Using Transposon Sequencing to Identify Vulnerabilities in Staphylococcus aureus

Abstract

Antibiotic resistant infections cost thousands of lives in the United States every year. Resistance exists for every known antibiotic, making the development new antibiotics crucial to human health. One technique that has become instrumental in prioritizing targets for antibiotic development is transposon sequencing (Tn-Seq). Tn-Seq is a powerful high-throughput technology that connects bacterial genes with phenotypes and can be used to identify genes that are essential for bacterial survival. In the early years of Tn-Seq, results from a single representative strain were largely assumed to reflect the entire species. However, researchers are now showing that gene essentiality varies among members of a species and that this variability can have direct implications for antibiotic susceptibility. Here I describe the use of Tn-Seq to more thoroughly characterize Staphylococcus aureus gene reliance. We have generated a compendium of core essential genes shared by five strains from across the S. aureus phylogeny, including three strains of methicillin-resistant S. aureus (MRSA), a leading cause of antibiotic resistance-associated mortality in the United States. To better understand antibiotic resistance, we have also developed a new analytical approach that uses Tn-Seq data to identify genes whose overexpression confers a fitness advantage in a given condition. We applied this method to a range of data from antibiotic-treated samples and recovered many clinically-reported mechanisms of resistance as well as new mechanisms that may provide insights into how antibiotics work and how bacteria overcome them. Given the baseline differences in gene reliance between strains of S. aureus, we wondered whether antibiotic resistance factors would be shared across strains. We used daptomycin, a common antibiotic for the treatment of MRSA, as a case study to compare resistance factors and vulnerabilities across the five aforementioned strains of S. aureus. We found that the genes modulating antibiotic susceptibility were largely shared, even in cases where the strains relied on the genes to differing degrees in favorable growth conditions. Together, these studies provide the most nuanced characterization of S. aureus gene reliance to date and emphasize the utility of Tn-Seq for investigating antibiotic resistance.