Prophage acquisition by Staphylococcus aureus contributes to the expansion of Staphylococcal immune evasion
biorxiv(2023)
摘要
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 (ϕSa3int). Human-adapted S. aureus often 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 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 ϕSa3int 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 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 silico analysis of the S. aureus genome confirmed the insertion of a ∼43.8 kb ϕSa3int 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 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 help 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
### Competing Interest Statement
The authors have declared no competing interest.
* ### Glossary
Active lysogen
: bacterial strain harboring identifiable prophage sequence and releasing reinfecting phage particles.
Double lysogen
: bacterial cell harbouring two complete/intact prophages
Lysogen
: bacterial cell containing one or more prophages within its genome
Lysogenic conversion
: phenotypic change in a host bacterium caused by the integration of a prophage.
Lysogeny
: state of phage integration into the bacterial genome.
Non-lysogen
: bacterial strains lacking any identifiable prophage sequence in their genome
Passive lysogen
: bacterial strains harboring identifiable prophage sequence but not releasing actively reinfecting phage particles
Productive induction
: excision of prophage from the bacterial chromosome followed by release of phage particles either spontaneously or under external stress
Prophage
: temperate phage DNA integrated in the bacterial genome.
Single lysogen
: bacterial cell harbouring only one complete/intact prophage
Temperate phage
: bacterial virus that can integrate into a bacterial genome (or be maintained extra-chromosomally), become stabilized in this way, and, upon receiving a cue, can excise and propagate.
Transduction
: process of horizontal gene transfer, wherein a region of a bacterial genome is packaged into phage particles that, upon release and entrance into a new host, is inserted into the genome of the latter.
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