Comprehensive Virulence Profiling and Evolutionary Analysis of Specificity Determinants in Staphylococcus aureus Two-Component Systems

In the Staphylococcus aureus genome, a set of highly conserved two-component systems (TCSs) composed of histidine kinases (HKs) with their cognate response regulators (RRs) sense and respond to environmental stimuli, which drive the adaptation of the bacteria. This study investigates the complex interplay between TCSs in S. aureus USA300, a predominant Methicillin-Resistant S. aureus (MRSA) strain, revealing shared and unique virulence regulatory pathways and genetic variations mediating signal specificity within TCSs. Using TCS-related mutants from the Nebraska Transposon Mutant Library, we analyzed the effects of inactivated TCS HKs and RRs on the production of various virulence factors, in vitro infection abilities, and adhesion assays. We found that the TCSs influence on virulence determinants was not associated with their phylogenetic relationship, indicating divergent functional evolution. Using the cocrystalized structure of the DesK-DesR from B. subtilis and modelled structures of the 4 NarL TCSs in S. aureus , we identified interacting residues, revealing specificity determinants and conservation within the same TCS, even from different strain backgrounds. The interacting residues were highly conserved within strains but varied between species due to selection pressures and coevolution of cognate pairs. This study unveils the complex interplay and divergent functional evolution of TCSs, highlighting their potential for future experimental exploration of phosphotransfer between cognate and non-cognate recombinant HK and RRs. IMPORTANCE Given the widespread conservation of Two-Component Systems (TCSs) in bacteria and their pivotal role in regulating metabolic and virulence pathways, they present a compelling target for anti-microbial agents—especially in the face of rising multi-drug resistant infections. Harnessing TCSs therapeutically necessitates a profound understanding of their evolutionary trajectory in signal transduction, as this underlies their unique or shared virulence regulatory pathways. Such insights are critical for effectively targeting TCS components, ensuring an optimized impact on bacterial virulence and mitigating the risk of resistance emergence via the evolution of alternative pathways. Our research offers an in-depth exploration of virulence determinants controlled by TCSs in S. aureus, shedding light on the evolving specificity determinants that orchestrate interactions between their cognate pairs.