Comparative Tn-Seq reveals common daptomycin resistance determinants inStaphylococcus aureusdespite strain-dependent differences in essentiality of shared cell envelope genes

Abstract

Antibiotic-resistant Staphylococcus aureus remains a leading cause of antibiotic resistance-associated mortality in the United States. Given the reality of multi-drug resistant infections, it is imperative that we establish and maintain a pipeline of new compounds to replace or supplement our current antibiotics. A first step towards this goal is to prioritize targets by identifying the genes most consistently required for survival across the S. aureus phylogeny. Here we report the first direct comparison of gene essentiality across multiple strains of S. aureus via transposon sequencing. We show that mutant fitness varies by strain in key pathways, underscoring the importance of using more than one strain to differentiate between core and strain-dependent essential genes. Despite baseline differences in gene importance, several pathways, including the lipoteichoic acid pathway, become consistently essential under daptomycin exposure, suggesting core vulnerabilities that can be exploited to resensitize daptomycin-nonsusceptible isolates. We also demonstrate the merit of using transposons with outward-facing promoters capable of overexpressing nearby genes for identifying clinically-relevant gain-of-function resistance mechanisms. Together, the daptomycin vulnerabilities and resistance mechanisms support a mode of action with wide-ranging effects on the cell envelope and cell division. This work adds to a growing body of literature demonstrating the nuanced insights gained by comparing Tn-Seq results across multiple bacterial strains.Author summaryAntibiotic-resistant Staphylococcus aureus kills thousands of people every year in the United States alone. To stay ahead of the looming threat of multidrug-resistant infections, we must continue to develop new antibiotics and find ways of making our current repertoire of antibiotics more effective, including by finding pairs of compounds that perform best when administered together. In the age of next-generation sequencing, we can now use transposon sequencing to find potential targets for new antibiotics on a genome-wide scale, identified as either essential genes or genes that become essential in the presence of an antibiotic. In this work, we created a compendium of genes that are essential across a range of S. aureus strains, as well as those that are essential in the presence of the antibiotic daptomycin. The results will be a resource for researchers working to develop the next generation of antibiotic therapies.

Structure and reconstitution of a hydrolase complex that may release peptidoglycan from the membrane after polymerization

“…We created a high-density transposon library, which comprises six sublibraries, in S. aureus USA300 by phage-based transposition as previously described 9 , 10 , 34 . The transposon library was treated with beta-lactams (8 μg mL −1 for mecillinam, 0.4 μg mL −1 for cefoxitin, and 0.1 μg mL −1 for oxacillin) at 37°C.…”

A decade of advances in transposon-insertion sequencing

“…4) and can contribute to developing a better understanding of how resistance emerges, as well as guide the development of new strategies to target resistant bacteria. Traditional TIS experiments performed by culturing transposon libraries with inhibitory but sublethal concentrations of antibiotics for several generations have been used to define a comprehensive non-essential gene complement involved in intrinsic resistance for many clinically important pathogens, including the notorious ESKAPE species (Enterococcus faecium, S. aureus, K. pneumoniae, Acinetobacter baumannii, P. aeruginosa and Enterobacter species) 18,[42][43][44] . These studies have shown that while antibiotics may have specific targets (for example, in cell wall synthesis, DNA replication or protein synthesis), the bacterial response to antibiotics is actually distributed across the genome.…”

High‐throughput transposon sequencing highlights the cell wall as an important barrier for osmotic stress in methicillin resistant Staphylococcus aureus and underlines a tailored response to different osmotic stressors

“…We therefore focused our further analysis on the S. aureus salt stress response and conducted two additional TN‐seq screens to confirm the robustness of this approach (Figure a). TN‐seq screens were performed using a previously published library (Coe et al, ; Santiago et al, ) with six transposon variants and the library containing only one transposon variant used in the first TN‐seq experiment (Supporting Table for overview). The libraries were propagated for 17 generations in either LB or in medium containing 0.5 M NaCl (Figure b).…”

“…A previously published highly‐saturated S. aureus USA300 library with a mix of six different transposons was used (Coe et al, ; Santiago et al, ). In addition, a new transposon library with only the blunt transposon was produced using the same techniques as described (Santiago et al, ) containing approximately 1.2 million individual clones.…”