Recombinant Staphylococcus aureus UPF0478 protein SAR1817 (SAR1817), partial

What is UPF0478 protein SAR1817 and why is it significant in S. aureus research?

UPF0478 protein SAR1817 is an uncharacterized protein from Staphylococcus aureus subsp. aureus MRSA252. It belongs to the UPF0478 protein family (InterPro: IPR009293) . While its specific function remains largely unknown, studying such uncharacterized proteins is important for understanding S. aureus biology and potential virulence mechanisms. S. aureus is a major human pathogen causing a wide range of clinical infections, including bacteremia, infective endocarditis, and skin and soft tissue infections . Characterizing novel proteins like SAR1817 may reveal new targets for antimicrobial development or vaccine design.

What structural information is available for SAR1817?

The structure of SAR1817 has been computationally modeled using AlphaFold. According to the RCSB PDB (AF_AFQ6GFW9F1), the model has a global pLDDT (predicted Local Distance Difference Test) score of 79.13, indicating a confident structure prediction . The protein consists of 163 amino acids and appears to have regions of both high and moderate confidence in the structural prediction. It is important to note that this is a computed model with no experimental verification. Regions with pLDDT scores between 70-90 are considered confidently predicted but may still contain inaccuracies compared to experimental structures .

What expression systems are recommended for recombinant production of SAR1817?

For recombinant production of S. aureus proteins like SAR1817, E. coli expression systems are commonly employed due to their ease of use and high yield. Based on similar S. aureus protein expression studies, BL21(DE3) or Rosetta strains are recommended host cells, particularly when codon optimization is performed for the heterologous expression. Expression vectors containing strong inducible promoters like T7 (pET vectors) with appropriate affinity tags (His6, GST, or MBP) facilitate efficient purification. Expression conditions typically involve induction with 0.1-1.0 mM IPTG at lower temperatures (16-25°C) to promote proper folding of S. aureus proteins and reduce inclusion body formation.

What purification strategies are effective for obtaining high-purity SAR1817 for structural and functional studies?

A multi-step purification approach is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) for His-tagged SAR1817 or glutathione affinity for GST-tagged protein

• Intermediate purification: Ion exchange chromatography based on the theoretical isoelectric point of SAR1817

• Polishing: Size exclusion chromatography to remove aggregates and obtain monodisperse protein

Typical buffer conditions should maintain protein stability:

• Lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, protease inhibitors

• Purification buffers: Similar composition with decreasing salt gradient for ion exchange

• Storage buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

Protein purity should be assessed by SDS-PAGE, with expected molecular weight around 18-20 kDa based on its 163 amino acid sequence .

What quality control parameters should be monitored during SAR1817 production?

Several quality control parameters should be assessed:

For structural studies like X-ray crystallography, additional dynamic light scattering analysis is recommended to confirm monodispersity before crystallization attempts.

How reliable is the AlphaFold model of SAR1817 and what experimental approaches could validate it?

The AlphaFold model of SAR1817 (AF_AFQ6GFW9F1) has a pLDDT score of 79.13, categorizing it as a "confident" prediction but not "very high confidence" (which requires >90) . To validate this computational model, several experimental approaches are recommended:

Cross-validation using multiple techniques provides higher confidence in the structural model.

What computational approaches beyond AlphaFold could provide insights into SAR1817 function?

Several computational approaches can complement the AlphaFold structural model:

• Molecular dynamics simulations: To examine conformational flexibility and identify potential binding pockets

• Structural homology searches: Using DALI, VAST, or PDBeFold to identify structural homologs that might suggest function

• Evolutionary coupling analysis: To identify co-evolving residues that may indicate functional sites

• Binding site prediction: Using algorithms like FTSite, SiteMap, or CASTp to identify potential ligand binding pockets

• Protein-protein interaction prediction: Using tools like HADDOCK or ClusPro to model potential protein partners

• Electrostatic surface analysis: To identify charged patches that might indicate nucleic acid or substrate binding regions

• Conservation mapping: Using ConSurf to map evolutionary conservation onto the protein structure

Integrating these computational analyses with laboratory experiments would provide a more comprehensive understanding of SAR1817's potential functions.

What approaches could identify potential binding partners or substrates of SAR1817?

To identify potential binding partners or substrates, employ a multi-faceted approach:

• Pull-down assays: Using tagged recombinant SAR1817 as bait to capture interacting proteins from S. aureus lysates, followed by mass spectrometry identification

• Yeast two-hybrid screening: To identify protein-protein interactions, particularly if SAR1817 functions as a regulatory protein

• Bacterial two-hybrid systems: More appropriate for bacterial proteins like SAR1817

• Co-immunoprecipitation: Using anti-SAR1817 antibodies to pull down protein complexes

• Crosslinking mass spectrometry: To capture transient interactions

• Thermal shift assays: To screen for small molecule ligands that stabilize SAR1817

• Surface plasmon resonance or bio-layer interferometry: To quantify binding affinities of identified partners

• Differential scanning fluorimetry: To screen metabolite libraries for potential substrates

For substrate identification, metabolomics approaches comparing wild-type and SAR1817 knockout strains could reveal accumulated metabolites indicating potential enzyme function.

How can genetic approaches be used to elucidate the function of SAR1817 in S. aureus?

Several genetic approaches can help determine SAR1817 function:

• Gene knockout/deletion: Creating a ΔSAR1817 strain using allelic exchange methods to observe phenotypic changes

• Conditional expression systems: For essential genes where knockout may be lethal

• Complementation studies: Reintroducing SAR1817 to confirm phenotype restoration

• Point mutations: Of predicted critical residues to confirm their functional importance

• Reporter gene fusions: To understand expression patterns under different conditions

• Transcriptomics (RNA-seq): Comparing wild-type and knockout strains to identify affected pathways

• Transposon mutagenesis screens: To identify synthetic lethal or synthetic sick interactions

• CRISPR interference (CRISPRi): For partial knockdown of expression

Each approach provides different insights into the protein's function, and combining multiple methods increases confidence in the findings.

What high-throughput screening methods could reveal SAR1817's role in S. aureus biology?

Several high-throughput approaches could elucidate SAR1817's role:

• Phenotype microarrays: Testing ΔSAR1817 mutants across hundreds of growth conditions to identify specific requirements

• Fitness profiling: Using transposon sequencing (Tn-seq) to measure relative fitness of SAR1817 mutants under various stresses

• Chemical genomics: Screening for compounds with differential activity against wild-type versus ΔSAR1817 strains

• Protein microarrays: To identify interacting proteins or small molecules

• Metabolomics profiling: Using LC-MS/MS to compare metabolite profiles between wild-type and mutant strains

• CRISPR-based screening: To identify genetic interactions

• Automated microscopy: For morphological phenotyping of mutants

Data from these approaches should be integrated with bioinformatics analysis to develop testable hypotheses about SAR1817 function.

Could SAR1817 play a role in S. aureus virulence or antibiotic resistance?

While the specific function of SAR1817 remains uncharacterized, it could potentially contribute to S. aureus virulence or antibiotic resistance. To investigate this possibility:

• Infection models: Compare virulence of wild-type and ΔSAR1817 strains in appropriate animal models (murine bacteremia, skin infection, etc.)

• Antibiotic susceptibility testing: Determine if deletion affects minimum inhibitory concentrations of various antibiotics

• Biofilm formation assays: Assess if SAR1817 influences biofilm development, which contributes to antibiotic tolerance

• Stress resistance testing: Evaluate response to oxidative stress, pH changes, and antimicrobial peptides

• Host cell interaction studies: Examine adhesion, invasion, and survival within host cells

• Immune evasion assays: Test resistance to neutrophil killing and complement activation

S. aureus employs numerous virulence factors including toxins, immune evasion proteins, and adhesins . If SAR1817 regulates any of these, its deletion could significantly impact pathogenicity.

How does SAR1817 expression change under different infection-relevant conditions?

To understand how SAR1817 expression responds to infection-relevant conditions, several approaches are recommended:

• qRT-PCR analysis: Measure SAR1817 transcript levels under various stresses (oxidative, nitrosative, pH, antimicrobial peptides)

• Reporter gene constructs: Fusing the SAR1817 promoter to luciferase or fluorescent proteins to monitor expression

• Proteomics: Quantitative mass spectrometry to measure protein levels under different conditions

• RNA-seq: Examining transcriptome-wide changes including SAR1817

• In vivo expression technology (IVET): To identify if SAR1817 is specifically induced during infection

• Single-cell analysis: To determine if expression is heterogeneous within the population

Experimental conditions should mimic relevant host environments:

• Low pH (phagolysosome-like)

• Nutrient limitation (iron, manganese restriction)

• Oxidative stress (H₂O₂, HOCl exposure)

• Host factors (serum, antimicrobial peptides)

• Biofilm versus planktonic growth

How does SAR1817 compare to similar proteins in other Staphylococcal species?

A comparative analysis of SAR1817 homologs across Staphylococcal species can provide evolutionary insights:

• Sequence alignment: Using BLAST and multiple sequence alignment tools to identify conserved residues

• Phylogenetic analysis: Constructing trees to understand evolutionary relationships

• Synteny analysis: Examining conservation of genomic context around the SAR1817 locus

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

• Structural comparison: Of predicted or experimentally determined structures

• Functional complementation: Testing if homologs from other species can complement a SAR1817 knockout

• Closely related pathogenic species (S. epidermidis, S. lugdunensis)

• Less pathogenic staphylococci (S. saprophyticus)

• Divergent staphylococcal species

• Analysis of presence/absence in different S. aureus lineages (MRSA vs. MSSA)

How might SAR1817 be evaluated as a potential vaccine antigen against S. aureus?

Evaluating SAR1817 as a vaccine antigen would require a systematic approach:

• Antigen conservation analysis: Sequence comparison across diverse S. aureus clinical isolates to ensure broad coverage

• Surface accessibility prediction: Computational and experimental verification of exposure on bacterial surface

• Immunogenicity assessment: Testing humoral and cellular immune responses in animal models

• Protective efficacy studies: Challenge experiments in appropriate animal models

• Adjuvant optimization: Testing different adjuvant formulations to enhance immunogenicity

• Cross-protection evaluation: Against different S. aureus strains

• Toxicity and safety testing: In appropriate animal models

S. aureus vaccine development has faced significant challenges, with several candidates failing in clinical trials . Previous vaccine approaches targeted surface proteins, capsular polysaccharides, or toxins . If pursuing SAR1817 as a vaccine antigen, combining it with other antigens may be more effective, as multi-component vaccines have shown more promise than single-antigen approaches .

What methodological approaches would be suitable for studying potential interactions between SAR1817 and host immune factors?

To investigate SAR1817 interactions with host immune components:

• ELISA-based binding assays: To detect direct binding to host factors

• Surface plasmon resonance: For quantitative binding kinetics measurements

• Cell-based assays: Using immune cells (neutrophils, macrophages) to assess functional effects

• Phagocytosis assays: To determine if SAR1817 affects bacterial uptake by phagocytes

• Complement activation studies: To measure effects on classical, alternative, or lectin pathways

• Cytokine profiling: Measuring immune cell responses to purified SAR1817

• Ex vivo infection models: Using human blood or tissue samples

• Immunoprecipitation from infected cells: To identify host binding partners

These approaches should be conducted with proper controls, including SAR1817 mutants with disrupted predicted functional domains and appropriate negative control proteins.

How could advanced structural biology techniques elucidate SAR1817's potential role in protein-protein interactions?

Advanced structural biology techniques to study SAR1817 protein interactions include:

• X-ray crystallography of complexes: Co-crystallization with identified binding partners

• Cryo-electron microscopy: Particularly useful for larger complexes

• NMR spectroscopy: For mapping interaction interfaces through chemical shift perturbations

• Hydrogen-deuterium exchange mass spectrometry: To identify regions protected upon complex formation

• Cross-linking mass spectrometry: To identify proximity relationships between interacting proteins

• Small-angle X-ray scattering (SAXS): For low-resolution envelopes of complexes in solution

• Förster resonance energy transfer (FRET): For studying interactions in real-time

• Analytical ultracentrifugation: To determine stoichiometry and binding affinities

Integration of these methods with computational approaches like molecular dynamics simulations and protein-protein docking would provide comprehensive understanding of interaction mechanisms.

How does research on SAR1817 fit into the broader context of S. aureus transcriptional regulation studies?

Research on SAR1817 should be contextualized within the complex transcriptional regulatory network of S. aureus:

• Comparison with known transcriptional regulators: Such as the SaeRS two-component system, which controls expression of important virulence factors

• Regulatory network mapping: Using ChIP-seq if SAR1817 functions as a transcription factor, or RNA-seq comparing wild-type and mutant strains

• Integration with existing regulon data: From studies of global regulators like SarA, Agr, and Rot

• Analysis of promoter elements: To identify potential binding sites for known regulators that might control SAR1817

• Epistasis experiments: With other regulatory mutants to establish hierarchy in regulatory networks

S. aureus employs numerous transcriptional regulators to adapt to changing environments during infection. SA1804, for example, has been identified as a novel transcriptional regulator involved in mediating invasion and cytotoxicity, acting in a SaeRS-dependent manner . Understanding where SAR1817 fits within these regulatory networks could provide insights into its physiological role.

What experimental design considerations are important when studying proteins of unknown function like SAR1817?

When investigating uncharacterized proteins like SAR1817, several experimental design considerations are critical:

• Comprehensive controls: Including multiple negative controls and positive controls where possible

• Validation across multiple techniques: Confirming findings using orthogonal approaches

• Reproducibility assessment: Biological and technical replicates with appropriate statistical analysis

• Strain background considerations: Testing in multiple S. aureus strain backgrounds

• Growth condition variability: Examining phenotypes under diverse conditions

• Complementation studies: To confirm that phenotypes are specifically due to SAR1817 disruption

• Domain-level analysis: Creating truncated or point mutants to map functional regions

• Avoiding over-interpretation: Clearly distinguishing direct from indirect effects

For statistical analysis, appropriate methods should be selected based on data distribution, with consideration of multiple testing corrections when performing high-throughput analyses .

How might SAR1817 research contribute to understanding S. aureus metabolism and adaptation during infection?

Investigating SAR1817's role in S. aureus metabolism and adaptation requires:

• Metabolomic profiling: Comparing wild-type and SAR1817 mutant strains under infection-relevant conditions

• Isotope labeling studies: To track specific metabolic pathways potentially affected by SAR1817

• Transcriptomic analysis: Focusing on metabolic gene expression changes

• Growth phenotyping: In different carbon sources and nutrient-limited conditions

• Biofilm metabolic studies: As biofilms represent a distinct metabolic state with altered gene expression

• In vivo metabolic analysis: Using animal infection models

• Oxygen adaptation studies: Given S. aureus' ability to grow in both aerobic and anaerobic environments