Recombinant Staphylococcus aureus UPF0747 protein SAR1153 (SAR1153), partial

What is known about the structural characteristics of SAR1153 protein?

SAR1153 belongs to the UPF0747 protein family, an uncharacterized group that shares structural similarities with other regulatory proteins in S. aureus. While specific structural data on SAR1153 remains limited, comparative analysis suggests it may share homology with SarA-like proteins. SarA is a 14.5-kDa regulatory protein encoded by the sarA gene within the sar locus, which comprises three overlapping transcripts (sarP1, sarP3, and sarP2) . Similar to the 13.6-kDa SarR protein that binds to sar promoters, SAR1153 may potentially function as a DNA-binding protein involved in gene regulation .

Methodologically, researchers investigating SAR1153 structure should consider:

• X-ray crystallography or NMR spectroscopy for detailed structural analysis

• Bioinformatic approaches comparing sequence homology with characterized S. aureus regulatory proteins

• Structural prediction modeling using transformer-based AI architectures similar to those employed in ProtET protein modification research

How does SAR1153 compare to other characterized regulatory proteins in S. aureus?

Current research suggests possible functional similarities between SAR1153 and other S. aureus regulatory proteins like SarR, which modulates the expression of virulence determinants. SarR exhibits binding affinity to specific promoter regions, particularly the sar promoters, thereby influencing transcription of sar genes and subsequent SarA protein production . While the specific binding targets of SAR1153 remain to be fully characterized, its classification in the UPF0747 family suggests potential involvement in analogous regulatory networks.

To establish comparative functional similarities, researchers should consider:

• DNA-binding assays using recombinant SAR1153 protein with known S. aureus promoter regions

• Transcriptional analysis in wild-type versus SAR1153 deletion mutants

• Immunoprecipitation studies to identify protein-DNA and protein-protein interactions

What are the recommended methods for expressing and purifying recombinant SAR1153 protein?

For optimal expression and purification of recombinant SAR1153 protein, researchers should implement protocols similar to those successfully employed for other S. aureus regulatory proteins. Based on methodologies used for similar proteins, the following approach is recommended:

• Clone the SAR1153 gene into an expression vector (e.g., pET system for E. coli expression)

• Transform the construct into an appropriate E. coli expression strain (BL21 derivatives often yield good results)

• Induce protein expression with IPTG under optimized conditions (temperature, time, concentration)

• Lyse cells and purify using affinity chromatography (His-tag or GST-tag approaches)

• Verify purity using SDS-PAGE and Western blotting

• Confirm functionality through DNA-binding assays

This approach mirrors successful purification strategies for SarR protein, which was initially identified through DNA-specific column chromatography containing sar promoter fragments .

What experimental approaches are most effective for determining the biological function of SAR1153?

Determining the biological function of an uncharacterized protein like SAR1153 requires a comprehensive multi-method approach:

• Genetic manipulation: Generate SAR1153 deletion mutants using allelic replacement techniques similar to those employed for sarR gene knockout, where the target gene is replaced with an antibiotic marker (e.g., ermC) . This allows for phenotypic comparison between wild-type and mutant strains.

• Transcriptomic analysis: Employ RNA-seq to compare gene expression profiles between wild-type and SAR1153 mutant strains, identifying differentially expressed genes that may represent regulatory targets.

• DNA-binding studies: Conduct electrophoretic mobility shift assays (EMSA) and DNase footprinting to identify specific DNA sequences bound by recombinant SAR1153 protein.

• Protein interaction studies: Implement pull-down assays, yeast two-hybrid screens, or co-immunoprecipitation to identify protein partners that may provide functional insights.

• Phenotypic characterization: Assess virulence factor production, antibiotic resistance profiles, and growth characteristics in the mutant strain compared to wild-type.

• Whole genome analysis: Utilize next-generation sequencing approaches similar to those employed in comprehensive S. aureus genome analyses to identify genomic contexts and variations that may influence SAR1153 function .

How might SAR1153 contribute to S. aureus virulence and pathogenicity?

Based on our understanding of regulatory proteins in S. aureus, SAR1153 may potentially contribute to virulence regulation through several mechanisms:

• Transcriptional regulation: Similar to SarR, which modulates sarP1 transcription and subsequent SarA protein expression , SAR1153 may regulate expression of virulence-associated genes.

• Stress response coordination: Many uncharacterized proteins in pathogens play roles in adaptation to host environments and stress conditions.

• Biofilm formation: Regulatory proteins often influence biofilm development, which contributes significantly to S. aureus persistence and antibiotic resistance.

• Immune evasion: SAR1153 might participate in pathways that modulate host immune responses.

To investigate these possibilities, researchers should consider phenotypic analyses comparing wild-type and SAR1153 mutant strains in:

• Animal infection models

• Biofilm formation assays

• Growth under various stress conditions

• Immune cell interaction studies

What bioinformatic approaches can reveal potential functions of SAR1153?

Advanced bioinformatic analyses can provide valuable insights into potential functions of uncharacterized proteins like SAR1153:

• Comparative genomics: Analyze conservation and genomic context of SAR1153 across different S. aureus strains and related species.

• Protein domain prediction: Identify conserved domains that might suggest DNA-binding, enzymatic, or other functional capabilities.

• Protein-protein interaction networks: Use established databases and prediction tools to identify potential functional partners.

• AI-driven approaches: Implement advanced machine learning models similar to ProtET, which uses transformer-based architecture and contrastive learning to predict protein functions and modifications .

• Phylogenetic analysis: Determine evolutionary relationships with characterized proteins to infer potential functions.

• Structural prediction: Use AlphaFold or similar tools to predict three-dimensional structure and functional sites.

How can gene knockout studies of SAR1153 be optimized for S. aureus?

For effective SAR1153 gene knockout studies in S. aureus, researchers should implement the following optimized protocol:

• Vector selection: Use temperature-sensitive shuttle vectors like pCL52.1, which allows for efficient allelic replacement in S. aureus .

• Construct design: Create a construct containing flanking regions of SAR1153 with an antibiotic resistance marker (e.g., ermC) inserted between them to replace the target gene .

• Transformation method: Optimize electroporation parameters specifically for the S. aureus strain being used, as transformation efficiency varies significantly between strains.

• Selection strategy: Implement a double selection strategy, first growing transformants at permissive temperature (30°C) with antibiotic selection, then shifting to non-permissive temperature (42°C) to force integration .

• Screening approach: Use PCR, Southern blotting, and DNA sequencing to confirm successful allelic replacement .

• Phenotypic verification: Implement transcriptomic and proteomic analyses to confirm knockout effects and identify compensatory mechanisms.

• Complementation: Reintroduce the SAR1153 gene in trans to confirm that observed phenotypes are specifically due to SAR1153 deletion.

What is the potential role of SAR1153 in S. aureus antimicrobial resistance mechanisms?

The potential role of SAR1153 in antimicrobial resistance requires investigation, particularly given S. aureus's clinical significance as a pathogen with extensive resistance mechanisms:

• Transcriptional regulation: Similar to other regulatory proteins in S. aureus, SAR1153 may modulate expression of resistance genes either directly or indirectly.

• Stress response modulation: SAR1153 might participate in coordinating cellular responses to antibiotic stress.

• Biofilm contribution: If involved in biofilm development, SAR1153 could indirectly contribute to the inherent resistance properties of biofilm-embedded bacteria.

• Metabolic adaptation: Regulatory proteins often modulate metabolic pathways that can influence susceptibility to antimicrobials.

Research methodologies to investigate these possibilities should include:

• Antimicrobial susceptibility testing comparing wild-type and SAR1153 mutant strains

• Transcriptomic analysis under antibiotic stress conditions

• Biofilm formation assays with antibiotic challenges

• Analysis of resistance gene expression patterns

• Whole genome sequence analysis to identify mutations or variations in SAR1153 associated with resistant phenotypes

How should researchers design experiments to investigate SAR1153 interaction with host immune factors?

To effectively investigate SAR1153 interactions with host immune factors, researchers should implement the following experimental design:

• Recombinant protein production: Express and purify SAR1153 following protocols similar to those used for other S. aureus proteins, ensuring proper folding and biological activity .

• Cellular interaction studies:

  • Expose various immune cell types (neutrophils, macrophages, dendritic cells) to purified SAR1153

  • Measure activation markers, cytokine production, and functional responses

  • Compare responses to wild-type and SAR1153 mutant bacterial strains

• Lymphocyte proliferation assays: Implement methodologies similar to those used for S. aureus-cure-associated proteins, assessing lymphocyte proliferation and cytokine production (particularly focusing on IL-17A production) in response to SAR1153 stimulation .

• Type 3 immunity assessment: Given the importance of type 3 immunity in S. aureus infection response, evaluate IL-17A production by various T cell subsets (CD4+, CD8+, γδ T cells) following SAR1153 exposure .

• In vivo models: Develop appropriate animal models to assess immune responses to SAR1153 administration or infection with SAR1153 mutant versus wild-type strains.

• Immunoproteomics approach: Use methodologies similar to those that identified S. aureus-cure-associated proteins to investigate if SAR1153 is recognized by antibodies from individuals who have cleared S. aureus infections .

What considerations are important when designing DNA-binding studies for SAR1153?

When designing DNA-binding studies for SAR1153, researchers should consider several key factors:

• Protein preparation: Ensure recombinant SAR1153 is properly folded and active. Consider testing multiple expression systems and purification methods to optimize functional protein yield.

• Target DNA selection:

  • Based on the example of SarR binding to sar promoters , initially test SAR1153 binding to known regulatory regions in S. aureus

  • Implement a systematic approach testing promoter regions of virulence genes, stress response genes, and metabolic pathways

  • Consider genome-wide approaches such as ChIP-seq to identify binding sites without prior hypotheses

• Binding assay selection:

  • Electrophoretic mobility shift assays (EMSA) for initial binding assessment

  • DNase footprinting to identify specific binding sequences

  • Surface plasmon resonance (SPR) for quantitative binding kinetics

  • Fluorescence anisotropy for solution-based binding analysis

• Controls and validation:

  • Include known DNA-binding proteins (e.g., SarA, SarR) as positive controls

  • Use non-specific DNA sequences as negative controls

  • Validate in vitro findings with in vivo approaches like ChIP-qPCR

• Binding condition optimization:

  • Test various buffer compositions, pH conditions, and salt concentrations

  • Evaluate the impact of potential cofactors or protein partners

  • Consider the influence of DNA topology (linear vs. supercoiled)

What approaches can resolve contradictory data about SAR1153 function in different S. aureus strains?

Resolving contradictory data about SAR1153 function across different S. aureus strains requires a systematic approach:

• Comprehensive strain characterization:

  • Perform whole genome sequencing of all strains used in contradictory studies

  • Analyze SAR1153 sequence variations and genomic context

  • Determine strain phylogeny and classification (MLST typing)

  • Characterize phenotypic differences beyond SAR1153 function

• Standardized experimental conditions:

  • Implement identical growth conditions, media composition, and experimental protocols

  • Use multiple complementary methodologies to assess function

  • Control for growth phase effects by analyzing multiple timepoints

• Cross-laboratory validation:

  • Exchange strains between laboratories reporting contradictory results

  • Implement blinded experimental designs

  • Establish collaborative projects to standardize protocols

• Genetic complementation strategies:

  • Create SAR1153 deletion mutants in multiple strain backgrounds

  • Complement with SAR1153 variants from different strains

  • Analyze the effect of specific sequence variations through site-directed mutagenesis

• Host interaction studies:

  • Evaluate strain-specific differences in SAR1153 function during host cell interaction

  • Assess impact in various infection models

  • Consider host-specific factors that might influence SAR1153 activity

How can structural biology approaches be applied to understand SAR1153 function?

Structural biology offers powerful approaches to elucidate SAR1153 function:

• X-ray crystallography workflow:

  • Express and purify SAR1153 in high yield (10-20 mg/ml)

  • Screen crystallization conditions systematically

  • Collect diffraction data at synchrotron facilities

  • Solve structure using molecular replacement (if homologous structures exist) or experimental phasing methods

• NMR spectroscopy approach:

  • Express isotopically labeled SAR1153 (15N, 13C)

  • Collect multi-dimensional NMR spectra

  • Assign resonances and calculate solution structure

  • Perform dynamics measurements to identify flexible regions

• Cryo-electron microscopy:

  • Particularly valuable if SAR1153 forms complexes with other proteins or DNA

  • Optimize sample preparation and grid conditions

  • Collect and process data to generate 3D reconstructions

• Computational structure prediction:

  • Implement AI-driven approaches similar to those used in ProtET

  • Use AlphaFold2 or RoseTTAFold to generate predicted models

  • Validate predictions with experimental data

• Structure-function analysis:

  • Identify potential functional sites based on structural features

  • Design site-directed mutagenesis studies targeting these sites

  • Correlate structural features with DNA-binding properties

• Ligand and interaction studies:

  • Perform structural studies with potential DNA targets or protein partners

  • Use techniques like HDX-MS to map interaction interfaces

  • Implement computational docking to predict binding modes

What are the optimal conditions for analyzing SAR1153 expression during different growth phases?

To analyze SAR1153 expression across different growth phases, researchers should implement:

• Growth condition standardization:

  • Define precise media composition and growth parameters

  • Establish reproducible growth curves for each S. aureus strain studied

  • Sample at multiple defined points (early/mid/late exponential, early/late stationary)

• RNA extraction and analysis:

  • Implement rapid RNA stabilization upon sampling (e.g., RNAlater or flash freezing)

  • Use specialized extraction protocols optimized for gram-positive bacteria

  • Perform RT-qPCR with carefully validated reference genes

  • Consider northern blotting to identify potential transcript variants

• Protein expression analysis:

  • Generate specific antibodies against SAR1153 or use epitope tagging approaches

  • Perform western blotting with quantitative analysis

  • Consider proteomic approaches (LC-MS/MS) for broader context

• Promoter activity measurement:

  • Construct reporter fusions (e.g., SAR1153 promoter driving GFP expression)

  • Measure activity using flow cytometry or plate reader approaches

  • Analyze data with appropriate normalization for cell density

• Single-cell analysis:

  • Implement fluorescent reporters for single-cell tracking

  • Use time-lapse microscopy to monitor expression dynamics

  • Apply flow cytometry to assess population heterogeneity

*Exact OD values may vary by strain and should be calibrated accordingly

What methodological approaches can determine if SAR1153 plays a role in biofilm formation?

To investigate SAR1153's potential role in biofilm formation, implement the following methodological approaches:

• Genetic manipulation studies:

  • Create SAR1153 deletion mutants using allelic replacement techniques

  • Generate complemented strains to confirm phenotype specificity

  • Consider creating SAR1153 overexpression strains

• Static biofilm assays:

  • Crystal violet staining in microtiter plates under various conditions

  • Confocal laser scanning microscopy with fluorescent strains

  • Scanning electron microscopy for detailed structural analysis

• Dynamic biofilm models:

  • Flow cell systems to assess biofilm development under shear stress

  • Microfluidic devices for real-time microscopic observation

  • Drip flow reactors for air-liquid interface biofilms

• Matrix composition analysis:

  • Quantify extracellular DNA, proteins, and polysaccharides

  • Implement immunochemical detection of specific matrix components

  • Use enzymatic treatments to assess matrix component contributions

• Gene expression studies:

  • Analyze expression of known biofilm-associated genes in wild-type vs. mutant

  • Perform RNA-seq on biofilm vs. planktonic cells

  • Use reporter constructs to monitor spatiotemporal gene expression in biofilms

• Anti-biofilm intervention testing:

  • Assess biofilm susceptibility to antimicrobials

  • Evaluate dispersal in response to various signals

  • Test host defense factor effects on biofilm integrity

What are the most promising future research directions for understanding SAR1153 function?

Future research on SAR1153 should focus on:

• Comprehensive functional characterization:

  • Determine DNA-binding specificity and target genes

  • Establish regulatory networks influenced by SAR1153

  • Identify protein interaction partners

• Clinical relevance assessment:

  • Investigate SAR1153 expression in clinical isolates

  • Correlate sequence variations with virulence or resistance phenotypes

  • Assess potential as a biomarker or therapeutic target

• Host-pathogen interaction studies:

  • Evaluate SAR1153's role during infection processes

  • Determine impact on immune response similar to studies of S. aureus-cure-associated proteins

  • Assess contribution to persistence and chronic infection

• Structural biology applications:

  • Resolve three-dimensional structure

  • Identify druggable sites for potential inhibitor development

  • Characterize DNA-binding mechanisms

• Systems biology integration:

  • Place SAR1153 within broader regulatory networks

  • Develop predictive models of its function under various conditions

  • Implement machine learning approaches to predict impacts of sequence variations

• Technological innovations:

  • Apply emerging AI-based protein design approaches like ProtET

  • Develop high-throughput functional screening methods

  • Implement CRISPR-based approaches for precise genetic manipulation

How can researchers effectively integrate SAR1153 findings into broader S. aureus virulence models?

To integrate SAR1153 research into broader S. aureus virulence models:

• Systematic literature integration:

  • Place findings in context of established virulence regulatory networks

  • Identify connections to major regulators (Agr, SarA, SaeRS)

  • Map potential interactions with known pathogenicity mechanisms

• Network modeling approaches:

  • Develop computational models incorporating SAR1153 regulatory effects

  • Validate model predictions with targeted experiments

  • Refine models iteratively based on experimental findings

• Multi-omics integration:

  • Combine transcriptomic, proteomic, and metabolomic data

  • Identify system-wide impacts of SAR1153 modulation

  • Establish causative relationships through temporal analyses

• Host-relevant context:

  • Evaluate SAR1153 function in infection models ranging from cellular to animal

  • Assess impact on interactions with specific host tissues and immune components

  • Consider host-specific factors that might influence SAR1153 activity

• Collaborative research networks:

  • Establish standardized methodologies across laboratories

  • Create repositories of well-characterized strains and genetic constructs

  • Develop shared databases of experimental results