<i>In Vivo</i> Gene Expression Profiling of Staphylococcus aureus during Infection Informs Design of Stemless Leukocidins LukE and -D as Detoxified Vaccine Candidates

Staphylococcus aureus is a clinically important bacterial pathogen that has become resistant to treatment with most routinely used antibiotics. Alternative strategies, such as vaccination and phage therapy, are therefore actively being investigated to prevent or combat staphylococcal infections. Vaccination requires that vaccine targets are expressed at sufficient quantities during infection so that they can be targeted by the host's immune system. While our knowledge of <i>in vitro</i> expression levels of putative vaccine candidates is comprehensive, crucial <i>in vivo</i> expression data are scarce and promising vaccine candidates during <i>in vitro</i> assessment often prove ineffective in preventing S. aureus infection. Here, we show how a newly developed high-throughput quantitative reverse transcription-PCR (qRT-PCR) assay monitoring the expression of 84 staphylococcal genes encoding mostly virulence factors can inform the selection and design of effective vaccine candidates against staphylococcal infections. We show that this assay can accurately quantify mRNA expression levels of these genes in several host organs relying only on very limited amounts of bacterial mRNA in each sample. We selected two highly expressed genes, <i>lukE</i> and <i>lukD</i>, encoding pore-forming leukotoxins, to inform the design of detoxified recombinant proteins and showed that immunization with recombinant genetically detoxified LukED antigens conferred protection against staphylococcal skin infection in mice. Consequently, knowledge of <i>in vivo</i>-expressed virulence determinants can be successfully deployed to identify and select promising candidates for optimized design of effective vaccine antigens against S. aureus. Notably, this approach should be broadly applicable to numerous other pathogens. <b>IMPORTANCE</b> Vaccination is an attractive strategy for preventing bacterial infections in an age of increased antimicrobial resistance. However, vaccine development frequently suffers significant setbacks when candidate antigens that show promising results in <i>in vitro</i> experimentation fail to protect from disease. An alluring strategy is to focus resources on developing bacterial virulence factors that are expressed during disease establishment or maintenance and are critical for bacterial in-host survival as vaccine targets. While expression profiles of many virulence factors have been characterized in detail <i>in vitro</i>, our knowledge of their <i>in vivo</i> expression profiles is still scarce. Here, using a high-throughput qRT-PCR approach, we identified two highly expressed leukotoxins in a murine infection model and showed that genetically detoxified derivatives of these elicited a protective immune response in a murine skin infection model. Therefore, <i>in vivo</i> gene expression can inform the selection of promising candidates for the design of effective vaccine antigens.