The metallophore staphylopine is essential for survival of Staphylococcus epidermidis in human synovial fluid

Due to extended life expectancies, prosthetic joint infections are an increasing burden on healthcare institutions worldwide. The most commonly isolated causative agents are staphylococci, though the mechanisms underpinning survival and proliferation in synovial fluid are still not fully understood. In this study, we aimed to identify genes important for survival in synovial fluid using a transposon mutant library and RNAseq. We produced a transposon mutant library, containing approximately 57,000 unique insertion mutants, in Staphylococcus epidermidis strain 846. This library was grown in Muller Hinton broth or processed human synovial fluid samples, and Transposon-directed Insertion Sequencing (TraDIS) was used to identify genes involved in survival in synovial fluid. This identified importance of the his, pur and cnt operons. These genes were also upregulated in both Staphylococcus epidermidis 846 and the model Staphylococcus epidermidis strain RP62A when exposed to human synovial fluid. All these key pathways contribute to production of the metallophore staphylopine. To confirm staphyopine production is essential for survival in synovial fluid, a defined transposon insertion in the gene encoding for staphylopine export (cntE) mutant was used. This demonstrated impaired survival in synovial fluid compared to the wild type. RT-qPCR also showed that cntE was more highly expressed after exposure to infected synovial fluid (where metals will be depleted) than non-infected fluid. In conclusion, TraDIS and RNASeq both identified the importance of staphylopine for survival in human synovial fluid. This suggests an opportunity for exploitation for therapeutic or diagnostic use. Author SummaryStaphylococcus epidermidis is a common cause of prosthetic joint infection, however accurate diagnosis remains difficult. In this work we explored the genetic basis of Staphylococcus epidermidis survival in human synovial fluid using a large transposon mutant library, and identified which genes were differentially expressed upon exposure to the fluid. We found crossover between the datasets, which pointed to the importance of the metal acquisition compound staphylopine. The expression levels of the gene required for staphylopine export were shown to be dependent on the infection status of the individual samples were obtained from. We also found the gene cluster to be conserved in a range of staphylococcal species isolated from cases of prosthetic joint infection. Our work provides a valuable resource in the from of a large transposon mutant library, and provides a greater understanding of the requirements for staphylococcal survival in human synovial fluid, providing potential biomarkers for future diagnostic development.