A high-throughput cytotoxicity screening platform reveals <i>agr</i>-independent mutations in bacteraemia-associated <i>Staphylococcus aureus</i> that promote intracellular persistence.

Abstract

<i>Staphylococcus aureus</i> infections are associated with high mortality rates. Often considered an extracellular pathogen, <i>S. aureus</i> can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing <i>S. aureus</i> cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model<i>,</i> we have developed a platform called <i>InToxSa</i> (<u>in</u>tracellular <u>tox</u>icity of <i><u>S. a</u>ureus</i>) to quantify intracellular cytotoxic <i>S. aureus</i> phenotypes. Studying a panel of 387 <i>S</i>. <i>aureus</i> bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in <i>S. aureus</i> clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in <i>ausA</i>, encoding the aureusimine non-ribosomal peptide synthetase, reduced <i>S. aureus</i> cytotoxicity, and increased intracellular persistence. <i>InToxSa</i> is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant <i>S. aureus</i> pathoadaptive mutations that promote intracellular residency.