Molecular Cross-talk between Sa3int Phages and their Staphylococcus aureus Host

Abstract

Staphylococcus aureus is a major opportunistic pathogen that asymptomatically colonizes the human nasal cavity but also causes severe infections. Temperate phages play a central role in S. aureus genetic diversity and pathogenicity. Among them, Sa3int phages are the most prevalent in human nasal isolates and contribute to virulence by encoding highly human-specific immune evasion factors. Their dynamic regulatory switch, controlling expression of both the hlb integration locus and the phage-encoded virulence genes, plays a crucial role during infection. While the genomic organization and the lysogenic-lytic transition are well characterized in Sa3int phages, the regulatory mechanisms during the lytic life cycle and their interplay with host factors remain poorly understood. In this work, the dynamics of the Sa3int phage and its transcriptional patterns across diverse S. aureus strain backgrounds were investigated. Distinct strain-specific differences in phage transfer frequencies were observed, enabling classification of the strains into high- and low-transfer groups. Phage replication during the lytic cycle was identified as the key driver of host-dependent variations, while differences in adsorption, prophage integration, and prophage excision were ruled out. Furthermore, substantial variations in the expression of late genes associated with phage assembly, DNA packaging, and host cell lysis were detected. The essential promoter region P23, located upstream of the late gene cluster, was identified and its regulatory characteristics were elucidated. Through this analysis, SAOUHSC_02200 was discovered and characterized as the late transcriptional regulator (Ltr) of lytic late genes. Both P23 activity and ltr expression were found to vary in a host-dependent manner and to be modulated by the alternative sigma factor SigB and its downstream effector SpoVG. Further, the influence of the global regulator and DNA-binding protein SarA on the phage life cycle was examined. Phage production of the Sa3int phage Φ13 and the Sa5int phage Φ11 was shown to be promoted by SarA. Analyses of distinct stages of the phage life cycle revealed that SarA alters the glycosylation pattern of wall teichoic acid (WTA), thereby enhancing adsorption of Φ11. Additionally, a DNA-protective effect of SarA was observed, reflected by reduced activation of the SOS response. Finally, phage genome replication of Φ13 was shown to be promoted by SarA, likely through its function as a DNA-structure protein. Together, a complex regulatory network was revealed in which phage-encoded factors and host global regulators interact to coordinate phage gene expression, replication, and subsequent bacterial virulence.