Recombinant Staphylococcus aureus UPF0342 protein SAS1766 (SAS1766)

What is the role of SAS1766 in Staphylococcus aureus pathogenicity?

SAS1766 belongs to the UPF0342 protein family found in Staphylococcus aureus, a leading cause of both healthcare- and community-associated infections globally. While the specific function of SAS1766 has not been fully characterized, it likely contributes to the bacterial survival and virulence mechanisms. Similar to identified immunogenic proteins in S. aureus, SAS1766 may play a role in host-pathogen interactions during infection. Research on S. aureus virulence factors has identified numerous surface-associated and secreted proteins that contribute to pathogenicity, including cell surface-associated β-lactamases, lipoproteins, lipases, autolysins, and ABC transporter lipoproteins . Understanding these proteins, including SAS1766, is essential for developing effective vaccines and therapeutics against S. aureus infections.

What expression systems are most effective for producing recombinant SAS1766?

For recombinant expression of S. aureus proteins like SAS1766, E. coli-based expression systems are commonly employed due to their high yield and ease of genetic manipulation. The methodology typically involves:

• Gene cloning into an expression vector with an appropriate promoter

• Transformation into a suitable E. coli strain (e.g., BL21(DE3))

• Induction of protein expression (typically using IPTG for T7 promoter systems)

• Cell lysis and protein purification

For optimal expression, consider these methodological parameters:

When designing recombinant protein expression studies, it's important to define your variables clearly, similar to the experimental design principles outlined for scientific research . This includes identifying your independent variables (expression conditions) and dependent variables (protein yield, solubility, and activity).

How can I verify the identity and purity of recombinant SAS1766?

Verification of recombinant SAS1766 should follow a multi-method approach:

• SDS-PAGE analysis for molecular weight confirmation and initial purity assessment

• Western blotting using antibodies against the protein or fusion tag

• Mass spectrometry for precise molecular weight and peptide mapping

• N-terminal sequencing to confirm the correct start of the protein

• Circular dichroism to assess secondary structure elements

For functional verification, activity assays specific to the predicted function of the protein should be developed. Similar to approaches used for other S. aureus proteins, two-dimensional gel electrophoresis (2DGE) coupled with MALDI-TOF analysis provides robust identification . When reporting purity, quantitative assessment using densitometry of SDS-PAGE gels or HPLC analysis should be included, with >95% purity typically required for structural and functional studies.

What experimental design is optimal for studying the immunogenicity of SAS1766?

Based on established methodologies for S. aureus immunogen studies, a comprehensive experimental design for investigating SAS1766 immunogenicity should include:

• Expression and purification of recombinant SAS1766 with verification of structural integrity

• Animal model selection:

  • Mouse models for initial immunogenicity screening

  • Rabbit models for larger-scale antibody production and more complex infection models

The experimental design should follow the five key steps outlined in scientific methodology :

• Clearly define variables (SAS1766 as independent variable, immune response as dependent variable)

• Formulate specific, testable hypotheses about immunogenic properties

• Design appropriate treatments (various adjuvants, dosing schedules)

• Assign subjects to experimental groups using proper randomization

• Establish robust measures of immune response

A successful example of such an approach can be seen in the study of the recombinant five-antigen S. aureus vaccine (rFSAV), which demonstrated protection in S. aureus lethal sepsis and pneumonia mouse models by inducing comprehensive cellular and humoral immune responses .

For immunogenicity assessment, measure both humoral and cellular responses:

How should I design experiments to investigate potential synergistic effects between SAS1766 and other S. aureus antigens?

When investigating synergistic effects between SAS1766 and other S. aureus antigens, a factorial experimental design is most appropriate. This approach allows for the systematic evaluation of multiple antigens alone and in combination.

Methodological considerations:

• Select complementary antigens based on:

  • Known virulence factors (e.g., α-hemolysin, staphylococcal enterotoxin B, surface proteins like SpA, IsdB-N2, and MntC)

  • Different subcellular locations (surface vs. secreted)

  • Different functional roles in pathogenesis

• Design a multi-arm study with:

  • Individual antigen groups

  • Various combination groups

  • Appropriate controls (adjuvant-only, irrelevant protein)

• Readout parameters should include:

  • Antibody titers to individual components

  • Functional assays (opsonophagocytosis, neutralization)

  • Protection in relevant animal models

  • Cytokine profiles to assess immune polarization

• Analysis approach:

  • Use statistical methods specifically designed to detect synergistic effects

  • Apply isobologram analysis to quantify synergy

  • Consider principal component analysis for complex immune response data

This approach parallels the successful development of the five-antigen S. aureus vaccine (rFSAV), which demonstrated broad immune protection against epidemiologically relevant S. aureus strains .

What are the best approaches to study the structure-function relationship of SAS1766?

Investigating the structure-function relationship of SAS1766 requires a multi-disciplinary approach combining structural biology, biochemistry, and molecular biology techniques:

• Structural determination:

  • X-ray crystallography: Optimal for high-resolution structure

  • Nuclear Magnetic Resonance (NMR): For solution structure and dynamics

  • Cryo-electron microscopy: Particularly if SAS1766 forms complexes

• Functional mapping through site-directed mutagenesis:

  • Alanine scanning of conserved residues

  • Domain deletion or swapping experiments

  • Targeted modifications based on structural predictions

• Binding partner identification:

  • Pull-down assays with host cell extracts

  • Yeast two-hybrid screening

  • Surface plasmon resonance for binding kinetics

• Computational approaches:

  • Molecular dynamics simulations

  • Homology modeling based on UPF0342 family proteins

  • Protein-protein interaction predictions

When designing mutagenesis experiments, a systematic approach similar to that used in experimental design research should be employed , with clear definition of variables, hypothesis testing, and appropriate controls. The approach should resemble methodologies used to characterize other S. aureus virulence factors, such as those identified as immunogens during chronic infections .

How can recombination analysis be applied to understand the evolution of SAS1766 across S. aureus lineages?

Understanding the evolution of SAS1766 across S. aureus lineages requires sophisticated recombination analysis methodologies. Based on approaches used for studying S. aureus genomic evolution , the following methodology is recommended:

• Sequence collection and alignment:

  • Gather SAS1766 sequences from diverse S. aureus lineages

  • Include sequences from early diverging lineages (like ST93)

  • Perform multiple sequence alignment using MUSCLE or MAFFT

• Recombination detection:

  • Implement detection algorithms like Gubbins or RDP4

  • Identify breakpoints and potential donor sequences

  • Calculate recombination/mutation ratios (r/m) to quantify impact

• Phylogenetic analysis:

  • Construct pre- and post-recombination phylogenies

  • Use maximum-likelihood methods with appropriate evolutionary models

  • Bootstrap analysis for statistical support (minimum 100 replicates)

• Functional impact assessment:

  • Map recombination events to protein domains

  • Analyze selection pressures using dN/dS ratios

  • Correlate recombination events with phenotypic changes

The importance of this approach is highlighted by findings that some S. aureus lineages have been heavily impacted by recombination, with large parts of their genomes showing specific relationships with other groups . For example, ST93 has segments showing greater similarity to ST59/ST121 than to ST8, suggesting recombination has played a significant role in its evolution.

What methods are most effective for investigating the immunological memory response to SAS1766 in the context of chronic S. aureus infections?

Investigating immunological memory responses to SAS1766 in chronic S. aureus infections requires sophisticated immunological techniques and carefully designed longitudinal studies:

• Clinical sample collection protocol:

  • Serial sampling at defined intervals (pre-infection, acute phase, 14, 28, 42 days post-infection, and during chronic phase)

  • Collection of both serum and cellular components

  • Detailed clinical metadata including infection site, duration, and treatment

• Antibody analysis methodology:

  • Isotype-specific ELISAs to track IgG, IgM, IgA responses over time

  • Avidity measurements to assess antibody maturation

  • Epitope mapping to identify immunodominant regions

  • Functional assessment through opsonophagocytic and neutralization assays

• Memory B-cell analysis:

  • Antigen-specific B-cell ELISPOT assays

  • Flow cytometry with fluorescently-labeled SAS1766

  • Single-cell sorting and BCR sequencing to track clonal evolution

• T-cell memory assessment:

  • Antigen-specific T-cell stimulation assays

  • Cytokine profiling (Th1, Th2, Th17, and Treg responses)

  • TCR sequencing to identify expanded clones

This approach parallels methods used to identify S. aureus proteins recognized by the immune system during chronic biofilm infections , where 2D gel electrophoresis and immunoblotting with sera from infected animals followed by MALDI-TOF analysis successfully identified in vivo-expressed S. aureus antigens. Despite recognition by the immune system, chronic biofilm infections can persist, suggesting immune evasion mechanisms that should be considered when studying SAS1766.

How can I address solubility issues when expressing recombinant SAS1766?

Solubility challenges are common when expressing recombinant bacterial proteins like SAS1766. A systematic troubleshooting approach should include:

• Expression system modifications:

  • Test multiple E. coli strains (BL21, Rosetta, Origami)

  • Consider alternative expression hosts (yeast, insect cells)

  • Implement cold-shock or heat-shock expression protocols

• Fusion partner strategy:

  • Test solubility-enhancing tags: MBP, GST, SUMO, TrxA

  • Position tags at either N- or C-terminus to determine optimal configuration

  • Include TEV or PreScission protease sites for tag removal

• Expression condition optimization matrix:

• Refolding strategies (if inclusion bodies are unavoidable):

  • Gradual dialysis from denaturing conditions

  • On-column refolding with decreasing denaturant gradients

  • Pulsatile dilution methods

  • Addition of chaperones or folding enhancers

This methodological approach follows experimental design principles by systematically testing independent variables (expression conditions) against the dependent variable (protein solubility) , while implementing strategies that have proven successful for other S. aureus proteins studied in research settings.

What approaches should be used to resolve contradictory data in SAS1766 functional studies?

When facing contradictory data in SAS1766 functional studies, a structured analytical approach is essential:

• Critical evaluation of methodological differences:

  • Protein preparation methods (tags, purification approach)

  • Experimental conditions (buffers, temperature, pH)

  • Detection systems and their sensitivity

  • Animal models or cell lines used

• Independent verification:

  • Reproduce key experiments using standardized protocols

  • Employ alternative methodological approaches for the same endpoint

  • Collaborate with independent laboratories for validation

• Systematic investigation of variables:

  • Design factorial experiments to test multiple variables simultaneously

  • Implement statistical design of experiments (DoE) approaches

  • Develop dose-response curves rather than single-point measurements

• Integration of complementary techniques:

  • Combine in vitro, ex vivo, and in vivo approaches

  • Correlate structural data with functional outcomes

  • Apply systems biology approaches to place contradictory findings in context

When analyzing S. aureus virulence factors, contradictions often arise due to strain variability and growth conditions. This has been observed in studies of S. aureus biofilm antigens, where proteins recognized by the immune system were still associated with persistent infections, highlighting the complexity of host-pathogen interactions . Using transcriptomic and proteomic approaches simultaneously, as demonstrated in ST93 S. aureus studies , can help resolve such contradictions.

What are the most promising research directions for exploring SAS1766 as a potential vaccine component?

Based on successful approaches with other S. aureus antigens, the most promising research directions for exploring SAS1766 as a vaccine component include:

• Antigen optimization strategies:

  • Epitope mapping to identify immunodominant regions

  • Structure-based design of stable, highly immunogenic constructs

  • Development of chimeric antigens combining SAS1766 with other immunogens

• Combination vaccine approaches:

  • Systematic testing of SAS1766 with established S. aureus vaccine candidates

  • Exploration of synergistic antigen combinations

  • Investigation of multi-component formulations similar to the five-antigen S. aureus vaccine (rFSAV)

• Adjuvant and delivery system innovation:

  • Novel adjuvant screening for optimal immune polarization

  • Nanoparticle or virus-like particle delivery platforms

  • Mucosal delivery systems for targeted immunity

• Translational research pathway:

  • Preclinical efficacy against diverse clinical isolates

  • Development of correlates of protection

  • Bridging studies between animal models and human immunity

The development of a five-antigen S. aureus vaccine (rFSAV) provides an important precedent, having demonstrated broad immune protection when challenged with epidemiologically relevant S. aureus strains . This approach generated comprehensive cellular and humoral immune responses and decreased bacterial loads, inflammatory cytokine expression, and pathology after challenge. Similar methodological approaches should be applied to evaluate SAS1766, with particular attention to its ability to induce protective immunity against diverse S. aureus strains.

How might genomic approaches advance our understanding of SAS1766 function in different S. aureus lineages?

Advanced genomic approaches offer powerful tools for elucidating SAS1766 function across S. aureus lineages:

• Comparative genomics methodology:

  • Whole-genome sequencing of diverse clinical isolates

  • SAS1766 sequence and synteny analysis across lineages

  • Identification of co-evolving genes suggesting functional relationships

  • Correlation with virulence phenotypes and clinical outcomes

• Transcriptomic profiling approaches:

  • RNA-seq under infection-relevant conditions

  • Single-cell transcriptomics during host-pathogen interactions

  • Dual RNA-seq to capture both pathogen and host responses

  • Comparison of expression patterns across lineages

• Functional genomics strategies:

  • CRISPR interference for conditional knockdown

  • Transposon mutagenesis to identify genetic interactions

  • Complementation studies across lineages to assess functional conservation

  • TraDIS or TnSeq approaches to measure fitness contributions

• Population genomics analysis:

  • Investigation of selection pressures using dN/dS ratios

  • Assessment of recombination impacts on SAS1766 evolution

  • Phylogenetic analyses to track protein evolution alongside lineage diversification

These approaches mirror successful methodologies applied to understand S. aureus genomic evolution, such as those used to study ST93 lineage, which identified extensive recombination events and their impact on genome structure . For SAS1766 specifically, examining its conservation and variation across the early diverging and recombinant lineages would provide insights into its evolutionary importance and potential functional constraints.