Recombinant Staphylococcus aureus UPF0349 protein SAR0901 (SAR0901)

What is the structural characterization of Recombinant Staphylococcus aureus UPF0349 protein SAR0901?

SAR0901 belongs to the UPF (Uncharacterized Protein Family) class of proteins in S. aureus. While the specific crystal structure of SAR0901 is not fully determined, similar S. aureus proteins have been successfully crystallized using vapor diffusion at room temperature. For example, successful crystallization of S. aureus protein domains has been achieved using 21-24% (wt/wt) monomethyl polyethylene glycol 5,000 in 100 mM sodium cacodylate at pH 6.5 . The structure determination typically employs molecular replacement techniques using related protein coordinates, with refinement procedures to achieve accurate modeling. X-ray crystallography remains the gold standard for determining the three-dimensional structure of such proteins, allowing for analysis of functional domains and potential interaction sites.

How does SAR0901 compare with other characterized S. aureus proteins?

SAR0901 should be evaluated in comparison with well-characterized S. aureus proteins such as staphylococcal protein A (SpA), iron surface determinant B (IsdB), and manganese transport protein C (MntC), which serve as virulence factors . These proteins have established roles in pathogenesis, with SpA enhancing staphylococcal virulence through mechanisms including immune evasion . Unlike proteins with known functions such as α-hemolysin (Hla) and staphylococcal enterotoxin B (SEB) that act as secreted factors, the UPF0349 family requires additional functional characterization to determine its specific role in S. aureus pathophysiology.

What expression systems are recommended for producing recombinant SAR0901?

For recombinant production of S. aureus proteins, E. coli-based expression systems are commonly employed. The methodological approach typically involves:

• Gene synthesis or PCR amplification of the SAR0901 gene from S. aureus genomic DNA

• Cloning into an expression vector with an appropriate affinity tag (His-tag is commonly used)

• Transformation into an expression strain like BL21(DE3)

• Induction of protein expression using IPTG or auto-induction media

• Purification via affinity chromatography followed by size-exclusion chromatography

These methods align with approaches used for other recombinant S. aureus proteins, such as those incorporated into multi-antigen vaccines . For proteins with potential disulfide bonds, consideration should be given to specialized E. coli strains like Origami or SHuffle.

What are the optimal conditions for assessing SAR0901 function in virulence models?

When designing experiments to assess potential virulence functions of SAR0901, researchers should consider both in vitro and in vivo approaches:

In vitro models:

• Bacterial adhesion to host cells (e.g., epithelial cells, endothelial cells)

• Biofilm formation assays

• Immune cell interaction studies (neutrophil/macrophage activation)

• Protease activity measurements if proteolytic functions are suspected

In vivo models:
For animal models, researchers can reference established S. aureus infection protocols. Studies with other S. aureus antigens have employed both sepsis and pneumonia mouse models . When designing such experiments, researchers should include:

• Wild-type controls

• SAR0901 deletion mutants

• Complemented strains to confirm phenotype specificity

• Assessment of bacterial loads in tissues

• Inflammatory cytokine measurements

• Histopathological analysis

These approaches allow for comprehensive evaluation of potential roles in virulence, similar to methodologies used for characterized S. aureus proteins.

How should researchers design experiments to identify potential interaction partners of SAR0901?

To identify protein-protein interactions involving SAR0901, researchers should employ multiple complementary approaches:

• Pull-down assays: Using recombinant tagged SAR0901 as bait to capture interacting proteins from bacterial or host cell lysates

• Yeast two-hybrid screening: For identifying direct protein-protein interactions

• Co-immunoprecipitation: Using antibodies against SAR0901 to isolate protein complexes

• Surface plasmon resonance: For quantitative measurement of binding kinetics

• Bacterial two-hybrid systems: Particularly useful for membrane proteins

• Cross-linking mass spectrometry: To identify transient interactions

These methods should be followed by validation through biochemical assays and functional studies. Analysis of protein-protein interactions requires consideration of the surface area of interaction, which for many protein complexes ranges from 1400-1700 Ų, with equal contributions from both interacting molecules .

What advanced structural biology techniques are most appropriate for analyzing SAR0901 conformational changes?

For advanced structural analysis of SAR0901, researchers should consider:

Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

• Provides information on protein dynamics and solvent accessibility

• Can identify regions involved in ligand binding or protein-protein interactions

• Particularly valuable for examining conformational changes upon binding events

Nuclear Magnetic Resonance (NMR) spectroscopy:

• Allows for structure determination in solution

• Provides dynamics information at atomic resolution

• Can detect weak transient interactions

Cryo-electron microscopy:

• Particularly useful if SAR0901 forms larger complexes

• Enables visualization without crystal formation

• Can capture different conformational states

Molecular dynamics simulations:

• Complements experimental data

• Provides insights into conformational flexibility

• Can predict potential binding sites and mechanisms

These advanced techniques should be implemented in conjunction with biochemical validation to establish structure-function relationships, following approaches similar to those used in structure-based computational studies of other bacterial proteins .

How can researchers effectively employ multi-omics approaches to understand SAR0901 in the context of S. aureus pathogenesis?

A comprehensive multi-omics approach to studying SAR0901 should include:

Transcriptomics:

• RNA-seq under various infection-relevant conditions

• Analysis of co-expressed genes to identify functional networks

• Examination of regulatory elements controlling SAR0901 expression

Proteomics:

• Quantitative proteomics to measure SAR0901 expression levels

• Post-translational modification analysis

• Protein interaction network mapping

Metabolomics:

• Identification of metabolic pathways affected by SAR0901 expression/deletion

• Measurement of key metabolites during infection

Integration of multi-omics data:
The following data integration table outlines how researchers should synthesize findings:

This integrated approach allows researchers to place SAR0901 within the broader context of S. aureus biology and pathogenesis.

How should researchers address the challenges of functional redundancy when studying SAR0901?

S. aureus possesses numerous virulence factors with potentially overlapping functions, complicating the functional characterization of proteins like SAR0901. Researchers should:

• Create multiple mutant strains:

  • Single SAR0901 deletion mutant

  • Combined deletion with functionally related genes

  • Complementation with wild-type and mutated versions

• Employ varied infection models:

  • Different tissue/organ systems

  • Various immune states

  • Multiple S. aureus genetic backgrounds

• Utilize comprehensive phenotypic screening:

  • High-throughput assays testing multiple conditions

  • Stress response analysis

  • Host cell interaction panels

• Apply statistical approaches for detecting subtle phenotypes:

  • Mixed models accounting for strain background effects

  • Time-course analyses rather than single timepoints

  • Competition assays with wild-type strains

These approaches help overcome the challenges presented by functional redundancy, as demonstrated in studies of other S. aureus proteins where single-gene knockout phenotypes may be subtle due to compensatory mechanisms .

What are the best practices for analyzing contradictory data regarding SAR0901 function?

When faced with contradictory data regarding SAR0901 function, researchers should:

• Systematically evaluate experimental conditions:

  • Growth conditions (media, temperature, oxygen levels)

  • Strain backgrounds and genetic variations

  • Host cell types or animal models used

• Consider protein expression levels:

  • Natural expression levels vs. recombinant overexpression

  • Post-translational modifications

  • Protein folding and stability

• Implement orthogonal validation techniques:

  • Different methodological approaches to test the same hypothesis

  • Independent laboratory verification

  • Consulting with experts in specific techniques

• Perform meta-analysis:

  • Systematic review of all available data

  • Statistical analysis of combined datasets

  • Identification of variables that may explain discrepancies

• Report comprehensively:

  • Document all experimental conditions

  • Acknowledge limitations

  • Present alternative interpretations

This methodical approach to resolving contradictions follows principles of good research practice in complex biological systems, similar to approaches used in advanced qualitative research methods .

How can researchers evaluate SAR0901 as a potential vaccine antigen candidate?

To evaluate SAR0901 as a potential vaccine antigen, researchers should follow a systematic approach:

• Conservation analysis:

  • Sequence conservation across clinical S. aureus isolates

  • Structural epitope preservation

  • Assessment of antigenic drift potential

• Immunogenicity screening:

  • B-cell epitope prediction and validation

  • T-cell epitope mapping

  • Analysis of MHC binding potential

• Animal immunization studies:

  • Antibody response characterization (titer, isotype, duration)

  • Functional antibody assays (opsonophagocytosis, neutralization)

  • T-cell response assessment

• Protection assays:

  • Challenge studies in multiple infection models

  • Assessment of bacterial loads, survival, and pathology

  • Evaluation of immune correlates of protection

This approach mirrors successful evaluation strategies used for other S. aureus antigens in vaccine development, such as the five-antigen vaccine incorporating Hla, SEB, SpA, IsdB-N2, and MntC . Researchers found that multi-antigen formulations provided broader protection against diverse S. aureus strains than single antigens, suggesting that SAR0901 might be most valuable as part of a multi-component vaccine.

What methodologies are most appropriate for determining the immunomodulatory effects of SAR0901?

To characterize potential immunomodulatory effects of SAR0901, researchers should employ:

• Immune cell response assays:

  • Dendritic cell activation and maturation

  • T-cell proliferation and cytokine production

  • B-cell activation and antibody production

  • Neutrophil functional assays (NETosis, phagocytosis)

• Cytokine profiling:

  • Multiplex cytokine analysis following SAR0901 exposure

  • Temporal analysis of inflammatory mediators

  • Comparison with known immunomodulatory S. aureus proteins

• Signaling pathway analysis:

  • Investigation of TLR activation

  • NF-κB pathway monitoring

  • MAPK signaling evaluation

  • Inflammasome activation assessment

• In vivo immunomodulation:

  • Tissue-specific immune cell recruitment

  • Local vs. systemic immune responses

  • Duration of immunomodulatory effects

These methodologies would help determine whether SAR0901 has immunomodulatory properties similar to other S. aureus proteins, such as SpA, which can contribute to immune evasion through interaction with B-cell receptors .