Recombinant Staphylococcus aureus Uncharacterized hydrolase SAS1339 (SAS1339)

What is SAS1339 and how is it classified among S. aureus hydrolases?

SAS1339 is an uncharacterized hydrolase identified in Staphylococcus aureus strain MSSA476. It belongs to the broader family of serine hydrolases, which play diverse roles in regulating host-pathogen interactions in S. aureus. Recent proteomic studies have identified several previously uncharacterized serine hydrolases in S. aureus, including ten fluorophosphonate-binding hydrolases (FphA-J) that mostly lack human homologs . While SAS1339 has not been extensively characterized, it likely shares functional and structural features with other S. aureus serine hydrolases that contribute to bacterial physiology and virulence .

What expression systems are available for producing recombinant SAS1339?

Recombinant SAS1339 can be produced using various expression systems, including E. coli, yeast, baculovirus, or mammalian cell systems . The choice of expression system depends on research objectives, required protein modifications, and downstream applications. E. coli expression systems are commonly used for initial characterization due to their simplicity and high yield, while mammalian expression systems may better preserve native protein folding and post-translational modifications. For functional studies requiring properly folded protein, baculovirus expression in insect cells may provide a balance between yield and proper folding .

How does SAS1339 compare to other characterized S. aureus hydrolases?

While specific comparative data for SAS1339 is limited, insights can be drawn from research on other S. aureus hydrolases. The recently identified fluorophosphonate-binding hydrolases (Fphs) show diverse functional characteristics despite belonging to the same enzymatic class. For example, FphB functions as a virulence factor localized to the bacterial surface and processes short-chain lipid esters , while FphH shows evidence of functional links to other Fph proteins, with its absence triggering upregulation of FphE and FphD in certain S. aureus strains . SAS1339 may share similar enzymatic mechanisms with these proteins while potentially having distinct substrate specificity and cellular localization patterns that contribute to its specific role in S. aureus biology.

What are the potential biological functions of SAS1339 based on homology to known S. aureus hydrolases?

Based on homology to characterized S. aureus hydrolases, SAS1339 may function in one or more of the following biological processes:

• Cell wall metabolism and maintenance

• Lipid processing and metabolism

• Host-pathogen interactions during infection

• Biofilm formation and maintenance

• Resistance to host defense mechanisms

Similar hydrolases like FphB have been shown to respond to host-derived factors and play important roles during the early colonization phase of infection . Others, like FphF, are highly expressed during the bacterial life cycle and may be involved in essential metabolic processes . The specific function of SAS1339 would require experimental determination through targeted studies.

How can activity-based protein profiling be optimized for studying SAS1339 function?

Activity-based protein profiling (ABPP) can be optimized for studying SAS1339 by adapting approaches used for other S. aureus serine hydrolases:

• Selection of appropriate probe: Fluorophosphonate (FP) probes, such as FP-TMR, have successfully identified active serine hydrolases in S. aureus . For SAS1339-specific studies, probe design may need to be tuned based on the enzyme's catalytic properties.

• Growth condition optimization: Since the expression of many S. aureus hydrolases varies with growth conditions, multiple conditions should be tested, including biofilm-promoting conditions, which have revealed the activity of previously uncharacterized hydrolases .

• Competitive labeling: To identify specific inhibitors or substrates, competitive ABPP can be employed, where potential inhibitors compete with the activity probe for binding to the active site .

• Combined genetic and chemical approaches: Integration of knockout/knockdown studies with ABPP can reveal functional redundancy among related hydrolases, as observed with FphH and other Fph proteins .

• In vivo ABPP: To understand SAS1339 activity during infection, ABPP can be performed on bacteria recovered from infection models.

What are the methodological challenges in determining SAS1339 substrate specificity?

Determining substrate specificity for uncharacterized hydrolases like SAS1339 presents several methodological challenges:

• Lack of predictive models: Without structural data or well-characterized homologs, predicting substrate specificity is difficult. Some S. aureus hydrolases like FphB process short-chain lipid esters , but SAS1339 may have different specificities.

• Heterogeneous expression: S. aureus hydrolases often show heterogeneous expression within bacterial populations , complicating bulk biochemical assays.

• Redundant functions: Functional redundancy among related hydrolases may mask phenotypes in single-gene knockout studies, as observed with FphH .

• Context-dependent activity: The activity of S. aureus hydrolases can be regulated in response to host-derived factors , requiring recreation of appropriate conditions for substrate identification.

• Limited commercial substrate libraries: Custom substrate libraries may need to be synthesized based on predicted biochemical properties.

A systematic approach combining proteomic screens, structural predictions, and diverse substrate panels under various growth conditions would be necessary to overcome these challenges.

How might SAS1339 contribute to S. aureus pathogenesis and antibiotic resistance?

SAS1339's potential contribution to S. aureus pathogenesis and antibiotic resistance can be investigated through several research avenues:

• Infection models: Testing SAS1339 knockout strains in various infection models, similar to studies that revealed FphB's importance during early colonization in intravenous infection models .

• Host factor response: Investigating whether SAS1339 activity is regulated in response to host-derived factors, as seen with other S. aureus hydrolases .

• Surface localization: Determining if SAS1339 is surface-localized, which would facilitate interactions with host components and potential roles in colonization, as observed with FphB .

• Biofilm contribution: Since many S. aureus hydrolases are active during biofilm growth , SAS1339 may contribute to biofilm formation or maintenance, which is associated with antibiotic resistance.

• Enzymatic modification of antibiotics: Some bacterial hydrolases can modify antibiotics, contributing to resistance. Testing SAS1339's activity against various antibiotics could reveal such functions.

Research suggests that serine hydrolases in S. aureus may have functions at the host-pathogen interface that are difficult to capture with in vitro model systems, which explains why they might have escaped identification in phenotypic screens .

What structural features of SAS1339 can be predicted through bioinformatic analysis?

Bioinformatic prediction of SAS1339 structural features can provide valuable insights despite limited experimental data:

• Catalytic triad identification: Sequence analysis can identify the conserved serine-histidine-aspartate catalytic triad characteristic of serine hydrolases.

• Domain organization: Comparison with characterized hydrolases can reveal domain organization and potential substrate-binding regions.

• Surface exposure prediction: Algorithms predicting membrane association or secretion signals can indicate whether SAS1339 is likely intracellular, membrane-associated, or secreted.

• Structure prediction: Modern AI-based structure prediction tools can generate structural models that may reveal potential substrate-binding pockets.

• Evolutionary conservation: Analysis of conservation across S. aureus strains and related species can identify functionally important regions.

For comparison, structure prediction of Fph proteins has indicated varying features such as FphH's lack of a well-defined acyl binding pocket compared to larger Fph proteins , which influences substrate specificity and function.

What protocols are recommended for purification and characterization of recombinant SAS1339?

The purification and characterization of recombinant SAS1339 can follow established protocols for bacterial hydrolases with appropriate modifications:

• Expression optimization:

  • Test multiple expression systems (E. coli, yeast, baculovirus, mammalian cells)

  • Optimize induction conditions (temperature, inducer concentration, time)

  • Consider fusion tags (His, GST, MBP) to enhance solubility and facilitate purification

• Purification strategy:

  • Affinity chromatography using fusion tags

  • Ion exchange chromatography based on predicted isoelectric point

  • Size exclusion chromatography for final polishing

  • Consider detergent extraction if membrane-associated

• Activity assays:

  • Fluorogenic substrate screening (para-nitrophenyl esters of varying chain lengths)

  • Activity-based probe labeling with FP-TMR

  • pH and temperature optima determination

  • Metal ion and inhibitor sensitivity profiling

• Structural characterization:

  • Circular dichroism for secondary structure assessment

  • Thermal stability assays

  • Crystallization trials for X-ray structure determination

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

How can genetic knockout or knockdown approaches be used to study SAS1339 function?

Genetic manipulation approaches to study SAS1339 function should consider the following methodological considerations:

• Knockout strategy selection:

  • Allelic replacement for complete gene deletion

  • Transposon mutagenesis for initial screening

  • CRISPR-Cas9 systems adapted for S. aureus

  • Antisense RNA for knockdown if knockout is lethal

• Strain selection:

  • Use multiple S. aureus strains (e.g., MSSA476, USA300, Newman) to account for strain-specific effects

  • Consider clinical isolates to enhance relevance

• Phenotypic characterization:

  • Growth curves under various conditions

  • Biofilm formation assays

  • Antibiotic susceptibility testing

  • Host cell interaction models

  • In vivo infection models with tissue-specific analysis

• Compensatory mechanism assessment:

  • Monitor expression of related hydrolases in the SAS1339 mutant

  • Create double or triple mutants to address functional redundancy

  • Use ABPP to monitor global serine hydrolase activity profiles in mutants

Studies with FphH mutants revealed upregulation of other hydrolases, suggesting compensatory mechanisms that might mask phenotypes in single mutants . Similar considerations should be applied when studying SAS1339.

What imaging techniques are suitable for localizing SAS1339 in S. aureus cells?

Several imaging techniques can be employed to determine the subcellular localization of SAS1339:

• Fluorescent fusion proteins:

  • C- or N-terminal GFP/mCherry fusions with careful functional verification

  • Photoactivatable fluorescent proteins for super-resolution microscopy

  • Split-GFP complementation to minimize functional disruption

• Immunofluorescence microscopy:

  • Generation of specific antibodies against purified SAS1339

  • Optimization of fixation and permeabilization for S. aureus

  • Co-staining with markers for different cellular compartments

• Activity-based probe imaging:

  • Fluorescent activity-based probes like FP-TMR

  • Click chemistry-compatible probes for post-labeling functionalization

  • Super-resolution microscopy for precise localization

• Electron microscopy:

  • Immunogold labeling for transmission electron microscopy

  • Correlative light and electron microscopy

• Fractionation controls:

  • Complement imaging with subcellular fractionation and western blotting

  • Activity assays on fractions to confirm functional localization

Studies of FphB have shown heterogeneous distribution in bacterial populations and localization at the bacterial surface , highlighting the importance of single-cell analysis and population studies when investigating S. aureus hydrolases.

What experimental design is recommended for evaluating SAS1339 as a potential therapeutic target?

Evaluation of SAS1339 as a therapeutic target requires a systematic experimental approach:

How should researchers interpret structural homology between SAS1339 and other bacterial hydrolases?

When interpreting structural homology between SAS1339 and other bacterial hydrolases, researchers should consider:

• Catalytic core conservation: The serine-histidine-aspartate catalytic triad is highly conserved among serine hydrolases, but similar catalytic mechanisms don't necessarily indicate similar biological functions or substrate preferences.

• Substrate-binding pocket variations: Even closely related hydrolases can have significant differences in substrate-binding regions. For example, FphH lacks a well-defined acyl binding pocket compared to larger Fph proteins , which affects its substrate specificity.

• Evolutionary context: Phylogenetic analysis can reveal whether homology reflects shared ancestry or convergent evolution to similar functions.

• Domain architecture: Beyond the catalytic domain, presence of additional domains or motifs can indicate specialized functions or localizations.

• Surface properties: Electrostatic surface potential and hydrophobicity patterns can provide insights into potential protein-protein interactions or membrane associations.

Homology analysis should be complemented with experimental validation, as sequence or structural similarity alone may not predict functional equivalence in the cellular context.

What statistical approaches are recommended for analyzing SAS1339 activity data across different experimental conditions?

Robust statistical approaches for analyzing SAS1339 activity data should include:

• Experimental design considerations:

  • Include appropriate technical and biological replicates (minimum n=3)

  • Use randomized block designs to control for batch effects

  • Include positive and negative controls in each experiment

• Data normalization strategies:

  • Normalize to total protein concentration when comparing across samples

  • Consider internal standards for enzymatic assays

  • Use housekeeping enzymes as references when appropriate

• Statistical tests:

  • ANOVA with post-hoc tests for multi-condition comparisons

  • Consider non-parametric alternatives if normality assumptions are violated

  • Use repeated measures designs for time-course experiments

  • Apply correction for multiple comparisons (e.g., Bonferroni, Benjamini-Hochberg)

• Advanced methods for complex datasets:

  • Principal component analysis for multivariate data

  • Linear mixed-effects models to account for random factors

  • Bayesian analysis for integrating prior knowledge with new data

• Effect size reporting:

  • Report fold changes and confidence intervals in addition to p-values

  • Calculate and report standardized effect sizes (e.g., Cohen's d)

When analyzing heterogeneous enzyme expression within bacterial populations, as observed with some S. aureus hydrolases , single-cell analytical approaches and appropriate population statistics should be employed.

How might systems biology approaches enhance our understanding of SAS1339's role in S. aureus physiology?

Systems biology approaches can significantly enhance understanding of SAS1339's role through:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from wild-type and SAS1339 mutant strains

  • Correlate SAS1339 expression with global metabolic networks

  • Identify co-regulated genes and proteins suggesting functional relationships

• Network analysis:

  • Construct protein-protein interaction networks including SAS1339

  • Identify metabolic pathways potentially affected by SAS1339 activity

  • Map genetic interactions through synthetic lethality screens

• Temporal dynamics:

  • Study expression and activity changes across growth phases and infection stages

  • Model dynamic responses to environmental and host-derived signals

  • Capture heterogeneous expression patterns within bacterial populations

• Host-pathogen interaction modeling:

  • Integrate SAS1339 activity data with host response measurements

  • Model contribution to biofilm formation dynamics

  • Simulate infection scenarios with varying SAS1339 expression levels

Such approaches may reveal why serine hydrolases like SAS1339 have "largely escaped identification in in vitro phenotypic screens" despite their importance, possibly due to "functional redundancy or functions at the host-pathogen interface that are difficult to capture with in vitro model systems" .

What are promising strategies for developing selective inhibitors of SAS1339?

Development of selective SAS1339 inhibitors could follow several strategic approaches:

• Structure-based design:

  • Utilize homology models or experimental structures

  • Focus on unique features of the substrate-binding pocket

  • Design transition-state analogs specific to SAS1339's catalytic mechanism

• Fragment-based screening:

  • Screen fragment libraries against purified SAS1339

  • Use thermal shift assays, NMR, or X-ray crystallography to detect binding

  • Elaborate fragments that bind to unique pockets

• Activity-based probe development:

  • Modify fluorophosphonate scaffolds with various recognition elements

  • Test selectivity profiles across all S. aureus serine hydrolases

  • Develop competition assays for rapid inhibitor screening

• Natural product screening:

  • Test plant extracts and microbial metabolites for inhibitory activity

  • Focus on sources with historical antimicrobial properties

  • Fractionate active extracts to identify active compounds

• Allosteric modulator development:

  • Identify non-active site binding pockets

  • Screen for compounds that modify enzyme dynamics rather than directly blocking the active site

  • Explore potential for species-selective allosteric regulation

The fact that S. aureus serine hydrolases "mostly lack human homologs" provides an opportunity for developing highly selective inhibitors with minimal off-target effects on human proteins.