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Search for New Antibiotics Reveals Links Between Metabolism and Virulence

 

By: Carol A. Rouzer, VICB Communications
Published:  November 21, 2016

 

Metabolic stress resulting from virulence factor production acts in concert with an inhibitor of Fe-S cluster assembly to suppress growth of fermenting Staphylococcus aureus

 

 

The growing problem of antibiotic resistance among major pathogenic bacteria has spurred interest in the discovery of new drugs that have novel targets and mechanisms of action. Thus, the recent observations by Vanderbilt Institute of Chemical Biology members Eric Skaar and Gary Sulikowski that a novel compound [VU003882 ('882, Figure 1)] inhibits the growth of fermenting Staphylococcus aureus was of particular interest. Although '882 was originally discovered in a high-throughput screen for compounds that modulate bacterial heme biosynthesis, further studies demonstrated that its effect on heme homeostasis was distinct from its growth inhibitory activity. This led the Skaar and Sulikowski lab researchers to launch an effort to determine '882's mechanism of action. They now report that '882 blocks the assembly of iron-sulfur clusters, key cofactors for many enzymes of intermediary metabolism. Their results also reveal an interesting link between virulence regulation and metabolism in fermenting S. aureus [J. E. Choby, et al. (2016) Cell Chem. Biol., published online November 17, DOI:10.1016/j.chembiol.2016.09.012].

 

 

FIGURE 1. Chemical structure of VU0038882 ('882).

 

Although ubiquitous in the environment, S. aureus is capable of causing particularly devastating infections of the skin, soft tissues, bone, heart, and blood. Its impact on human health is magnified by its ability to produce multiple virulence factors and its facility in acquiring antibiotic resistance. S. aureus is capable of surviving in both the presence and absence of oxygen, and can readily switch from aerobic respiration to anaerobic fermentation as dictated by its environment. This enables it to form abscesses, enclosed areas of infection characterized by poor circulation and low oxygen tension. It also increases S. aureus's antibiotic resistance, because many antibiotics use the energy of the membrane potential generated during aerobic respiration to enter the bacterial cell. Thus, a drug that targets fermenting bacteria, such as '882, would be an important addition to the antibacterial armamentarium.

 

The investigators began their research on '882's mechanism of action using the Newman strain of S. aureus, as it was the strain used in their original studies of the compound. By growing the bacteria in the presence of '882, they were able to isolate seven '882-resistant strains. Genomic sequencing of these strains revealed that all carried a mutation in the saePQRS operon, which encodes the SaeRS. It comprises mulitple proteins, including SaeS, a histidine kinase that is activated in response to components from the host immune system. SaeS autophosphorylates a histidine residue and then transfers the phosphate to SaeR, a transcription factor. Phosphorylation activates SaeR, which then binds to target promoters, inducing transcription of the associated genes. In addition to virulence factors, SaeR promotes the expression of genes for SaeP and SaeQ, proteins encoded by the saePQRS operon that regulate the activity of the system.

 

FIGURE 2. Diagrammatic representation of the SaeRS system. Exposure to neutrophil products stimulates SaeS to autophosphorylate a histidine residue. The phosphate group is then transferred to SaeR, a transcription factor that triggers the expression of genes regulated by target promoters. This results in the transcription of multiple virulence factors in addition to the SaeP and SaeQ regulatory proteins that bind to SaeS, and serve to downregulate the activity of the system. Figure reproduced under the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License from Q. Liu, et al. (2016) Genes., 7, 81. Copyright 2016.

 

 

The presence of mutations in saePQRS in all '882-resistant strains of S. aureus Newman suggested that turning off expression of this system was key to the resistance. This hypothesis was further supported after additional attempts to isolate '882-resistant strains yielded bacteria with mutations in other proteins that interact with the SaeRS system. This led the investigators to note that the Newman strain carries an L18P mutation in the saeS gene that encodes a constitutively active protein. Thus, it appeared that mutations conveying '882 resistance did so by turning off the abnormally high level of SaeRS activation in this strain. Consistently, Newman strains genetically modified to eliminate SaeRS expression (ΔsaeRS) or to return SaeS function to normal (saeSP18L) were resistant to '882, as was the USA300LAC (LAC) strain that had been isolated from an infected patient and carried a wild-type saeS gene. 


Fermentation is a less efficient energy-producing pathway than aerobic respiration. Thus, under anaerobic conditions, it is advantageous for bacteria to conserve energy as much as possible. This led the investigators to hypothesize that constitutive SaeRS activity leading to the unnecessary production of virulence factors is deleterious to fermenting bacteria. In support of this hypothesis, they found that the parental Newman strain of S. aureus grew less well anaerobically than the LAC strain or Newman strains containing the ΔsaeRS or saeSP18L mutations. This finding suggested that metabolic stress resulting from constitutive SaeRS activity contributes to '882 sensitivity in the parental Newman strain.
     

Despite these new insights into the mechanism of action of '882, the researchers had still not identified its target. In search of new clues, they used microarray analysis to search for transcriptional differences between parental Newman strain bacteria and bacteria containing the ΔsaeRS mutation grown in the presence of '882. As expected, the two strains differed with regard to expression of virulence factors, but they also differed in levels of expression of multiple enzymes involved in intermediary metabolism. Among the affected genes was rimJ, which encodes a ribosomal protein N-acetyltranferase and was overexpressed in the parental Newman strain. They confirmed that suppression of rimJ expression in the Newman strain conveys '882 resistance, leading to the hypothesis that excessive rimJ expression results in overconsumption of acetyl-CoA, and that this contributes to '882 sensitivity. Consistent with this hypothesis, they found four other acetyl-CoA consuming enzymes that were overexpressed in Newman strain bacteria. Repression of the expression of two of these enzymes conveyed '882 resistance. Adding pantothenate, a precursor of CoA also conveyed '882 resistance to Newman strain bacteria.

 

These results were consistent with the hypothesis that sensitivity to '882 was tied in some way to metabolic stress. But they still did not reveal the target of the compound. Thus, the researchers tried a more direct approach. They synthesized a biotin-conjugated analog of '882 and used it with streptavidin beads to pull down '882-associated proteins from S. aureus lysates. This approach yielded four proteins: SufB, SufC, SufD, and SufS, all of which are components of the Suf iron-sulfur (Fe-S) cluster assembly system (Figure 3). Fe-S clusters are important cofactors in a large number of enzymes critical to intermediary metabolism. The researchers confirmed that '882 binds directly to SufC with a dissociation constant of 4 μM. Consistently, they also discovered that exposure of bacteria to '882 leads to reduced activity of aconitase, an Fe-S cluster enzyme, and that this reduction was more pronounced when the bacteria were grown under anaerobic conditions. Additional experiments demonstrated that '882 had no effect on the amount of aconitase protein in the cells, and it did not affect aconitase activity in cells exposed to protein synthesis inhibitors. The latter findings suggested that '882 acts to disrupt Fe-S cluster assembly on newly synthesized protein. Also consistent with the hypothesis that '882's mechanism of action is to block SufC, the investigators found that LAC strains deficient in Suf proteins other than SufC showed reduced aconitase activity and did not grow well in medium lacking leucine and isoleucine, amino acids requiring Fe-S cluster proteins for their biosynthesis. In both cases, exposure to '882 exacerbated these conditions in the bacteria.

 

FIGURE 3. Diagrammatic representation of the role of Suf proteins in Fe-S cluster assembly. SufS provides sulfur from cysteine to the SufBCD complex with the aid of the SufE sulfur transfer shuttle. Fe2-S2 or Fe4-S4 complexes are then assembled by the SufBCD complex. Figure reproduced according to the ACS Author Choice License from E. S. Boyd, et al. (2014) Biochemistry, 53, 5834. Copyright 2014, American Chemical Society.

 

 

Together the data suggest that '882 acts by inhibiting Fe-S cluster assembly, thereby blocking the synthesis of a large number of enzymes required for normal metabolism. The result is metabolic stress that is worsened under anaerobic conditions that limit the bacterial cell's metabolic repertoire. Sensitivity to '882 is further increased when additional metabolic stressors are applied to the cell. This is most evident in the case of the Newman strain, in which constitutive SaeRS activity leads to wasteful production of virulence factors (Figure 4). The researchers note that the Suf Fe-S cluster assembly system is not found in eukaryotes, so targeting this system should be possible with low toxicity to humans. In addition, it is interesting to speculate that '882 or compounds like it, may exhibit increased efficacy in vivo under conditions that lead to a strong SaeRS-mediated virulence factor production response.

 

 

FIGURE 4. Proposed mechanisms of toxicity of '882. '882 binds to SufC, a key component of the SufBCD Fe-S cluster assembly complex. As a result, it inhibits Fe-S cluster assembly, resulting in reduced activity of a large number of enzymes, such as aconitase, that are required for intermediary metabolism in fermenting bacteria. In addition, constitutive activity of the SaeRS two component system, as seen in the Newman strain of S. aureus, leads to excessive production of virulence factors, draining key metabolic reservoirs and increasing the metabolic stress.

 

 

 

View Cell Chemical Biology article: A Small-Molecule Inhibitor of Iron-Sulfur Cluster Assembly Uncovers a Link between Virulence Regulation and Metabolism in Staphylococcus aureus

 

 

 

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