Recombinant Staphylococcus saprophyticus subsp. saprophyticus Sensor protein vraS (vraS)

Basic Research Questions

• What is the VraS protein in Staphylococcus saprophyticus and how does it function?

  The VraS protein in S. saprophyticus is a histidine protein kinase that forms part of the VraSR two-component regulatory system. This transmembrane sensor undergoes autophosphorylation at a conserved histidine residue (analogous to H156 in S. aureus) in response to cell wall damage. Once phosphorylated, VraS transfers its phosphoryl group to its cognate response regulator VraR, which then mediates transcriptional changes to coordinate a cellular response to cell wall stress .

  The VraSR system functions as a "sentinel" mechanism, rapidly detecting damage to cell wall peptidoglycan and initiating adaptive responses that enhance antibiotic resistance. The system is particularly responsive to antibiotics targeting cell wall peptidoglycan biosynthesis, such as beta-lactams and vancomycin . VraS not only phosphorylates VraR but also controls its dephosphorylation, providing tight regulation of the signaling pathway .

• What methods are used to express and purify recombinant VraS protein from S. saprophyticus?

  Recombinant VraS protein from S. saprophyticus can be expressed and purified using protocols adapted from studies on S. aureus VraS. The standard methodology involves:

  1. PCR amplification of the vraS gene from S. saprophyticus genomic DNA

  2. Cloning into an expression vector (typically pET-based) with an appropriate affinity tag

  3. Expression in E. coli BL21(DE3) or similar strains under IPTG induction

  4. For functional studies, expression of a truncated version lacking the transmembrane domain (e.g., VraS[64-347] in S. aureus) improves solubility while retaining kinase activity

  5. Purification by affinity chromatography followed by size exclusion chromatography

  It's important to note that purification of full-length VraS with its transmembrane domain often requires detergent solubilization, while the cytoplasmic domain can be purified under native conditions for in vitro phosphorylation studies .

• How does the VraSR system contribute to antibiotic resistance in S. saprophyticus?

  The VraSR system contributes to antibiotic resistance in S. saprophyticus through several mechanisms:

  1. Upon detection of cell wall damage by antibiotics, VraS initiates a phosphotransfer signaling cascade that ultimately results in upregulation of genes involved in cell wall biosynthesis and repair

  2. This response helps the bacterium survive the antibiotic challenge by reinforcing its cell wall structure

  3. VraSR activation leads to expression of genes necessary for cell wall biosynthesis and proteolytic quality control

  4. In S. saprophyticus isolated from urinary tract infections, resistance to multiple antibiotics is common (58% of isolates are multidrug-resistant), potentially involving VraSR-mediated responses

  5. The VraSR system likely contributes to the intrinsic resistance of S. saprophyticus to certain antibiotics and the acquisition of resistance to others through adaptive responses

Advanced Research Questions

• What are the experimental approaches for investigating VraS phosphotransfer mechanisms in S. saprophyticus?

  Investigating VraS phosphotransfer mechanisms requires sophisticated biochemical and genetic approaches:

  1. In vitro autophosphorylation assays: Using purified recombinant VraS cytoplasmic domain with [γ-32P]ATP to detect and quantify autophosphorylation activity

  2. Site-directed mutagenesis: Creating targeted mutations in the VraS histidine residue (equivalent to H156 in S. aureus) to confirm the phosphorylation site

  3. Phosphotransfer assays: Incubating phosphorylated VraS with purified VraR and monitoring phosphotransfer using SDS-PAGE and autoradiography

  4. Phosphorylation kinetics: Measuring the rates of VraS autophosphorylation and phosphotransfer to VraR under various conditions to understand regulatory mechanisms

  5. Dephosphorylation assays: Assessing VraS phosphatase activity toward phosphorylated VraR to understand the complete signaling cycle

  6. Chromosomal vraS mutations: Engineering site-specific mutations in the S. saprophyticus chromosome to correlate in vitro findings with in vivo phenotypes

• How do structural features of VraS inform its function in sensing cell wall damage?

  The structural features of VraS that inform its sensing function include:

  1. Transmembrane domain: Contains the sensor region that detects cell wall peptidoglycan damage, likely through conformational changes induced by alterations in the cell wall structure

  2. HAMP domain: Transmits signals from the transmembrane sensor region to the cytoplasmic kinase domain, facilitating conformational changes that activate the kinase

  3. Dimerization and histidine phosphotransfer (DHp) domain: Contains the conserved histidine residue (H156 in S. aureus) that undergoes autophosphorylation and mediates dimerization of VraS molecules

  4. Catalytic and ATP-binding (CA) domain: Binds ATP and catalyzes phosphorylation of the conserved histidine in the DHp domain

  Structural studies suggest that cell wall damage is detected by the extracellular/transmembrane portions of VraS, triggering conformational changes that activate the cytoplasmic kinase domain. This activation leads to autophosphorylation and subsequent phosphotransfer to VraR, initiating the cellular response to cell wall stress .

• What is the relationship between VraSR signaling and biofilm formation in S. saprophyticus?

  The relationship between VraSR signaling and biofilm formation in S. saprophyticus is complex and not fully elucidated, but several connections can be inferred:

  1. Biofilm formation is highly prevalent in S. saprophyticus isolates (65-91% of isolates), suggesting its importance for bacterial survival and virulence

  2. The VraSR system responds to cell wall damage, which can occur during attachment to surfaces and biofilm formation processes

  3. Cell wall stress may trigger VraSR-mediated responses that alter cell surface properties conducive to biofilm formation

  4. In S. saprophyticus, biofilm formation appears to be predominantly ica-independent, as only four strains carried a complete ica gene cluster (icaADBCR)

  5. Instead, biofilm formation in S. saprophyticus involves various matrix components including proteins, polysaccharides, and eDNA in different combinations

  6. The VraSR system may regulate alternative biofilm-associated genes in S. saprophyticus, contributing to its adaptation to different environments

  Table 1: Biofilm Matrix Phenotypes in S. saprophyticus

  Matrix Type	Components	Prevalence	Potential VraSR Involvement
  S	Polysaccharide	Varies	May regulate non-ica polysaccharide genes
  PS	Protein-polysaccharide	Common	Likely regulates cell wall associated proteins
  PD	Protein-eDNA	Common	May respond to eDNA-induced cell wall stress
  P	Protein	Common	Regulates cell surface proteins
  PDS	Protein-eDNA-polysaccharide	Less common	Complex regulation of multiple components

• How do mutations in the vraS gene affect antibiotic resistance profiles in S. saprophyticus?

  Based on studies in S. aureus and extrapolation to S. saprophyticus, vraS mutations can significantly alter antibiotic resistance profiles:

  1. Mutations at the phosphorylation site (equivalent to H156A in S. aureus) would disrupt the phosphotransfer signaling cascade, potentially reducing resistance to cell wall-active antibiotics

  2. Chromosomal vraS mutations in S. aureus lead to decreased upregulation of the cell wall stress stimulon after antibiotic exposure, suggesting similar effects would occur in S. saprophyticus

  3. S. saprophyticus isolates show varying resistance to antibiotics, with highest resistance rates to erythromycin but sensitivity to linezolid and vancomycin

  4. The interaction between vraS mutations and mecA gene presence (found in 21% of S. saprophyticus isolates) would further modulate resistance profiles

  5. Altered VraS signaling would likely impact S. saprophyticus biofilm formation, which is an important contributor to antibiotic resistance

  6. Target site mutations in VraS could potentially be exploited for developing novel antimicrobial strategies against S. saprophyticus infections

• What comparative genomic approaches can reveal the evolution of the VraSR system in S. saprophyticus versus other staphylococci?

  Comparative genomic approaches to study VraSR evolution include:

  1. Phylogenetic analysis: Constructing phylogenetic trees based on vraS and vraR sequences from multiple staphylococcal species to determine evolutionary relationships

  2. Synteny analysis: Examining the conservation of gene order around the vraRS operon in different species

  3. Domain architecture comparison: Analyzing the conservation of functional domains within VraS across species

  4. Selection pressure analysis: Calculating dN/dS ratios to identify regions under positive or purifying selection

  5. Horizontal gene transfer detection: Identifying signatures of horizontal gene transfer that may have influenced vraS evolution

  The VraRS TCS is highly conserved in the low-G+C Gram-positive family Firmicutes, suggesting fundamental importance . In S. saprophyticus, genomic adaptations in the VraSR system may reflect its specialized niche as a uropathogen. Comparative analysis with S. aureus VraS can reveal unique features that may contribute to S. saprophyticus-specific antibiotic responses and biofilm formation capacities .

• What techniques can be used to monitor VraSR-mediated transcriptional responses in S. saprophyticus under antibiotic stress?

  Several techniques can be employed to monitor VraSR-mediated transcriptional responses:

  1. RNA-Seq: Global transcriptome analysis to identify all genes differentially expressed following antibiotic exposure, comparing wild-type and vraS mutant strains

  2. Quantitative RT-PCR: Targeted analysis of specific VraSR-regulated genes before and after antibiotic exposure

  3. Promoter-reporter fusions: Constructing fusions between VraSR-regulated promoters and reporter genes (GFP, luciferase) to monitor transcriptional activity in real-time

  4. Chromatin immunoprecipitation (ChIP-seq): Identifying direct VraR binding sites across the S. saprophyticus genome after antibiotic stress

  5. Proteomics: Mass spectrometry-based approaches to identify changes in protein abundance in response to antibiotics, mediated by VraSR

  These approaches can reveal the specific genes and pathways regulated by VraSR in S. saprophyticus compared to other staphylococci, potentially identifying unique adaptation mechanisms related to its role as a uropathogen and biofilm former .