The Extracytoplasmic Linker Peptide of the Sensor Protein SaeS Tunes the Kinase Activity Required for Staphylococcal Virulence in Response to Host Signals

Abstract

Bacterial pathogens often employ two-component systems (TCSs), typically consisting of a sensor kinase and a response regulator, to control expression of a set of virulence genes in response to changing host environments. In Staphylococcus aureus, the SaeRS TCS is essential for in vivo survival of the bacterium. The intramembrane-sensing histidine kinase SaeS contains, along with a C-terminal kinase domain, a simple N-terminal domain composed of two transmembrane helices and a nine amino acid-long extracytoplasmic linker peptide. As a molecular switch, SaeS maintains low but significant basal kinase activity and increases its kinase activity in response to inducing signals such as human neutrophil peptide 1 (HNP1). Here we show that the linker peptide of SaeS controls SaeS’s basal kinase activity and that the amino acid sequence of the linker peptide is highly optimized for its function. Without the linker peptide, SaeS displays aberrantly elevated kinase activity even in the absence of the inducing signal, and does not respond to HNP1. Moreover, SaeS variants with alanine substitution of the linker peptide amino acids exhibit altered basal kinase activity and/or irresponsiveness to HNP1. Biochemical assays reveal that those SaeS variants have altered autokinase and phosphotransferase activities. Finally, animal experiments demonstrate that the linker peptide-mediated fine tuning of SaeS kinase activity is critical for survival of the pathogen. Our results indicate that the function of the linker peptide in SaeS is a highly evolved feature with very optimized amino acid sequences, and we propose that, in other SaeS-like intramembrane sensing histidine kinases, the extracytoplasmic linker peptides actively fine-control their kinases.

VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System

“…SaeRS has been shown to be activated by several antibiotics and ␣-defensins (17,21), including human neutrophil peptide 1 (HNP-1), resulting in increased transcription of class I target genes such as coa (15)(16)(17). This occurs when extracellular HNP-1 interacts with an extracellular loop of SaeS (13). As an additional approach to the constitutive SaeS used above, we hypothesized that HNP-1 could be used to stimulate SaeS to levels that would bypass the need for VfrB in coa promoter expression.…”

SaeRS Is Responsive to Cellular Respiratory Status and Regulates Fermentative Biofilm Formation in Staphylococcus aureus

“…The mechanistic details underlying the integration of internal and external signals, as well as repressing and activating signals, by Sae are currently unclear. However, recent studies have postulated an intriguing theory wherein intramembrane HKs, such as SaeS, could achieve signal integration using a trip wire-like model (51,52). In this model, the conformations adopted by the N-terminal domain govern the kinase activity of the histidine kinase.…”

The SaeRS Two-Component System Is a Direct and Dominant Transcriptional Activator of Toxic Shock Syndrome Toxin 1 in Staphylococcus aureus

“…The His 6 -tagged proteins were purified using nickel column chromatography using a previously described protocol (26). The purified SaeR and SaeS c were dialyzed in buffers consisting of 10 mM Tris-HCl, 138 mM NaCl, and 2.7 mM KCl (pH 7.5) and 50 mM Tris-HCl, 50 mM KCl, and 1 mM MgCl 2 (pH 8.0), respectively (27), and concentrated up to 10-fold using Amicon centrifugal filter devices (Millipore). The protein concentration was determined by the bicinchoninic acid assay (Thermo Scientific), and purified proteins were stored in buffers containing 10% glycerol at Ϫ80°C until used.…”