A high-throughput cytotoxicity screening platform revealsagr-independent mutations in bacteraemia-associatedStaphylococcus aureusthat promote intracellular persistence

Abstract

Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses and causing host cell death. Classical methods for assessing S. aureus 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, we have developed a platform called InToxSa (Intracellular Toxicity of S. aureus) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S. aureus bacteraemia isolates, and combined with comparative, statistical and functional genomics, our platform identified mutations in S. aureus 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 ausA, encoding the aureusimine non ribosomal peptide synthetase, reduced S. aureus cytotoxicity and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.

The AusAB non-ribosomal peptide synthase inStaphylococcus aureuspreferentially incorporates exogenous phenylalanine and tyrosine into the aureusimine natural products

“…Aureusimines are enigmatic natural products and, while AusAB appears to play a role in the virulence of intracellular S. aureus [8, 9], the exact targets of these molecules are poorly understood. Naturally occurring phevalin was first isolated from Streptomyces sp., and exhibited calpain inhibitory activity [16].…”

“…The high degree of conservation of the aus AB gene cluster across all sequenced S. aureus strains, suggests an important biological function for the NRPS. Two studies propose a role for the AusAB for intracellular S. aureus virulence [8, 9]. Furthermore, the dipeptide aldehyde form of phevalin (i.e.…”