Prophage acquisition byStaphylococcus aureuscontributes to the expansion of Staphylococcal immune evasion
Authors: Nepal Roshan , Houtak Ghais , Bouras George , Ramezanpour Mahnaz , Feizi Sholeh , Shaghayegh Gohar , Shearwin Keith , Psaltis Alkis James , Wormald Peter-John , Vreugde Sarah 
Published: April 2023

AbstractStaphylococcus aureuscolonizes 30% of the human population, but only a few clones cause severe infections.S. aureus’virulence varies and partly depends on the presence of prophages, viral DNA embedded in theS. aureuscore genome, such as hlb-converting prophage (ϕSa3int). Human-adaptedS. aureusoften harbours a ϕSa3int group of prophages preferentially integrated into their β-hemolysin (hlb) gene that encodes human immune evasion cluster (IEC) genes. Exotoxins and immune modulatory molecules encoded by this prophage can inhibit human innate immunity increasingS. aureuspathogenicity. This study aims to investigate the genomic and phenotypic plasticity ofS. aureusand changes in its extracellular proteome after the acquisition of ϕSa3int prophage.To achieve this, we usedS. aureusstrains isolated from the sinus cavities of a patient with severe chronic rhinosinusitis (CRS) at two different time points (S. aureusSA222 andS. aureusSA333) and hybrid sequenced the strains using short-read Illumina and long-read Oxford nanopore technology.In silicoanalysis showed the presence of a ϕSa3int prophage in the later isolate but not in the earlier isolate while most of the core genes remained identical. Using mitomycin C, we induced the ϕSa3int prophage, and transduced it into the Sa3int-prophage-free SA222 isolate to obtain a laboratory generated ‘double lysogen’. We confirmed the successful lysogenisation with culture methods (spot assay, blood agar) and also by sequencing. We compared growth kinetics, biofilm biomass and metabolic activity between parent and the lysogen by establishing growth curves, crystal violet and resazurin assays. Exoproteins were identified and quantified using mass spectrophotometry.Integration of ϕSa3int prophage in SA222 down-regulated the beta-hemolysin expression of the lysogen. In silicoanalysis of theS. aureusgenome confirmed the insertion of a ∼43.8 kb ϕSa3int prophage intohlbgene. Insertion of prophage DNA did not alter the growth kinetics, biofilm formation, adhesion to primary human nasal epithelial cells and the metabolic activity in a biofilm. However, the acquisition of ϕSa3int prophage significantly changed the expression of various secreted proteins, both bacterial and prophage-encoded. Altogether, thirty-eight exoproteins were significantly differentially regulated in the laboratory created lysogen, compared to its recipient strain SA222. Among these proteins, there was significant upregulation of 21 exoproteins (55.3 %) including staphylokinase (sak), SCIN (scn), and intercellular adhesion protein B (icaB) and downregulation of 17 exoproteins (44.7 %), including β-hemolysin (hlb/sph) and outer membrane porin (phoE). Most of the upregulated proteins were involved in immunomodulation that helpS. aureusescape human innate immunity and help cause chronic infection. These findings may contribute to the development of novel approaches to render S.aureussusceptible to the immune response by blocking prophage-associated defence mechanisms.HighlightsA ϕSa3int prophage preferentially integrates into the β-haemolysin gene (hlb) gene thereby disrupting the beta-hemolysin function.A ∼43.8 kb ϕSa3int prophage acquisition byS. aureushas no impact on its growth kinetics, biofilm formation and adhesion to primary human nasal epithelial cells (HNECs).The presence of a ϕSa3int group prophage likely enhancesStaphylococcus aureus’human immune evasion capability as the prophage encodes a complete set of immune evasion cluster (IEC) genes.Targeted identification of virulence factors in addition to species and strain identification may lead to better-personalized therapy as not allS. aureuscarry the same virulence genes.