The two-component system ArlRS is essential for wall teichoic acid glycoswitching in <i>Staphylococcus aureus</i>

<i>Staphylococcus aureus</i> is among the leading causes of hospital-acquired infections. Critical to <i>S. aureus</i> biology and pathogenesis are the cell wall-anchored glycopolymers wall teichoic acids (WTA). Approximately one-third of <i>S. aureus</i> isolates decorates WTA with a mixture of α1,4- and β1,4-<i>N</i>-acetylglucosamine (GlcNAc), which requires the dedicated glycosyltransferases TarM and TarS, respectively. Environmental conditions, such as high salt concentrations, affect the abundance and ratio of α1,4- and β1,4-GlcNAc WTA decorations, thereby impacting biological properties such as antibody binding and phage infection. To identify regulatory mechanisms underlying WTA glycoswitching, we screened 1,920 <i>S</i>. <i>aureus</i> mutants (Nebraska Transposon Mutant Library) by immunoblotting for differential expression of WTA-linked α1,4- or β1,4-GlcNAc using specific monoclonal antibody Fab fragments. Three two-component systems (TCS), GraRS, ArlRS, and AgrCA, were among the 230 potential hits. Using isogenic TCS mutants, we demonstrated that ArlRS is essential for WTA β1,4-GlcNAc decoration. ArlRS repressed <i>tarM</i> expression through the transcriptional regulator MgrA. In bacteria lacking <i>arlRS</i>, the increased expression of <i>tarM</i> correlated with the absence of WTA β1,4-GlcNAc, likely by outcompeting TarS enzymatic activity. ArlRS was responsive to Mg²⁺, but not Na⁺, revealing its role in the previously reported salt-induced WTA glycoswitch from α1,4-GlcNAc to β1,4-GlcNAc. Importantly, ArlRS-mediated regulation of WTA glycosylation affected <i>S. aureus</i> interaction with the innate receptor langerin and lysis by β1,4-GlcNAc-dependent phages. Since WTA represents a promising target for future immune-based treatments and vaccines, our findings provide important insight to align strategies targeting <i>S. aureus</i> WTA glycosylation patterns during infection.IMPORTANCE<i>Staphylococcus aureus</i> is a common colonizer but can also cause severe infections in humans. The development of antibiotic resistance complicates the treatment of <i>S. aureus</i> infections, increasing the need for antibiotic alternatives such as vaccines and therapies with bacterial viruses also known as phages. Wall teichoic acids (WTA) are abundant glycosylated structures of the <i>S. aureus</i> cell wall that have gained attention as a promising target for new treatments. Importantly, WTA glycosylation patterns show variation depending on environmental conditions, thereby impacting phage binding and interaction with host factors, such as antibodies and innate pattern-recognition receptors. Here, we show that the two-component system ArlRS is involved in the regulation of WTA glycosylation by responding to environmental changes in Mg²⁺ concentration. These findings may support the design of new treatment strategies that target WTA glycosylation patterns of <i>S. aureus</i> during infection.