Recombinant Staphylococcus aureus Probable tautomerase SAS1303 (SAS1303)

What is SAS1303 and what is its predicted function in Staphylococcus aureus?

SAS1303 is a probable tautomerase protein identified in Staphylococcus aureus, including the MSSA476 strain. As a putative tautomerase, it likely catalyzes the conversion between tautomeric forms of its substrates by facilitating the movement of a hydrogen atom within the same molecule, accompanied by a switch of adjacent single and double bonds. Tautomerases often play roles in bacterial metabolism, potentially contributing to bacterial fitness and survival in various environments.

The protein has been computationally modeled and is available in the AlphaFold Database (AF-Q6G9J6-F1), though it's important to note that there are currently no experimental data to verify the accuracy of this computed structure model . This computational prediction provides a starting point for structural studies, but researchers should approach these models with appropriate caution until experimental validation is available.

How should researchers approach the production of recombinant SAS1303 for in vitro studies?

When producing recombinant SAS1303, researchers should consider several methodological approaches:

• Expression system selection: E. coli BL21(DE3) is often suitable for bacterial protein expression. Consider using a pET vector system with an N-terminal His-tag for purification ease.

• Optimization protocol:

  • Test multiple expression conditions (temperature, IPTG concentration, induction time)

  • Screen for solubility in different buffer conditions

  • Evaluate protein stability with thermal shift assays

• Purification strategy:

  • Initial capture: Immobilized metal affinity chromatography (IMAC)

  • Secondary purification: Size exclusion chromatography to ensure monodispersity

  • Optional: Ion exchange chromatography for higher purity

• Quality control measures:

  • SDS-PAGE to verify size and purity

  • Mass spectrometry to confirm protein identity

  • Circular dichroism to assess secondary structure

  • Thermal stability assessment using differential scanning fluorimetry

The design of an effective expression and purification protocol should follow principles of good experimental design, with careful consideration of variables and appropriate controls as outlined in experimental methodology literature .

What structural information is currently available for SAS1303?

The structural information available for SAS1303 is currently limited to computational predictions. The protein structure has been predicted using AlphaFold and is available in the RCSB Protein Data Bank with identifier AF_AFQ6G9J6F1 . This computational model was released in the AlphaFold Database on December 9, 2021, and last modified on September 30, 2022.

When using this model for research purposes, it's crucial to:

• Examine the model confidence metrics provided by AlphaFold for different regions of the protein

• Validate structural predictions with experimental techniques when possible

• Compare the predicted structure with experimentally determined structures of homologous tautomerases

• Use the model as a hypothesis-generating tool rather than definitive structural information

Researchers should clearly acknowledge the computational nature of the structure when publishing results based on this model, as noted in the database entry: "There are no experimental data to verify the accuracy of this computed structure model" .

What experimental approaches are recommended for functional characterization of SAS1303?

Functional characterization of SAS1303 requires a systematic approach combining in vitro biochemical assays and in vivo studies:

In vitro enzymatic characterization:

• Substrate screening: Test potential tautomerase substrates including phenylpyruvate, p-hydroxyphenylpyruvate, and 2-hydroxymuconate.

• Enzyme kinetics: Determine key parameters for active substrates:

  Parameter	Method	Expected Output
  Km	Michaelis-Menten kinetics	Substrate affinity (μM-mM range)
  kcat	Steady-state kinetics	Catalytic rate (s⁻¹)
  kcat/Km	Calculated ratio	Catalytic efficiency (M⁻¹s⁻¹)
  pH optimum	Activity assays at varying pH	Optimal pH range

• Mechanism studies: Investigate catalytic mechanism through:

  • Site-directed mutagenesis of predicted catalytic residues

  • pH-rate profiles

  • Solvent isotope effects

  • Inhibition studies

Genetic approaches:

• Create knockout mutants in S. aureus to assess phenotypic changes

• Perform complementation studies to verify phenotype restoration

• Utilize transposon mutagenesis libraries to identify genetic interactions

These methodological approaches should follow rigorous experimental design principles, including appropriate controls, replication, and statistical analysis as outlined in experimental design literature .

How might researchers investigate potential roles of SAS1303 in S. aureus pathogenesis?

To investigate SAS1303's potential role in pathogenesis, researchers should employ a systematic experimental approach:

• Expression analysis during infection:

  • Measure SAS1303 expression levels during different stages of infection

  • Compare expression in different host environments (blood, tissue, biofilm)

  • Analyze regulation patterns in response to host defense mechanisms

• Infection models with SAS1303 knockout mutants:

  • Using principles of sound experimental design , establish:

    • Clearly defined dependent variables (bacterial load, host survival, etc.)

    • Controlled independent variables (inoculum dose, time points)

    • Appropriate controls (wild-type, complemented strains)

  • Test in multiple relevant infection models:

    • Murine sepsis model (similar to models used for rFSAV testing)

    • Pneumonia model

    • Skin infection model

• Host-pathogen interaction studies:

  • Assess immune cell responses to wild-type vs. SAS1303 mutants

  • Measure inflammatory cytokine expression

  • Quantify neutrophil recruitment and activation

  • Examine complement activation patterns, drawing on methodologies similar to those used for studying SdrE

• Comparative studies with known virulence factors:

  • Include established virulence factors as comparators

  • Consider virulence factors with known mechanisms, such as SdrE which binds complement factor H to evade immune response

This approach allows researchers to systematically evaluate whether SAS1303 contributes to S. aureus pathogenesis through metabolic adaptation, immune evasion, or other mechanisms.

What methodology is recommended for experimentally validating the predicted structure of SAS1303?

To experimentally validate the predicted AlphaFold structure of SAS1303 , researchers should employ a multi-technique approach:

Data from these complementary techniques should be integrated to generate a comprehensive structural understanding of SAS1303, with careful attention to discrepancies between predicted and experimental results.

What approaches should be used to identify potential protein-protein interactions involving SAS1303?

Identifying protein-protein interactions involving SAS1303 requires a combination of computational prediction and experimental validation techniques:

• Computational predictions:

  • Structural-based docking with potential partners

  • Homology-based prediction from known tautomerase interactions

  • Machine learning approaches using protein sequence features

• In vitro interaction screening:

  • Pull-down assays using tagged recombinant SAS1303

  • Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI) for quantitative binding analysis

  • Isothermal Titration Calorimetry (ITC) for thermodynamic characterization

• In vivo interaction identification:

  • Bacterial two-hybrid systems adapted for S. aureus

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity-labeling approaches (e.g., BioID or APEX2)

  • Chemical cross-linking followed by mass spectrometry (similar to approaches used to identify SdrE interactions)

• Validation of identified interactions:

  • Mutational analysis of interaction interfaces

  • Competition assays

  • Functional studies to assess biological relevance

For cross-linking mass spectrometry approaches, a methodology similar to that used to identify SdrE interactions with factor H could be adapted, where water-soluble and membrane-impermeable cross-linkers like BS³ are employed followed by LC-ESI-MS/MS analysis . This technique was successful in identifying functional interactions for other S. aureus proteins and could be applied to SAS1303.

How does SAS1303 compare to other characterized bacterial tautomerases in terms of structure and function?

When comparing SAS1303 to other bacterial tautomerases, researchers should consider the following analytical framework:

• Structural comparison:

  • Conduct phylogenetic analysis of tautomerase superfamily members

  • Perform structural alignment of the SAS1303 AlphaFold model with experimentally determined structures of related tautomerases

  • Analyze conservation patterns in catalytic residues and active site architecture

  • Identify unique structural features that may suggest specialized functions

• Functional comparison:

  • Compare substrate specificity profiles with characterized tautomerases

  • Analyze catalytic efficiency (kcat/Km) across similar enzymes

  • Examine pH and temperature optima variations

  • Investigate differences in regulatory mechanisms and expression patterns

• Evolutionary context:

  • Assess gene neighborhood analysis for functional associations

  • Compare genomic context across different bacterial species

  • Investigate horizontal gene transfer patterns of tautomerase genes

  • Examine presence/absence patterns across different S. aureus strains

A comprehensive comparative analysis may reveal whether SAS1303 represents a canonical tautomerase or possesses unique properties that could indicate specialized functions in S. aureus physiology or pathogenesis.

What considerations should be taken into account when designing experiments to assess potential immune evasion properties of SAS1303?

When designing experiments to investigate potential immune evasion properties of SAS1303, researchers should apply rigorous experimental design principles while drawing insights from studies of established S. aureus immune evasion factors like SdrE :

• Experimental design foundations:

  • Formulate specific, testable hypotheses about SAS1303's interaction with immune components

  • Clearly define independent variables (protein concentration, bacterial strains) and dependent variables (immune function metrics)

  • Implement appropriate controls, including other S. aureus proteins with and without known immune evasion functions

  • Consider both between-subjects and within-subjects designs for robust evaluation

• Host factor binding studies:

  • Adapt methodologies used for SdrE-factor H interaction studies :

    • Purified protein overlay techniques with fractionated immune components

    • Cross-linking followed by mass spectrometry to identify binding partners

    • Recombinant protein binding assays to quantify interactions

  • Test interactions with key immune components:

    • Complement proteins (C3, factor H, factor I)

    • Antimicrobial peptides

    • Pattern recognition receptors

• Functional immune evasion assays:

  • Complement activation studies:

    • C3b deposition assays on bacterial surfaces

    • Cofactor activity tests for complement regulation

    • Terminal complement complex formation assessment

  • Phagocytosis assays:

    • Neutrophil killing assays comparing wild-type and SAS1303 mutants

    • Opsonophagocytosis with human serum components

  • Inflammatory response modulation:

    • Cytokine production by immune cells

    • Neutrophil extracellular trap (NET) formation

• In vivo validation:

  • Use animal models with key immune components knocked out

  • Compare virulence of wild-type and SAS1303 mutants in different immune backgrounds

  • Consider tissue-specific immune responses

Researchers should be mindful that immune evasion mechanisms in S. aureus are often multifactorial, as exemplified by studies showing how SdrE-bound factor H exhibits cofactor functionality for factor I-mediated cleavage of C3b to iC3b, resulting in reduced C3-fragment deposition, decreased C5a generation, and reduced killing by polymorphonuclear cells .

How might SAS1303 be evaluated as a potential vaccine antigen against S. aureus?

Evaluation of SAS1303 as a potential vaccine antigen should follow a systematic approach similar to that used for other S. aureus antigens in successful multi-component vaccines like rFSAV :

• Antigen conservation analysis:

  • Sequence conservation assessment across diverse S. aureus clinical isolates

  • Structural epitope mapping and conservation analysis

  • Expression analysis during different stages of infection

• Immunogenicity screening:

  • Recombinant protein production with careful quality control

  • Assessment of antibody responses in animal models:

    • Titer measurement

    • Isotype profiling

    • Epitope mapping

  • T-cell response characterization:

    • Cytokine profiling

    • T-cell proliferation assays

    • Identification of T-cell epitopes

• Functional antibody assessment:

  • Neutralization of enzymatic activity

  • Opsonophagocytic killing assays

  • Complement deposition enhancement

  • In vitro function tests similar to those used for rFSAV components

• Protection studies in animal models:

  • Challenge studies in multiple infection models:

    • Lethal sepsis model

    • Pneumonia model

    • Skin and soft tissue infection model

  • Assessment of bacterial load reduction

  • Measurement of inflammatory markers

  • Survival analysis

• Combination studies:

  • Evaluation alongside established vaccine antigens like those in rFSAV (Hla, SEB, SpA, IsdB-N2, and MntC)

  • Assessment of additive or synergistic protection

  • Analysis of comprehensive immune responses to combined formulations

Taking lessons from the successful rFSAV approach, researchers should evaluate SAS1303's ability to induce both humoral and cellular immune responses that can effectively reduce bacterial loads and inflammatory pathology in relevant infection models .

What methodological approaches should be used to assess potential immune responses to SAS1303 in experimental models?

To rigorously assess immune responses to SAS1303 in experimental models, researchers should implement a comprehensive immunological evaluation strategy based on established methods used for other S. aureus antigens:

• Antibody response characterization:

  • Quantitative ELISA to measure total antigen-specific antibody titers

  • Isotype-specific ELISAs to profile IgG1, IgG2a/c, IgG3, IgA, and IgM responses

  • Avidity assays using chaotropic agents to assess antibody maturation

  • Epitope mapping using peptide arrays or phage display

• Cellular immunity assessment:

  • T-cell proliferation assays using labeled cells and antigen stimulation

  • Intracellular cytokine staining for T-cell polarization (Th1/Th2/Th17)

  • ELISpot assays for enumeration of cytokine-producing cells

  • Flow cytometric analysis of memory T-cell subsets

• Functional immune assays:

  • Opsonophagocytic killing assays with immune sera and neutrophils

  • Complement deposition assays on S. aureus surfaces

  • Neutralization of SAS1303 enzymatic activity by immune sera

  • Bacterial growth inhibition tests with immune sera

• In vivo protection experiments:

  Infection Model	Metrics	Timepoints	Analysis Method
  Sepsis	Bacterial load in organs, survival	24h, 48h, 72h	CFU counting, Kaplan-Meier
  Pneumonia	Lung bacterial burden, inflammation	24h, 48h	CFU counting, histopathology
  Skin infection	Abscess size, bacterial recovery	Days 1-7	Caliper measurement, CFU counting

• Mechanism of protection studies:

  • Passive transfer of immune sera to naïve animals

  • Adoptive transfer of T-cells from immunized animals

  • Experiments in knockout mice lacking specific immune components

  • Depletion of specific cell types or cytokines during challenge

These approaches should draw on the methodological framework established for other S. aureus vaccine antigens, such as those used in rFSAV studies where comprehensive cellular and humoral immune responses were measured along with their effect on bacterial loads, inflammatory cytokine expression, and inflammatory cell infiltration .