Recombinant Staphylococcus aureus Putative ATP:guanido phosphotransferase SAS0481 (SAS0481)

What is the functional classification of SAS0481 within S. aureus?

SAS0481 is classified as a putative ATP:guanido phosphotransferase in Staphylococcus aureus. This family of enzymes reversibly catalyzes the transfer of phosphate between ATP and various phosphagens . The protein likely belongs to the broader ATP:guanido phosphotransferase family that includes enzymes such as glycocyamine kinase, arginine kinase, and creatine kinase . The putative designation indicates that while sequence homology suggests this function, complete experimental validation may still be ongoing.

What are the predicted substrate specificity patterns for SAS0481?

Based on homology with other ATP:guanido phosphotransferases, SAS0481 likely catalyzes the transfer of phosphate from ATP to a specific guanidino-containing substrate. While the exact substrate remains to be definitively characterized, potential candidates include arginine or a related guanidino compound specific to S. aureus metabolism . Experimental determination of substrate specificity would require enzymatic assays with various potential substrates, monitoring the transfer of phosphate through techniques such as radiometric assays or coupled enzyme systems.

How does SAS0481 fit into S. aureus metabolic pathways?

SAS0481 likely plays a role in energy metabolism within S. aureus, similar to how creatine kinase functions in energy metabolism in vertebrates . As an ATP:guanido phosphotransferase, it may be involved in maintaining energy homeostasis by creating phosphagen reserves that can rapidly regenerate ATP during periods of high energy demand. This function would be particularly important during infection processes when the bacterium experiences fluctuating energy requirements due to host defense mechanisms and varying nutrient availability.

What methods are most effective for expressing and purifying recombinant SAS0481?

For optimal expression and purification of recombinant SAS0481, a systematic approach is recommended:

• Expression System Selection: E. coli BL21(DE3) or similar strains are recommended due to their reduced protease activity. For proteins experiencing folding difficulties, consider specialized strains like Rosetta or OrigamiB.

• Vector Design: Incorporate an N-terminal or C-terminal affinity tag (His6 or GST) with a precision protease cleavage site. Test both N and C-terminal placements as tag position can affect folding.

• Expression Conditions:

  • Test induction at different temperatures (16°C, 25°C, 37°C)

  • Vary IPTG concentrations (0.1-1.0 mM)

  • Consider auto-induction media for higher yields

• Purification Protocol:

  • Initial capture using affinity chromatography (Ni-NTA for His-tagged protein)

  • Ion exchange chromatography as an intermediate step

  • Size exclusion chromatography as a polishing step for homogeneity

• Protein Stability: Include reducing agents (DTT or β-mercaptoethanol) to protect the conserved catalytic cysteine residue that is characteristic of ATP:guanido phosphotransferases .

What are the implications of SAS0481 in S. aureus virulence and pathogenesis?

The potential role of SAS0481 in S. aureus virulence remains an active area of investigation. As an ATP:guanido phosphotransferase, it may contribute to bacterial survival under host-imposed stress conditions by maintaining energy homeostasis . Several lines of evidence suggest potential implications:

• Metabolic Adaptation: SAS0481 could enable S. aureus to adapt to the rapid metabolic changes required during infection progression.

• Immune Evasion: By facilitating rapid energy mobilization, SAS0481 might contribute to the bacterium's ability to survive phagocytosis or other host defense mechanisms.

• Intracellular Survival: Given the importance of intracellular survival for S. aureus virulence, proteins that support energy metabolism under stress conditions may be essential for persistence within host cells . The "Trojan horse hypothesis" suggests that S. aureus can disseminate throughout the body via macrophage migration within the blood supply, which requires metabolic adaptability .

• Potential Vaccine Target: Although not specifically mentioned in current vaccine efforts, metabolic enzymes that contribute to pathogen fitness in vivo may represent underexplored vaccine candidates .

How can structural biology approaches enhance our understanding of SAS0481 function?

Structural biology provides critical insights into SAS0481 function through multiple complementary approaches:

• X-ray Crystallography:

  • Co-crystallization with substrates (ATP and potential guanidino substrates)

  • Resolution of at least 2.5Å to identify active site architecture

  • Analysis of substrate-binding pocket specificity

• Cryo-Electron Microscopy:

  • Especially valuable if the protein forms larger complexes

  • Can provide insights into dynamic conformational changes during catalysis

• NMR Spectroscopy:

  • Investigation of protein dynamics during substrate binding

  • Mapping of residue-specific interactions with substrates

• Computational Approaches:

  • Molecular dynamics simulations to model conformational changes

  • Structure-based virtual screening to identify potential inhibitors

  • Homology modeling based on characterized ATP:guanido phosphotransferases, which contain specific C-terminal catalytic domains with beta-alpha-beta2-alpha repeats

Structural insights would clarify the substrate specificity, catalytic mechanism, and allow comparison with other members of the ATP:guanido phosphotransferase family, such as creatine kinase and arginine kinase .

What is the potential of SAS0481 as a vaccine antigen candidate?

The evaluation of SAS0481 as a vaccine antigen candidate should consider several factors based on current understanding of S. aureus vaccine development:

• Antigen Conservation: The degree of sequence conservation across clinically relevant S. aureus strains would determine the breadth of protection. Highly conserved enzymes like SAS0481 may offer broad coverage.

• Expression Levels During Infection: Research should confirm whether SAS0481 is expressed during different stages of infection and in relevant anatomical sites.

• Immunogenicity Considerations:

  • Most failed S. aureus vaccines have demonstrated significant immunogenicity yet failed in protective efficacy, suggesting that traditional immunogenicity measures are insufficient predictors of protection .

  • For example, both V710 (IsdB) and StaphVAX (CP5/CP8 conjugate) vaccines elicited opsonophagocytic responses but failed in phase 3 trials .

  • SA4Ag vaccine showed sustained humoral responses but also failed to confer protection .

• Adjuvant Selection:

  • Experience with AS03 adjuvant in S. aureus vaccines suggests that adjuvant benefits may be limited due to pre-existing immunity to S. aureus antigens .

  • Alternative adjuvants promoting T cell immunity might be more beneficial.

• Safety Considerations:

  • The V710 vaccine failure demonstrated increased mortality in vaccinated patients who developed S. aureus infections, highlighting safety concerns .

  • Antibodies against certain S. aureus proteins might inadvertently promote bacterial pathogenesis through mechanisms like the "Trojan horse hypothesis" .

What enzymatic assay systems are optimal for characterizing SAS0481 activity?

For comprehensive characterization of SAS0481 enzymatic activity, multiple complementary assays are recommended:

Table 1: Recommended Enzymatic Assay Systems for SAS0481 Characterization

Each assay system should include proper controls:

• Enzyme-free reaction controls

• Heat-inactivated enzyme controls

• Known ATP:guanido phosphotransferases (e.g., creatine kinase) as positive controls

How can researchers develop effective inhibitors against SAS0481?

Development of effective SAS0481 inhibitors follows a systematic research pipeline:

• Initial Screening Approaches:

  • Structure-based virtual screening using homology models or crystal structures

  • Fragment-based screening to identify building blocks that bind to active site regions

  • High-throughput biochemical assays using diverse chemical libraries

• Rational Design Strategy:

  • Target the ATP-binding pocket (competitive with respect to ATP)

  • Target the guanidino substrate binding site (competitive with respect to guanidino substrate)

  • Target protein-specific allosteric sites (non-competitive inhibition)

  • Design transition-state analogs that mimic the phosphoryl transfer reaction

• Lead Optimization Process:

  • Structure-activity relationship (SAR) studies

  • Improvement of physicochemical properties (solubility, stability)

  • Enhancement of selectivity against human ATP:guanido phosphotransferases

• Validation Experiments:

  • Determination of inhibition mechanism (competitive, non-competitive, uncompetitive)

  • X-ray crystallography to confirm binding mode

  • Cellular assays to verify target engagement in bacterial cells

  • Assessment of effects on S. aureus growth and survival

• Therapeutic Development Considerations:

  • Evaluation of synergy with established antibiotics

  • Assessment of resistance development frequency

  • Determination of efficacy in animal infection models

This approach aligns with emerging alternative therapeutic strategies for S. aureus infections, which are increasingly important given the high antibiotic resistance profiles observed .

What genomic approaches can identify the physiological role of SAS0481?

Multiple genomic approaches can elucidate the physiological role of SAS0481:

• Gene Knockout and Complementation:

  • CRISPR-Cas9 or allelic replacement to generate SAS0481 deletion mutants

  • Phenotypic characterization under various growth conditions

  • Complementation with wild-type gene to confirm phenotype specificity

  • Construction of catalytically inactive mutants (targeting conserved cysteine)

• Transcriptomic Analysis:

  • RNA-Seq to identify conditions under which SAS0481 is differentially expressed

  • Comparison of expression patterns during infection vs. laboratory growth

  • Co-expression network analysis to identify functionally related genes

• Proteomic Approaches:

  • Identification of protein interaction partners through pull-down assays

  • Comparative proteomics between wild-type and SAS0481 mutants

  • Post-translational modification analysis

• Metabolomic Profiling:

  • Analysis of metabolite levels in wild-type vs. SAS0481 mutants

  • Isotope labeling to track metabolic flux through pathways

  • Identification of accumulated or depleted metabolites that might represent substrates

• In vivo Infection Models:

  • Assessment of SAS0481 mutant virulence in different infection models

  • Competitive index experiments with wild-type strains

  • Tissue-specific transcriptomics during infection

How might SAS0481 contribute to vaccine development strategies against S. aureus?

SAS0481 could potentially contribute to S. aureus vaccine development through several approaches:

• As a Conjugate Carrier Protein:

  • Recent findings suggest that using S. aureus proteins as carrier proteins for glycoconjugate vaccines improves immunogenicity compared to using carrier proteins from unrelated bacteria .

  • For example, a CP5 capsular polysaccharide conjugated to S. aureus α-toxin (Hla) showed superior immunogenicity to conjugates using non-S. aureus carriers .

  • SAS0481 could be explored as a carrier protein for capsular polysaccharides (CP5/CP8).

• As Part of a Multi-Antigen Approach:

  • Failed vaccine candidates like StaphVAX, V710, and SA4Ag have demonstrated that single-antigen or limited-antigen approaches are insufficient .

  • Current promising candidates like rFSAV include multiple antigens (Hla, SEB, MntC, IsdB, and SpA) .

  • SAS0481 could complement existing antigen combinations, particularly if it represents a different functional category.

• Targeting Cellular Immunity:

  • A critical lesson from failed vaccines is the importance of cellular immunity in addition to humoral responses .

  • Vaccination approaches should assess T cell responses against SAS0481 epitopes.

  • Appropriate adjuvant selection would be crucial, as experience with AS03 adjuvant showed minimal enhancement compared to non-adjuvanted vaccines in S. aureus trials .

• Safety Considerations:

  • The V710 vaccine (targeting IsdB) was associated with increased mortality in vaccinated individuals who developed S. aureus infections .

  • Thorough safety assessment would be essential, including evaluation of whether anti-SAS0481 antibodies could inadvertently promote pathogenesis.

What role might SAS0481 play in S. aureus intracellular survival?

SAS0481, as a putative ATP:guanido phosphotransferase, may significantly contribute to S. aureus intracellular survival through several mechanisms:

• Energy Buffer System:

  • ATP:guanido phosphotransferases typically function in phosphagen systems that serve as energy buffers .

  • This function could be crucial during intracellular survival when bacteria face fluctuating energy availability.

  • Phosphagens would allow rapid ATP regeneration during periods of high energy demand, such as responding to host cell antimicrobial mechanisms.

• Relevance to Intracellular Persistence:

  • S. aureus can survive intracellularly, which contributes to treatment failures and recurrent infections .

  • The "Trojan horse hypothesis" suggests that S. aureus disseminates throughout the body via macrophage migration within the blood supply .

  • Proteins supporting metabolic adaptability during intracellular residence are likely critical for this persistence.

• Resistance to Host-Imposed Stress:

  • Intracellular bacteria face nutrient limitation, oxidative stress, and antimicrobial peptides.

  • Energy buffer systems would provide metabolic flexibility during these stress conditions.

  • This adaptation mechanism could complement other S. aureus virulence factors and immune-evasion strategies.

• Potential Therapeutic Target:

  • Inhibition of SAS0481 could potentially reduce intracellular survival.

  • This approach would complement current therapeutic strategies that often fail to address intracellular populations.

  • Combined approaches targeting both extracellular and intracellular S. aureus would address a major gap in current treatment modalities.

How does post-translational modification affect SAS0481 activity?

Post-translational modifications (PTMs) likely play significant roles in regulating SAS0481 activity. Research approaches should focus on:

• Identification of PTM Sites:

  • Mass spectrometry-based proteomics to map phosphorylation, acetylation, or other modifications

  • Site-directed mutagenesis of identified PTM sites to assess functional impact

  • Comparison of PTM patterns under different growth conditions

• Redox Regulation:

  • Given the importance of cysteine residues in ATP:guanido phosphotransferases, redox-based regulation through cysteine oxidation/reduction should be investigated .

  • Sensitivity to oxidative stress would be particularly relevant during host-pathogen interactions.

  • Assessment of activity under different redox conditions using defined buffer systems.

• PTM Enzymes:

  • Identification of S. aureus kinases, acetylases, or other enzymes responsible for SAS0481 modifications

  • Co-immunoprecipitation studies to detect transient enzyme-substrate interactions

  • Genetic manipulation of potential regulatory enzymes to assess impact on SAS0481 function

• Temporal Dynamics:

  • Tracking of PTM changes throughout growth phases and infection stages

  • Correlation of PTM status with enzymatic activity

  • Development of PTM-specific antibodies for immunoblotting studies

What is the impact of SAS0481 genetic variation across S. aureus strains?

Understanding SAS0481 genetic variation across S. aureus strains provides valuable insights into functional evolution and strain-specific adaptations:

• Comparative Genomic Analysis:

  • Analysis of SAS0481 sequence conservation across clinical and environmental isolates

  • Identification of polymorphic sites and their correlation with strain virulence

  • Assessment of selection pressures through Ka/Ks ratio analysis

• Structure-Function Relationships:

  • Mapping of variants onto protein structural models

  • Focus on variations in catalytic regions versus peripheral domains

  • Expression and characterization of variant proteins to assess functional differences

• Population Genetics:

  • Analysis of SAS0481 allele distribution across major S. aureus lineages

  • Assessment of horizontal gene transfer events involving SAS0481

  • Correlation with antibiotic resistance profiles and virulence characteristics

• Clinical Correlations:

  • Association of specific SAS0481 variants with infection outcomes

  • Comparison between commensal and infectious isolates

  • Evaluation of SAS0481 variation in persistent versus acute infections