two lipoate-protein ligases, LplA1 and LplA2, that enable the effective recovery of free LA. These have differential specificities; while LplA1 modifies GcvH and the E2 subunit of ODH (E2o), LplA2 has GcvH-L as its main target in vivo (Figure 1).20 Then, the amidotransferase LipL transfers the lipoyl moieties to the apoproteins. The presence of two ligases and two H-proteins is a specific feature of S. aureus, which is not commonly observed in other Gram-positive bacteria. The expansion of LA salvage by the incorporation of an additional ligase signifies a critical adaptive trait that is likely essential for its survival in diverse environments.20Listeria monocytogenes is another well-studied example of a Gram-positive pathogen that possesses two lipoate salvage enzymes, LmLplA1 and LmLplA2.21 However, only LmLplA1 seems to be essential during infection and attaches LA to the single GcvH protein of L. monocytogenes.22

The essentiality of LA biosynthesis and salvage in S. aureus was underscored using mutants in each pathway to interrogate their functional roles during invasive infection.15 Moreover, recent research has highlighted a correlation between the E2 subunit lipoylation of PDH (E2p) and the suppression of macrophage activation which results in promotion of bacterial virulence.23

Acquisition of LA by LplA1 and LplA2 is not essential for bacterial survival.15 However, it has been shown that E. coli lipoate-protein ligase also accepts the selenolipoic acid as substrate, which is efficiently ligated to the E2 subunits, resulting in nonfunctional proteins.24 In S. aureus, such a strategy would involve the combined action of LplA1/LplA2 and LipL, which would accept the LA analogues as substrates and transfer these to the E2 subunits of various dehydrogenase complexes. Similar to selenolipoic acid, such a modification is expected to result in impaired S. aureus viability (Figure 1).

In this study, we carried out a structure-based virtual screening approach for identifying LA analogues that chemically resemble the natural substrate. Docking scores suggested a binding preference of these compounds for S. aureus LplA2 over LA. Selected compounds were synthesized and validated experimentally, resulting in the identification of several antibacterial agents against S. aureus. We also studied the mechanism of action of the selected compounds and tested their effect on S. aureus virulence by using Caenorhabditis elegans as a model of infection.

Results

LpLA2 Homology Model Generation and Virtual Screening for Lipoic Acid Analogues

To identify potential substrate analogues for S. aureus LplA2, we carried out a structure-based virtual screening. As no X-ray crystal structure of the enzyme is available, a homology model was generated using the SWISS-MODEL server and the LplA2 X-ray crystal structure of E. faecalis (PDB ID 5IBY) as modeling template. Twelve of the 18 amino acids interacting with LA in the template structure are conserved in the S. aureus LplA2 sequence. The stereochemical quality of the protein model was assessed using PROCHECK. The Ramachandran plot revealed 88.5% of the 325 residues located in the most favored region compared to 92.9% of the template structure. Only one residue was found to be located in the disallowed region (Val187), which is located in a loop.

To assess whether the natural substrate LA fits into the active site of the homology model, the compound (R-enantiomer) was docked into the protein model using the molecular docking program GOLD. The top-ranked LA docking pose is positioned in the active site in a similar manner as LA cocrystallized with E. faecalis LpLA2 (Figure S1). The dithiolane cycle is rotated about 90° of the origin, which is due to the side chain of Glu47 (Ile42 in E. faecalis LpLA2) that requires a different orientation of that group.

To search for potential LplA2 substrate analogues, the ZINC database was queried for LA analogues, resulting in a total of 4731 compounds. All substances were docked into the active site using GOLD. Based on the analysis of the docking scores and visual inspection of docking poses of the top-scored compounds, three analogues were selected for synthesis and experimental validation (Figure 2). The substances contain a LA residue coupled with different amino acids and contain a free carboxyl functional group that is required for covalent binding to the lysine residues of the E2 subunits. The docking scores of the selected compounds (Lpl-004, Lpl-008, and Lpl-023) are significantly higher compared to LA (Table S1), thereby suggesting a potential preference of the compound over LA by the enzyme.

Figure 2.

Figure 2

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

Synthesis of Virtual Hits

Virtual screening hits Lpl-004, Lpl-008, and Lpl-023 were prepared according to Schemes 1 and 2. First, the building blocks 4, 8, and 10 were synthesized (Scheme 1). N-Fmoc protected aspartic acid monoester 1 was coupled with glycine ester 2, and the resulting intermediate 3 was N-deprotected to give dipeptide 4. Analogously, N-Fmoc glutamic acid monoester 5 was coupled with alanine ester 6, and the resulting intermediate 7 was transformed to a dipeptide 8. N-Fmoc glutamic acid monoester 5 was subjected to the esterification using in situ prepared O-tBu imidate to give diester 9. This was N-deprotected providing glutamic acid derivative 10.

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

Scheme 1

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

Scheme 2

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Then, building blocks 4, 8, and 10 were coupled with R-LA (11) providing intermediates 12–14 (Scheme 2). These were subjected to O-deprotection in acidic conditions to give compounds Lpl-004, Lpl-008, and Lpl-023.

Phenotypic Assays for Validation of Compound Activity

At first, the three synthesized compounds were tested in vitro against S. aureus USA300 in Roswell Park Memorial Institute (RPMI) medium, as well as against B. subtilis JH642 grown in Spizizen’s minimal medium (SMM). Initially, the compounds were screened at concentrations of 50 and 100 μg/mL. Lpl-004, Lpl-008, and Lpl-023 showed a strong inhibition of S. aureus growth while none of them affected B. subtilis at any concentration (Figure 3). Lpl-004 turned out to be the most potent compound at 50 μg/mL.

Figure 3.

Figure 3

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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).

In the next step, we determined the minimum inhibitory concentrations of Lpl-004, Lpl-008, and Lpl-023 that affected the growth of USA300 in RPMI medium. We observed growth inhibition at 2.5 μg/mL (6.6 μM) for Lpl-004; 10 μg/mL (24 μM) for Lpl-008; and 2.5 μg/mL (7.4 μM) for Lpl-023.

Next, we analyzed the potential binding modes of the most potent compounds Lpl-004 and Lpl-023 as predicted by molecular docking. Visual inspection of the Lpl-004 docking poses suggested that the compound binds in various conformations to the enzyme when considering just the three top-ranked conformations. While the positioning of the dithiolane cycle and the aliphatic chain is in line with the docked R-LA, the remaining part of the molecule shows different interaction patterns with the enzyme. The most prevalent binding mode within the 15 top-ranked docking poses involves the formation of two salt bridges with Arg172 and three hydrogen bonds with Gly78, Lys136, and Ser182 (Figure 4A). Docking poses of Lpl-023 showed less diverse conformations, and the top-ranked pose revealed a binding mode involving two carboxyl moieties to form a salt bridge with Arg172 as well as two hydrogen bonds with Asn128 and Ser182 (Figure 4B).

Figure 4.

Figure 4

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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.

Deciphering the Mode of Action for Lpl-004

S. aureus mutants incapable to lipoylate proteins can compensate this deficiency by incorporating the products generated by LA-dependent enzymes. In this case, they are unable to grow in minimal medium unless they are provided with acetate and branched-chain fatty acid precursors (BCFAPs) that allow them to bypass the lack of PDH and BKDH activity, respectively.15 To determine if the most potent compound Lpl-004 affects protein lipoylation, we assessed its effect on USA300 growth in the presence and absence of acetate and BCFAP. Addition of the lipoate-dependent enzyme products restored the growth of this strain in the presence of Lpl-004, confirming that the activity of dehydrogenase complexes was impaired (Figure 5A). As expected, the compound did not affect bacterial growth in the rich medium TSB (Figure S2). To explore whether Lpl-004 affects the activity of dehydrogenases by replacing LA at the conserved lysine residue, we assessed the WT strain growth in the presence of 25 nM LA and 50 or 100 μg/mL Lpl-004. In the presence of free LA and both test compound concentrations, we observed a reduced growth inhibition compared to Lpl-004 alone (Figure 5B). Moreover, we observed a competitive effect between LA and Lpl-004 in a dose-dependent manner (Figure S3).

Figure 5.

Figure 5

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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.

To confirm the mechanism of action of substrate analogue Lpl-004, we analyzed whole cell lysates of WT S. aureus. USA300 cells were incubated in RPMI medium with or without the addition of 20 μg/mL Lpl-004. Proteins were extracted by mechanical rupture and analyzed by performing anti-LA immunoblots. The Western blot showed the characteristic lipoylation pattern of the E2 subunits at both conditions (Figure 5C), indicating that in the presence of Lpl-004 these proteins are modified, which is recognized by anti-LA antibodies. The growth data indicates that the dehydrogenases are inactive (Figure 5A); a plausible scenario is that, in contrast to other LA analogues such as 6,8-dichlorooctanoate and 8-bromooctanoic acid (BrO),25 the antibodies recognize Lpl-004. To assess the recognition capability of the antibody, we analyzed the protein lipoylation pattern of whole cell lysates of S. aureus mutants defective in lipoate synthase (ΔlipA). Selenolipoate was included as a control based on previous demonstrations in E. coli. Here, selenolipoate is ligated to the lipoyl domains of E2 subunits by the lipoate-protein ligase LplA, resulting in their inactivation.24 In the absence of LA supplementation, the ΔlipA strain did not show the characteristic lipoylation pattern because it is unable to synthesize LA (Figure 5D). However, in the presence of either Lpl-004 or selenolipoate, the mutant showed E2 subunit labeling (Figure 5D). To exclude that the signal obtained by Western blot originated from free LA, which may arise from Lpl-004 sample degradation, we performed a bioassay using the LA auxotrophic strain E. coli KER296.26 This strain lacks lipoate synthase activity and is unable to thrive in a minimal medium unless it contains LA. The presence of Lpl-004 did not stimulate bacterial growth, so we confirmed the absence of free LA in the sample (Figure S4). Our results indicate that Lpl-004 binds to E2 subunits of dehydrogenase complexes and is recognized by anti-LA antibodies, similar to selenolipoate (Figure 5D).

Lipoate-Protein Ligase Activity Is Required for Lpl-004 Inhibition of S. aureus Growth

To gain insight into the mechanism of action of Lpl-004, we assessed its effect on single and double lipoate-protein ligase mutants. Both ΔlplA1 and ΔlplA2 single mutants were susceptible to Lpl-004, although the growth inhibition was less pronounced compared to the wild type strain (Figure 6A). The ΔlplA1 mutant appeared to be more susceptible to Lpl-004 compared to the ΔlplA2 mutant. Notably, the double mutant was able to grow in the presence of Lpl-004.

Figure 6.

Figure 6

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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.

In addition, we evaluated the E2 subunit binding ability of Lpl-004 in a ΔlipA ΔlplA1 ΔlplA2 strain by Western blot (Figure 6B). In contrast to the ΔlipA single mutant (Figure 5D), the bands corresponding to labeled proteins did not appear in the presence of Lpl-004 when both ligases are absent. Taken together, the growth data and Western blot analysis highlighted the importance of lipoate-protein ligase activity in the inhibitory effect of Lpl-004.

For comparison, we investigated the effect of the known LA analogue selenolipoate on S. aureus growth. WT and mutant strains lacking LplA1, LplA2, or both ligases were grown in the presence or absence of selenolipoic acid (Figure 7). As expected, selenolipoate affected the growth of S. aureus USA300 and the ΔlplA2 mutant, while we observed no significant growth inhibition of the ΔlplA1 mutant or S. aureus lacking both lipoate-protein ligases. These results indicate that growth inhibition by selenolipoic acid is mostly exerted by LplA1-mediated transfer to the dehydrogenase complexes, unlike the effect produced by Lpl-004, whose ligation appears to be mainly dependent on LplA2.

Figure 7.

Figure 7

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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.

Lpl-004 Diminishes S. aureus Virulence

It has been previously shown that lipoylated E2p acts as an immunosuppressant in USA300 and blocks macrophage activation, thereby promoting virulence.23 In contrast, a lipA-deficient strain that contains unmodified E2p was demonstrated to induce an increased pro-inflammatory cytokine production by macrophages, rendering these cells efficient at controlling infection.23 We wondered if Lpl-004, which is expected to replace LA in E2 subunits, is capable of generating an effect similar to that reported for the lipA mutant on S. aureus virulence. Previously, it has been shown that the S. aureus clinical isolate USA300 kills C. elegans.27 We have recently demonstrated that the nematodes do not contain lipoate-protein ligases;28 therefore, these should be unable to ligate Lpl-004 to their apoproteins. Taking advantage of the fact that the S. aureus WT strain treated with Lpl-004 can grow in rich medium, we decided to determine if the presence of the compound diminishes its pathogenesis using C. elegans as a model of infection. We tested the ability of the bacteria to kill nematodes in a survival assay. Usually, nematodes progress through four larval stages (L1 to L4) before maturing into egg-laying adults. A synchronized population of L4-stage worms was fed with the WT bacteria as a food supply, with or without the addition of Lpl-004, and changes in worm population were registered every 24 h. The survival plot shows that the lifespan of the worms fed with S. aureus USA300 treated with Lpl-004 significantly increased compared to the control worms (Figure 8A). The mean survival in the absence and in the presence of the compound in the culture medium was 72 and 144 h, respectively. As a control of the reduced virulence, we included S. aureus NE264 (ΔlipA) strain (Figure 8A). This mutant, unable to synthesize LA, was significantly attenuated in C. elegans killing compared with USA300 and showed a mean survival of 120 h. Feeding worms with dead E. coli OP50, alone or in combination with the compound, did not show significant differences in their lifespan during a 7-day study period (Figure S5).

Figure 8.

Figure 8

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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.

According to the assumed mechanism of action, we expected that the virulence of ΔlplA1 ΔlplA2 strains would not be affected, since in these bacteria Lpl-004 cannot be ligated to the E2 subunits. In agreement with our hypothesis, S. aureus lacking lipoate-protein ligases were resistant to the antivirulence effect of Lpl-004 (Figure 8B). The lifespan curve of worms fed with USA300 deficient in ligase activity showed a decrease in population for 50% in 72 and 84 h with and without Lpl-004, respectively.

Discussion

Targeting enzymatic pathways present in pathogens but absent in humans is an attractive approach to developing drugs with a unique mode of action and improved efficacy. Protein lipoylation is a widely observed post-translational modification found across a broad range of organisms, spanning from bacteria to mammals. Diverse organisms have developed two distinct mechanisms for protein lipoylation: either synthesis of the cofactor or uptake from the environment. The phenomenon of LA salvage is predominantly observed in bacteria and in a limited number of eukaryotes.29 Mammalians and other higher organisms are incapable of using free lipoate as a substrate;30,31 therefore, the development of substrate analogues entering the LA salvage pathway is a promising strategy. The pathogenic Gram-positive bacterium S. aureus is a LA prototroph for which LA salvage is not essential. It possesses two lipoate-protein ligases and employs a lipoyl-relay pathway for both LA biosynthesis and salvage. Therefore, LplA1 and/or LplA2 inhibition is expected to have no effect on bacterial growth. Using a virtual screen for identifying substrate analogues for LplA2, we obtained compounds showing growth inhibition at low micromolar concentrations (Figure 3). Our observations revealed that Lpl-004, Lpl-008, and Lpl-023 specifically inhibit the growth of S. aureus while leaving B. subtilis unaffected. B. subtilis possesses a single lipoate-protein ligase, LplJ, that shares 57% and 39% sequence identity with LplA1 and LplA2, respectively.15 Therefore, it can be assumed that LplJ is unable to utilize Lpl-004 as a substrate, which would cause resistance against Lpl-004. The fact that the three LA analogues do not impact B. subtilis viability suggests at least partial species selectivity. Therefore, Lpl-004-based drugs may have a reduced interference with the patient’s Gram-positive microbiota.

In experiments using a ΔlipA mutant of S. aureus to assess the impact of Lpl-004 on protein post-translational modification, we found that the E2 subunits were labeled (Figure 5). To substantiate this finding, we employed selenolipoate, a compound with a well-established mechanism of action in E. coli: it is taken up through the action of the lipoate-protein ligase and binds to the E2 subunits, suppressing their activity.24 In this work, we showed that selenolipoic acid also inhibits S. aureus growth, and Western blot analysis confirmed its binding to the E2 subunits (Figures 5 and 7). This observation strongly suggests the effective binding of Lpl-004 to the dehydrogenase complexes. However, further investigations are required to clearly prove binding of Lpl-004 to the E2 subunits, e.g., by mass spectroscopy.

The crucial role of lipoate-protein ligases in growth inhibition was demonstrated by the double mutant ΔlplA1 ΔlplA2 that conferred resistance against Lpl-004 (Figure 6). In addition, due to the differences in growth inhibition obtained for the single mutants, the action of Lpl-004 appears to be mediated primarily by LplA2. This coincides with the fact that the compounds Lpl-004, Lpl-008, and Lpl-023, containing two carboxyl moieties, are able to interact with LplA2 via several hydrogen bonds and salt bridges and resulted in 53–65% higher docking scores compared to LA. This suggests that the three compounds might have a higher affinity to LplA2 than the natural substrate. From these findings, it can be inferred that the inhibitory effect of this compound is mediated through lipoate-protein ligase activity. This was verified through Western blot analysis of a mutant which, in addition to lacking ligase activity, is also incapable of synthesizing LA, revealing the absence of labeled E2s when exposed to Lpl-004 (Figure 6B). This result aligns with the growth data observed for selenolipoate (Figure 7). By utilizing several mutants in LA synthesis and uptake, we were able to demonstrate the ability of Lpl-004 to bind to the E2 subunits, similar to what occurs with selenolipoate.

The LA analogue 8-bromooctanoic acid (BrO) has been proposed to disrupt LA scavenging in the malaria-causing parasite Plasmodium falciparum. It has been previously disclosed that the use of exogenous LA in mitochondria plays a crucial role in its survival and growth during both the blood and liver stages,32,33 and parasite growth is inhibited.34 This compound also showed growth inhibition of Trypanosoma cruzi,35 although this parasite lacks lipoate-protein ligases. The actual target of BrO in trypanosomes is the amidotransferase, causing a blockade of LA synthesis.36 Recently, two cell-permeable and potent inhibitors (C3, LAMe) targeting the LA salvage pathway have been described that effectively killed blood-stage P. falciparum.37 In contrast to BrO, which is required in concentrations close to the millimolar range to inhibit the growth of the parasite, C3 and LAMe are effective at low micromolar concentrations. However, these compounds did not show any antimicrobial effect on L. monocytogenes despite a high affinity for LmLplA1, similar to that observed for PfLpl1, and cocrystallization studies showed favorable binding of C3 to the active site.37 Here, we report several LA analogues with comparable activity against S. aureus. To the best of our knowledge, Lpl-004, Lpl-008, and Lpl-023 are the first LA salvage-targeting compounds with antibacterial activity.

Previous studies indicated that, in mammalian cells, the lipoylated E2p excreted by S. aureus interacts with TLR1/2 receptors, effectively dampening the host immune response and serving as an immunosuppressant for macrophages.23 Moreover, animals infected with S. aureus ΔlipA showed a greater activation of macrophages and a reduction in the bacterial content. This suggests that protein lipoylation plays a crucial role in promoting bacterial survival during infection and its interruption aligns with more effective infection control by the host. Here, we show that Lpl-004 demonstrates an in vivo efficacy in a C. elegans infection model. We observed that survival of worms fed with the S. aureus WT strain grown in the presence of Lpl-004 was significantly prolonged. The lifespan was comparable to that of the ΔlipA mutant, which is unable to lipoylate its proteins and which is known to have an attenuated virulence in mice.23 The presence of Lpl-004 significantly decreased the mortality rate of the nematodes despite the fact that the bacteria were subjected to culture conditions that support their viability. While the innate worm response to S. aureus infection operates independently of the Toll-like receptor,38 the infection mechanism may still share similarities. We hypothesize that the binding of Lpl-004 to E2p might impede the ability of the bacterium to evade the immune response of the host. In contrast, when the worms were fed with the mutant strain defective in both lipoate-protein ligases, no significant impact on nematode survival was observed, despite the presence of the compound. Based on the fact that C. elegans lacks lipoate-protein ligases,28 we additionally ruled out any effects of the compound on the host with experimental data involving worms fed with dead E. coli along with Lpl-004 as a food additive. Our findings demonstrate that the compound does not influence the dehydrogenase activity in C. elegans. The use of this infection model was crucial for evaluating the efficacy of Lpl-004 in reducing the virulence and for corroborating its proposed mechanism of action. This organism emerges as a highly advantageous tool due to its simplicity, cost-effectiveness, and ease of maintenance and manipulation in the laboratory. These findings not only highlight the valuable features of this model but also establish the groundwork for conducting assays of compounds targeted against S. aureus virulence.

Based on the experimental data presented in this paper, we propose a mechanism of action for Lpl-004. The synthetic compound undergoes ligation to GcvH or E2o through the activity of lipoate-protein ligases, with a predominant involvement of LplA2. Subsequently, LipL transfers the compound to other E2 subunits, resulting in their inactivation. As Lpl-004 still contains the LA moiety, the exact mechanism of action is unclear. We assume that the additional group attached to the LA moiety prevents the LA part from being optimally positioned to its reaction partner. Further studies are needed to investigate the mechanism of action in detail.

The impact of the inactivated E2s can be circumvented by supplementing with the products of lipoyl-dependent complexes as present in a rich medium, mirroring conditions that can occur in a mammalian host context. Although under these conditions S. aureus is capable of growing in the presence of Lpl-004, the modified E2 subunits eventually lead to a reduced virulence.

Conclusion

Targeting the LA salvage pathway might be a viable approach for the development of a novel type of antibiotic adjuvant that improves the efficacy of S. aureus therapeutics. Further investigations are required to fully confirm the proposed mechanism of action and improve the efficacy of the compound. This approach could also be exploited for enzyme cascades in other pathogens for developing novel types of treatment options.

Methods

Molecular Modeling

The sequence of LplA2 was obtained from the UniProt database (https://www.uniprot.org/). A structural model of LplA2 was generated using the SWISS-MODEL server,39 based on the X-ray crystal structure of Enterococcus faecalis lipoate-protein ligase A (PDB ID 5IBY). Missing hydrogen atoms were added using Protonate3D in MOE (Molecular Operating Environment, Chemical Computing Group ULC, Montreal, Canada), followed by energy minimization (Amber10:EHT force field with R-Field implicit solvation model) using a three-step procedure (Cα atoms, backbone, all atoms), each step including a tether of 0.5 and an RMS gradient of 5.

A set of 4731 LA analogues was downloaded from the ZINC database. Compounds were docked without further modifications into the active site of the LplA2 protein model using GOLD version 1.8 in combination with the ChemPLP scoring function (Cambridge Crystallographic Data Centre, Cambridge, UK). The search space was defined by a sphere of 10 Å radius centered on coordinates of LA atom C2 in the modeling template structure. For each compound, 30 docking poses were generated. The early termination option was switched off.

Bacteria Strains and Growth Conditions

S. aureus and B. subtilis strains (Table 1) were routinely grown in Lysogeny Broth (LB)40 at 37 °C. Roswell Park Memorial Institute (RPMI) medium supplemented with 0.05% vitamin-free Casamino acids (VF-CAA) was used as a minimal medium for S. aureus, and Spizizen salts,41 supplemented with 0.05% VF-CAA and 0.01% each of tryptophan and phenylalanine, were used as the minimal medium (SMM) for B. subtilis. Different supplements, including 5 mM sodium acetate and 0.1 mM of each branched-chain fatty acid precursor (BCFAP: isobutyric acid, isovaleric acid, and 2-methylbutiric acid), were added as needed. Antibiotics were added to the media at the following concentrations: kanamycin, 5 μg mL–1; erythromycin, 5 μg mL–1. The synthetic compounds Lpl-004, Lpl-008, and Lpl-023 were dissolved in ethanol and added to the cultures at the concentrations indicated in each experiment. Selenolipoate was dissolved in ethanol and added to the cultures to 30 μg/mL final concentration. To assay the growth of S. aureus and B. subtilis cultures in liquid media, strains were incubated overnight in RPMI and SMM, respectively, containing 1% VF-CAA, supplemented with acetate and BCFAP. Cells were centrifuged and washed five times with RPMI or SMM and were used to inoculate the fresh media described above, at an OD600 of 0.1–0.15. Cell growth was measured using a Bioscreen C with 200 μL per well with continuous and medium shaking. Bacterial growth data presented as means ± SD statistical significance was determined using a paired t test. p values of 0.1, 0.01, and 0.001 were taken to indicate the statistical significance.

Table 1. Overview of Strains Used in This Study.

bacterial strains characteristics reference
Bacillus subtilis
JH642 trpC2 pheA1 laboratory stock
Staphylococcus aureus
USA300   laboratory stock
NE264 lipA::Tn (42)
NE1257 lplA1::Tn (42)
NE266 lplA2::Tn (42)
FA-S912 AH-LAC with an in-frame deletion of lplA1 and lplA2 (15)
FA-S1178 AH-LAC with an in-frame deletion of lipA, lplA1, and lplA2 (15)
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Protein Extracts and Western Blots

For the preparation of crude cell lysates, S. aureus strains were grown overnight in RPMI supplemented with acetate and BCFAP at 37 °C. Cells were resuspended in fresh media of the same composition, with or without Lpl-004, and cultured at 37 °C. A 10 mL aliquot of each culture was harvested after growth for 6 h. The samples were centrifuged; the supernatant was discarded, and the bacterial pellets were stored at −20 °C until whole cell lysates were prepared. After frozen pellets were thawed on ice, the bacteria were suspended in 250 μL of lysis buffer (50 Tris–HCl pH 8.0, 1 mM phenyl-methylsulfonyl fluoride, PMSF) and transferred to screw cap microcentrifuge lysing tubes (Tarsons) containing 250 μL of 600 μm glass cell disruption beads (Sigma-Aldrich). Cells were lysed by vortexing in eight sequential steps, for 60 s, each separated by a 30 s incubation period on ice. After cell disruption, samples were centrifuged at 14,000 rpm for 10 min. 250 μL of the supernatant was collected in microcentrifuge tubes containing 50 μL of loading buffer and subsequently boiled for 5 min prior to storage at −20 °C. Protein concentration was determined using BSA as a standard. Samples were run in 12% SDS-PAGE. Coomassie staining was performed to evaluate protein patterns and equivalent loading of samples using Coomassie Brilliant blue R 250 stain reagent (Sigma-Aldrich). Amersham LMW Calibration kit (GE Healthcare) or Amersham ECL Rainbow Marker (Cytiva) were used as molecular weight markers. Proteins were transferred to a nitrocellulose membrane, and lipoylated E2 subunits were detected using rabbit antilipoate antibody (Calbiochem) and a secondary antirabbit immunoglobulin G conjugated to horseradish peroxidase (Bio-Rad). RNA polymerase beta antibody (Invitrogen) and a secondary antimouse immunoglobulin G conjugated to horseradish peroxidase (Bio-Rad) were used as a loading control. The bands were visualized by the use of the ECL Plus Western Blotting Detection System (GE).

Lipoic Acid Bioassay

The LA content was assayed by a disk microbiological method.43E. coli strain KER 296 (ΔlipA26) was grown to saturation in M9 medium containing 0.1% VF-CAA, 5 mM sodium acetate, and 5 mM succinate. Cells were washed with M9 medium and resuspended in the same medium to one-half of the original volume. The assay plates consisted of M9 minimal medium containing 2% agar and 0.01% 2,2,5-triphenyl-tetrazolium chloride. Four milliliters of top agar (M9 medium, 0.8% agar, and 0.01% 2,2,5-triphenyl-tetrazolium chloride) containing 200 μL of washed cells were poured into each assay plate. A sterile disk was placed in the center of each plate, and 10 μL of sample was spotted on the disk. Plates were incubated for 24 h at 37 °C. The dye becomes red when microorganisms are alive and thus are capable of reducing the dye to formazan and remains colorless in dead bacteria. The diameter of the red zone of growth was measured. The LA concentration was determined by extrapolation of the sample diameter using a calibration curve over a range of 0.1 to 10 ng/mL of LA standards.

C. elegans Maintenance and Growth Conditions

Infertile TJ1060 worms (spe-9; rrf-3) were grown in NGM44 seeded with nonpathogenic E. coli OP50 at 15 °C for maintenance or with dead E. coli at 25 °C for killing assays. E. coli dead cells were obtained by treating a culture in the exponential phase of growth with 50 μg/mL kanamycin. After overnight incubation, the culture was centrifuged and concentrated 50-fold.

Nematode killing assays were performed as previously described.45 Briefly, 20 μL of a 1:2 dilution of a S. aureus overnight culture, with or without the addition of 50 μg/mL Lpl-004, was spread on 3.5 cm TSA plates supplemented with the appropriate antibiotics and incubated at 37 °C for 4 h. Fourth larval stage worms (L4) were transferred to the assay plates seeded with either S. aureus strains or dead E. coli and incubated at 25 °C. Survival was monitored over time as indicated. A worm was considered dead when it failed to respond to gentle touch with a platinum wire. Worms were censored when they died as a result of getting stuck to the wall of the plate. Mean survival days and intervals of mean survival days with 95% confidence were calculated by Kaplan–Meier tests using GraphPad Prism 8.4.2. All assays were performed at least in triplicates.

Acknowledgments

We express our sincere gratitude to Dr. Francis Alonzo for kindly providing S. aureus strains FA-S912 and FA-S1178. M.C.M. is a member of the Carrera del Investigador Científico del Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina; A.S. is a doctoral fellow from the same institution. This work was supported by Ministerio de Ciencia Tecnología e Innovación Productiva (Grant EULACH 16/T02-0161), CONICET (Grant P-UE-0039 2016), and the German Federal Ministry for Education and Research (01DN18001). A.J., A.N., and K.G. thank EULAC Health JOINT CALL 2016-2017 project TALASA for funding.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.4c00148.

  • Docking scores for virtual hits, binding mode of LA in LplA2 of S. aureus and B. faecalis, antibacterial activity of compounds in rich medium, LA competition assay, LA bioassay, effect of Lpl-004 on C. elegans survival, general methods for synthesis of Lpl-004, Lpl-008, and Lpl-023, and synthesis of selenolipoic acid (PDF)

  • Structure of the LplA2 homology model and the five top-ranked docking poses for LA, Lpl-004, and Lpl-023 (ZIP)

Author Contributions

A.J., M.C.M., and B.W. conceived and designed the project. B.W. carried out the virtual screen. K.G. and A.N. synthesized the compounds. A.S. and M.C.M. carried out the biological experiments. A.S., M.C.M., and B.W prepared the original draft of the manuscript. A.N. and B.W. reviewed the manuscript. A.J., M.C.M., and B.W. acquired funding. All authors have read and agreed to the published version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

id4c00148_si_001.pdf (1.1MB, pdf)
id4c00148_si_002.zip (97.2KB, zip)

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Supplementary Materials

id4c00148_si_001.pdf (1.1MB, pdf)
id4c00148_si_002.zip (97.2KB, zip)

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