Prophage acquisition byStaphylococcus aureuscontributes to the expansion of Staphylococcal immune evasion

Abstract

Staphylococcus aureus colonizes 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 the S. aureus core genome, such as hlb-converting prophage (Sa3int). Human-adapted S. aureus often harbours a Sa3int group of prophages preferentially integrated into their beta-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 increasing S. aureus pathogenicity. This study aims to investigate the genomic and phenotypic plasticity of S. aureus and changes in its extracellular proteome after the acquisition of Sa3int prophage. To achieve this, we used S. aureus strains isolated from the sinus cavities of a patient with severe chronic rhinosinusitis (CRS) at two different time points (S. aureus SA222 and S. aureus SA333) and hybrid sequenced the strains using short-read Illumina and long-read Oxford nanopore technology. In silico analysis showed the presence of a Sa3int 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 Sa3int 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 Sa3int prophage in SA222 down-regulated the beta-hemolysin expression of the lysogen. In silico analysis of the S. aureus genome confirmed the insertion of a ~43.8 kb Sa3int prophage into hlb gene. 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 Sa3int 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-generated 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 beta-hemolysin (hlb/sph) and outer membrane porin (phoE). Most of the upregulated proteins were involved in immunomodulation that helps S. aureus escape human innate immunity and help cause chronic infection. These findings may contribute to the development of novel approaches to render S. aureus susceptible to the immune response by blocking prophage-associated defence mechanisms. Highlights: 1. A Sa3int prophage preferentially integrates into the beta-haemolysin gene (hlb) gene thereby disrupting the beta-hemolysin function. 2. A ~43.8 kb Sa3int prophage acquisition by S. aureus has no impact on its growth kinetics, biofilm formation and adhesion to primary human nasal epithelial cells (HNECs). 3. The presence of a Sa3int group prophage likely enhances Staphylococcus aureus' human immune evasion capability as the prophage encodes a complete set of immune evasion cluster (IEC) genes. 4. Targeted identification of virulence factors in addition to species and strain identification may lead to better-personalized therapy as not all S. aureus carry the same virulence genes.

Characterization and Preclinical In Silico Safety Assessment of Three Virulent Bacteriophages Targeting Carbapenem-Resistant Uropathogenic Escherichia coli

“…Well-characterized phages are required for therapeutic use to ensure the genomic safety of the therapeutic phage as some lysogenic phages encode toxin-producing genes like Shiga toxin (stx) by Stx phages, cholera toxin (ctx) by CTX phages. Recently AMR genes and other VFs are also reported in transducing phages (prophages), which modulate bacterial lifestyle, virulence and pathogenesis (Nepal et al, 2023;Nepal, Houtak, Wormald, et al, 2022;Philipson et al, 2018). In the environment, the presence of phage depends upon the availability of their host bacterium.…”