A synthetic lethal approach for compound and target identification in Staphylococcus aureus

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The majority of bacterial proteins are dispensable for growth in the laboratory, but nevertheless play important physiological roles. There are no systematic approaches to identify cell-permeable small molecule inhibitors of these proteins. We demonstrate a strategy to identify such inhibitors that exploits synthetic lethal relationships both for small molecule discovery and for target identification. Applying this strategy in Staphylococcus aureus, we have identified a compound that inhibits DltB, a component of the teichoic acid D-alanylation machinery, which has been implicated in virulence. This D-alanylation inhibitor sensitizes S. aureus to aminoglycosides and cationic peptides and is lethal in combination with a wall teichoic acid inhibitor. We conclude that DltB is a druggable target in the D-alanylation pathway. More broadly, the work described demonstrates a systematic method to identify biologically active inhibitors of important bacterial processes that can be adapted to numerous organisms. Small molecules with specific protein targets are powerful tools to interrogate biological pathways1, 2. In bacteria, antibiotics have been used as probes to study divisome assembly and peptidoglycan recycling3–5 , to investigate how cytoskeletal dynamics are coupled to cell wall biosynthesis6–9 , and to characterize intrinsic resistance mechanisms and stress response pathways, among other processes10. Typical antibiotics, which inhibit targets that are essential for viability under laboratory growth conditions, can be identified in screens for growth inhibition. Unfortunately, the vast majority of proteins in bacteria are dispensable for growth in the laboratory and no systematic approaches for identifying inhibitors of these targets have been established. Here we demonstrate a small molecule discovery strategy that exploits synthetic lethality both to identify bioactive compounds that inhibit physiologically important processes and to identify their targets. Using this strategy, we have identified a compound that inhibits D-alanylation of Staphylococcus aureus teichoic acids. Synthetic lethality describes a biological interaction in which a given gene is dispensable in a wild-type background, but not in a mutant background in which another gene has been inactivated. The phenomenon implies that the interacting genes have functions that converge on the same essential process11. Large scale deletion and transposon mutant libraries have been used to identify gene-gene synthetic lethal interactions in bacteria and yeast12– 15, but a similar principle can be exploited in high throughput screens to discover small molecules that selectively kill a mutant but not a wild-type strain. Such molecules potentially inhibit targets in the synthetic lethal interaction network of the mutant. This screening approach has been used to identify possible anti-cancer therapeutics but has found limited use in bacteria16 . Here, we used a synthetic lethal screening approach to identify compounds that selectively inhibited growth of aS. aureus mutant deficient for synthesis of wall teichoic acids (WTAs). WTAs are anionic polymers that are covalently attached to peptidoglycan in many Grampositive organisms17. In S. aureus, WTAs comprise up to 50% of the cell wall by mass, making a substantial contribution to cell envelope integrity. Among other functions, they coordinate peptidoglycan synthesis and autolysin activity with cell division, and are also required for host infection18–21. Due to the importance of WTAs in cell envelope physiology, we predicted that they would have numerous synthetic lethal interactions with other cell envelope components15. We confirmed this hypothesis by probing a transposon library with the natural product tunicamycin, a selective inhibitor of TarO, the first enzyme in the WTA biosynthetic pathway20. We found that several genes became indispensible when WTA biosynthesis was inhibited15 . We have now screened a library of small molecules to identify compounds that inhibit proteins in the WTA synthetic lethal network, and have developed a strategy to identify the targets of these small molecules. Like the screening approach, the target identification strategy makes use of synthetic lethality. We report that amsacrine, a small molecule discovered in our chemical screen, inhibits DltB, a membrane-embedded enzyme required for D-alanylation of teichoic acids22. This modification is involved in virulence of S. aureus. We show that amsacrine sensitizes cells to aminoglycosides and cationic antimicrobial peptides23, a phenotype expected for a D-alanylation inhibitor. Hence, our approach can identify biologically active molecules that inhibit pathways implicated in virulence, but which are not essential for survival in vitro. Both the chemical screening approach and the target identification strategy described here can be adapted for other pathways or bacteria to identify inhibitors for use as cellular probes of bacterial physiology and components of synthetic lethal antibacterial compound combinations. RESULTS The synthetic lethal network for WTAs A screen for compounds that inhibit growth of a mutant bacterial strain can be carried out in the absence of prior knowledge of synthetic lethal interactions, but genetic information provides a list of possible targets for any small molecules that are identified. We previously identified a number of synthetic lethal interactions with wall teichoic acids by using tunicamycin to probe a pooled transposon library, and a subset of these interactions were validated15. To develop a comprehensive list of targets that are synthetically lethal with depletion of WTAs, we prepared a larger transposon library by a phage-based mini-Tn delivery mutagenesis method24, subjected it to growth in the presence of tunicamycin, and analyzed the results by Tn-seq25. Transposon insertions in each gene in the presence or absence of tunicamycin were compared to identify genes in which reads were depleted by >80%. These results were combined with those obtained previously to identify potential synthetic lethal partners of WTAs. Null mutants of candidate genes were tested for sensitivity to tunicamycin to confirm synthetic lethality with depletion of WTAs. Figure 1 shows a schematic of the S. aureus cell envelope in which all confirmed synthetic lethal targets with respect to WTAs are highlighted in red. The targets are all membrane- or wall-associated proteins, and include components of the lipoteichoic acid biosynthetic pathway26, the four components of the Dalanylation pathway, the cell wall stress response system GraRSVraFG27, and Stk1, a serine/ threonine kinase that regulates cell envelope remodeling. Stk1 is known to phosphorylate GraRS, which in turn regulates the expression of dltABCD 28. Several membrane proteins of unknown function were also confirmed as synthetically lethal with WTA depletion through this work or the previous study15.