Recombinant Staphylococcus aureus UPF0271 protein SAR1684 (SAR1684), partial

What is the UPF0271 protein SAR1684 and how is it classified within the S. aureus proteome?

UPF0271 protein SAR1684 is an uncharacterized protein from Staphylococcus aureus strain MRSA252 that belongs to the Uncharacterized Protein Family 0271 (UPF0271). This protein consists of approximately 250 amino acids and represents one of many proteins in S. aureus with currently unknown specific functions. The UPF designation indicates that while the protein's sequence is known, its biological role, structure, and function remain largely undefined through experimental verification.

Within the S. aureus proteome, SAR1684 is classified among the accessory genome components that may vary between different strains, unlike core genome proteins present in all S. aureus isolates. The protein lacks the characteristic features of known virulence factors such as Staphylococcal Protein A (SpA), which has defined immunoglobulin-binding domains and superantigen properties . Unlike SpA, which contains five homologous domains (labeled A-E) with well-characterized IgG binding regions, SAR1684's domain organization and binding partners remain undefined.

The classification of SAR1684 is particularly important in the context of comparative genomics of S. aureus strains, as differences in accessory genome content often contribute to variations in pathogenicity and host adaptation. Researchers studying SAR1684 should note that its presence in MRSA252 may not necessarily translate to all clinical isolates.

What expression systems are most effective for producing recombinant SAR1684 protein for research purposes?

Multiple expression systems can be utilized for recombinant SAR1684 production, each with distinct advantages depending on research needs. The E. coli expression system represents the most commonly employed approach for initial characterization studies due to its rapid growth, high protein yields, and cost-effectiveness . For SAR1684 specifically, BL21(DE3) strains with T7 promoter-based vectors have demonstrated successful expression of the partial protein (amino acids 1-250).

For researchers requiring post-translational modifications or improved protein folding, yeast expression systems (particularly Pichia pastoris) offer advantages over bacterial systems. This approach may be particularly relevant when studying potential glycosylation patterns of SAR1684 that might affect its interactions with host immune components, similar to other staphylococcal proteins involved in immune evasion .

Baculovirus expression in insect cells represents an intermediate approach between prokaryotic and mammalian systems, offering proper protein folding with moderate yields. For the most authentic post-translational modifications, particularly when studying SAR1684 interactions with human host factors, mammalian expression systems (typically HEK293 or CHO cells) would be most appropriate, though these systems typically produce lower yields and require more specialized equipment and expertise.

The choice of expression system should be guided by the specific research question, with E. coli being suitable for structural studies and initial characterization, while mammalian systems may be necessary for functional studies involving host-pathogen interactions.

How do researchers distinguish between the biological functions of SAR1684 and other uncharacterized proteins in S. aureus?

Distinguishing the biological functions of SAR1684 from other uncharacterized proteins requires a systematic, multi-faceted approach. Researchers typically begin with comparative genomics and bioinformatics analyses, examining sequence conservation across different S. aureus strains and potential orthologues in other bacterial species. Protein domain prediction tools can identify potential functional motifs, though UPF0271 family proteins like SAR1684 often lack recognizable domains with known functions.

Gene expression profiling under various conditions (e.g., different growth phases, stress conditions, host cell exposure) provides critical insights into when SAR1684 is upregulated or downregulated. Correlation of expression patterns with those of characterized proteins can suggest functional relationships. This approach draws inspiration from studies of other S. aureus proteins, such as SpA, whose expression varies depending on growth phase and environmental conditions .

Genetic approaches including targeted gene deletion and complementation studies provide direct evidence of SAR1684's role in S. aureus biology. Phenotypic comparison of wild-type and ΔSAR1684 mutants across various assays (growth rate, biofilm formation, antibiotic resistance, host cell interactions) can reveal functional contributions. Similar approaches have been successful in characterizing SpA's role in immune evasion, where SpA knockout strains demonstrated increased susceptibility to phagocytosis .

Protein-protein interaction studies using techniques such as yeast two-hybrid, pull-down assays, or proximity labeling can identify binding partners of SAR1684, providing functional clues. Finally, structural determination through X-ray crystallography or cryo-EM can reveal structural similarities to proteins with known functions, even when sequence homology is limited.

What are the optimal conditions for maintaining SAR1684 stability during purification and storage?

Maintaining the stability of recombinant SAR1684 protein requires careful consideration of buffer conditions, temperature, and storage protocols. For initial purification, a phosphate-buffered saline (PBS, pH 7.4) supplemented with low concentrations (1-5 mM) of reducing agents such as dithiothreitol (DTT) or β-mercaptoethanol helps prevent oxidation of cysteine residues and subsequent protein aggregation. The addition of 10% glycerol serves as a cryoprotectant and further enhances protein stability.

Temperature control is critical throughout the purification process. SAR1684, like many bacterial proteins, demonstrates optimal stability when maintained at 4°C during purification steps. Limited exposure to room temperature during the purification workflow minimizes proteolytic degradation. The addition of protease inhibitor cocktails (e.g., PMSF, EDTA, leupeptin) is advisable, particularly when working with crude lysates.

For long-term storage, SAR1684 protein solutions should be divided into single-use aliquots (typically 50-100 μg) to avoid repeated freeze-thaw cycles. Storage at -80°C provides the best long-term stability, with protein activity generally preserved for at least 12 months under these conditions. For shorter-term storage (1-2 weeks), 4°C is acceptable if the buffer contains preservatives such as 0.02% sodium azide to prevent microbial growth.

Researchers should conduct stability assessments before lengthy experiments, particularly for functional studies. SDS-PAGE analysis of stored samples can confirm the absence of degradation products, while dynamic light scattering can detect potential aggregation. For activity-based experiments, comparative analysis with freshly purified protein provides a reference for potential activity loss during storage.

What analytical methods are most effective for characterizing the structure-function relationship of SAR1684?

Circular dichroism (CD) spectroscopy provides valuable information about secondary structural elements (α-helices, β-sheets) and thermal stability without requiring crystallization. Small-angle X-ray scattering (SAXS) offers insights into the protein's shape and conformational changes in solution. These techniques are particularly valuable for comparing wild-type SAR1684 with site-directed mutants to identify structurally important regions.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map solvent-accessible regions and conformational dynamics, which is especially useful for identifying potential binding interfaces. This approach draws inspiration from studies of SpA, where structural analysis revealed the three-helix bundle conformation of its binding domains .

Functional characterization should be guided by structural findings. Site-directed mutagenesis of predicted functional residues followed by activity assays can establish structure-function relationships. For proteins with unknown functions like SAR1684, screening for biochemical activities (proteolysis, nuclease activity, binding to host factors) based on structural similarities to characterized proteins is a productive approach.

Computational methods including molecular dynamics simulations can predict conformational changes and potential binding sites, generating hypotheses for experimental validation. The integration of these methods provides a comprehensive understanding of how SAR1684's structure relates to its biological function.

How can researchers effectively generate and characterize antibodies against SAR1684 for immunological studies?

Generating and characterizing high-quality antibodies against SAR1684 requires strategic approaches to ensure specificity and utility across multiple applications. The immunization strategy should begin with careful antigen design—either using the full-length protein or selecting unique epitope regions to minimize cross-reactivity with other S. aureus proteins. For SAR1684, in silico epitope prediction tools can identify surface-exposed regions with high antigenicity and low homology to other staphylococcal proteins.

When immunizing animals (typically rabbits for polyclonal antibodies or mice for monoclonal development), a prime-boost strategy with purified recombinant SAR1684 conjugated to a carrier protein such as keyhole limpet hemocyanin (KLH) enhances immunogenicity. The immunization schedule should include at least three booster injections at 2-3 week intervals for optimal antibody development.

Rigorous antibody validation is essential and should include:

• ELISA assays to determine antibody titers and specificity

• Western blot analysis using both recombinant SAR1684 and S. aureus lysates to confirm native protein recognition

• Immunoprecipitation to verify antibody functionality in solution-phase applications

• Immunofluorescence microscopy to assess utility in cellular localization studies

• Specificity testing against related S. aureus proteins

Cross-adsorption against lysates from SAR1684 knockout strains can reduce non-specific binding. For monoclonal antibody development, screening should prioritize clones recognizing native conformations rather than just denatured epitopes, ensuring utility across multiple applications.

Researchers should characterize the antibody's ability to recognize different strains of S. aureus, as sequence variations in SAR1684 across clinical isolates may affect epitope recognition. This approach parallels strategies used for SpA antibody development, where researchers needed to account for strain-specific variations .

How might SAR1684 contribute to S. aureus pathogenesis and immune evasion?

While the specific function of SAR1684 remains uncharacterized, several experimental approaches can elucidate its potential role in S. aureus pathogenesis and immune evasion. Given S. aureus' extensive arsenal of immune evasion proteins, including the well-characterized SpA , researchers should consider whether SAR1684 might complement existing evasion mechanisms or provide novel functions.

Comparative expression analysis of SAR1684 across different infection models (abscess formation, bacteremia, endocarditis) can indicate infection stages where the protein may be relevant. Upregulation during specific infection phases would suggest a specialized role in pathogenesis. Transcriptomic studies comparing expression in community-acquired versus hospital-acquired MRSA strains may reveal differential utilization of SAR1684 across lineages with varying virulence profiles.

Targeted gene deletion studies offer the most direct evidence of SAR1684's role in pathogenesis. Comparison of wild-type and ΔSAR1684 mutants in animal infection models can reveal virulence defects. Complementation studies with the wild-type gene should restore the original phenotype, confirming the specific contribution of SAR1684. These approaches parallel methods used to characterize SpA's role in abscess formation, where SpA knockout strains showed reduced abscess development that was restored upon complementation .

For potential immune evasion functions, in vitro assays measuring interactions with specific immune components are invaluable. These might include testing SAR1684's ability to:

• Inhibit complement activation pathways

• Interfere with phagocytosis by neutrophils or macrophages

• Modulate cytokine responses from immune cells

• Interact with pattern recognition receptors

If SAR1684 demonstrates immunomodulatory properties, researchers should determine whether it acts extracellularly (requiring secretion) or affects host cell signaling upon bacterial internalization. While SpA functions primarily through B-cell superantigen activity and Fc/Fab binding , SAR1684 may employ distinct mechanisms that contribute to S. aureus' multi-faceted approach to immune evasion.

What role might SAR1684 play in potential vaccine development strategies against S. aureus?

The potential utility of SAR1684 in vaccine development against S. aureus requires systematic evaluation within the broader context of staphylococcal vaccinology challenges. Unlike established vaccine targets such as SpA, which has a defined role in immune evasion and for which modified versions (SpA KKAA) have demonstrated vaccine potential , the uncharacterized nature of SAR1684 necessitates preliminary investigations before considering it as a vaccine candidate.

Initial assessment should determine SAR1684's conservation across clinically relevant S. aureus strains, as an effective vaccine target must be broadly expressed. Protein sequence analysis across genome databases can establish conservation levels and identify any hypervariable regions that might reduce cross-protective potential. This approach is particularly important given the substantial genetic diversity among S. aureus clinical isolates.

Evaluating the immunogenicity of SAR1684 is a critical second step. Researchers should assess whether the protein naturally elicits antibody responses during human infection by examining sera from patients recovering from different types of S. aureus infections. Low natural immunogenicity would suggest either immune evasion properties or limited exposure to the immune system, possibly due to low expression or subcellular localization.

If deemed sufficiently conserved and immunogenic, SAR1684's protective potential should be evaluated through active immunization studies in animal models. Purified recombinant protein, when administered with appropriate adjuvants, should demonstrate protection against subsequent challenge with virulent S. aureus strains. Passive immunization studies with anti-SAR1684 antibodies can further validate protective potential.

For vaccine formulation considerations, researchers might explore combining SAR1684 with established targets like the detoxified SpA KKAA, which has shown promising results in murine models . This multi-antigen approach addresses the challenge of S. aureus' redundant virulence mechanisms. Additionally, structure-guided modifications of SAR1684, similar to the amino acid substitutions made in SpA KKAA to abrogate superantigen activity , might enhance its vaccine potential.

How can SAR1684 research inform broader understanding of uncharacterized bacterial proteins?

Research on SAR1684 extends beyond its specific role in S. aureus biology to establish methodological frameworks for studying uncharacterized bacterial proteins more generally. The UPF0271 family, to which SAR1684 belongs, represents one of many protein families with conserved sequences across bacterial species but unknown functions, presenting a significant knowledge gap in bacterial genomics.

Systematic investigation of SAR1684 demonstrates how integrating computational predictions with experimental validation can uncover functions of uncharacterized proteins. Initial bioinformatic approaches—including structural prediction algorithms, genomic context analysis, and phylogenetic profiling—generate testable hypotheses about protein function. The methodological workflow developed for SAR1684 can serve as a template for studying other UPF proteins.

Comparative analysis of SAR1684 expression patterns across different S. aureus strains illustrates how transcriptomic data can reveal the biological context in which uncharacterized proteins operate. Correlation networks linking SAR1684 expression with known virulence factors can suggest functional associations, even before direct experimental evidence is available. This systems biology approach provides context that purely biochemical characterizations might miss.

The study of UPF0271 proteins like SAR1684 also highlights the value of structural biology in function prediction. Even when sequence homology fails to identify functional similarities, structural homology may reveal relationships to characterized proteins. This principle has proven valuable in many bacterial systems and underscores the importance of structural determination efforts for uncharacterized protein families.

Finally, SAR1684 research exemplifies how bacterial accessory genome components contribute to species adaptation and pathogenesis. While much attention focuses on characterized virulence factors, the substantial portion of bacterial genomes encoding proteins of unknown function likely contributes significantly to bacterial fitness and adaptation. Methodologies developed for SAR1684 analysis contribute to uncovering this "dark matter" of bacterial proteomes.

What CRISPR-Cas9 strategies are most effective for generating SAR1684 knockout strains of MRSA?

Generating precise SAR1684 knockout strains in MRSA requires specialized CRISPR-Cas9 approaches that address the unique challenges of genetic manipulation in S. aureus. Unlike model organisms, S. aureus possesses robust restriction-modification systems and lower transformation efficiencies, necessitating optimized protocols. For targeting SAR1684 specifically, researchers should consider several critical factors in their experimental design.

Guide RNA (gRNA) design represents the first crucial step, with target sites ideally located near the start codon to ensure complete functional disruption. For SAR1684, researchers should design at least three gRNAs targeting different regions of the gene to maximize success probability. Each gRNA must be evaluated for potential off-target effects using S. aureus-specific prediction tools rather than generic CRISPR design software. The gRNA efficiency can be pre-validated using in vitro cleavage assays before attempting bacterial transformation.

Vector selection significantly impacts transformation efficiency in S. aureus. Temperature-sensitive plasmids with inducible Cas9 expression, such as modified pMAD vectors, allow for controlled editing and subsequent plasmid curing. For delivery into MRSA strains, electroporation protocols must be optimized with cells harvested during mid-exponential phase and treated with lysostaphin to weaken cell walls immediately before electroporation.

Homology-directed repair templates should include homology arms of at least 500 bp flanking the SAR1684 gene. To prevent CRISPR re-cutting after successful editing, silent mutations should be introduced within the PAM sequence or gRNA target site of the repair template. For marker-free deletions, a two-step process using counterselectable markers (e.g., IPTG-inducible toxins) facilitates screening for edited clones.

Genetic verification of successful knockouts requires both PCR confirmation of the deletion and whole-genome sequencing to verify the absence of off-target modifications or compensatory mutations. Phenotypic verification through proteomic analysis (Western blotting) and functional assays completes the validation process. This comprehensive approach ensures the generation of clean genetic backgrounds for studying SAR1684 function.

How can structural biology techniques be optimized to resolve the three-dimensional structure of SAR1684?

Resolving the three-dimensional structure of SAR1684 presents several technical challenges that require strategic optimization of structural biology approaches. The uncharacterized nature of UPF0271 family proteins means researchers lack structural templates for molecular replacement methods, necessitating experimental phasing strategies.

For X-ray crystallography, protein sample preparation is critical. High-throughput screening of crystallization conditions should be complemented by systematic construct optimization, including:

• Terminal truncations to remove potential disordered regions

• Surface entropy reduction mutations (replacing clusters of high-entropy residues like Lys/Glu/Gln with alanines)

• Fusion to crystallization chaperones such as T4 lysozyme or BRIL

• Selenomethionine labeling for phase determination

Researchers might consider parallel pursuit of NMR spectroscopy, particularly if initial crystallization attempts prove challenging. For the 250-amino acid SAR1684, NMR would require isotopic labeling (13C, 15N) and potentially deuteration. TROSY-based experiments optimize signal detection for this size range, while automated assignment software accelerates structure determination. NMR also provides the advantage of revealing dynamic regions and potential binding interfaces through chemical shift perturbation experiments.

Cryo-EM represents another viable approach, particularly if SAR1684 forms higher-order assemblies or can be engineered to increase molecular weight (e.g., by multimerization domains or antibody fragment complexation). Recent advances in cryo-EM technology have pushed resolution boundaries for smaller proteins, making this increasingly feasible for proteins in SAR1684's size range.

Integrative structural biology combining multiple techniques often yields the most complete structural information. Low-resolution techniques (SAXS, hydrogen-deuterium exchange mass spectrometry) can provide complementary data when high-resolution methods face challenges. Computational approaches including AlphaFold2 predictions can guide experimental design and offer preliminary structural insights, though experimental validation remains essential.

This multi-faceted approach mirrors successful structural studies of other S. aureus proteins, including the determination of SpA's three-dimensional structure in complex with immunoglobulin fragments, which revealed its unique three-helix bundle conformation .

What comparative proteomics approaches would best elucidate the functional network of SAR1684 in S. aureus?

Elucidating the functional network of SAR1684 requires sophisticated comparative proteomics strategies that can reveal both direct interaction partners and broader pathway associations. An integrated approach combining multiple proteomics methodologies provides the most comprehensive understanding of SAR1684's functional context within S. aureus biology.

Affinity purification-mass spectrometry (AP-MS) serves as the foundation for identifying direct protein-protein interactions. For this approach, epitope-tagged SAR1684 (typically with FLAG or His6 tags) expressed in S. aureus allows for specific pulldown of interaction complexes. Quantitative comparison between bait purifications and controls using SILAC or TMT labeling distinguishes genuine interactions from background contaminants. Crosslinking mass spectrometry (XL-MS) further defines interaction interfaces at amino acid resolution, providing structural constraints for modeling complex formation.

Proximity-dependent labeling methods, particularly TurboID or APEX2 fused to SAR1684, offer advantages for capturing transient or weak interactions that might be lost during conventional purification. These approaches enable in vivo labeling of proximal proteins, providing a more physiological view of the interaction landscape. The labeled proteins can then be purified and identified by mass spectrometry.

Phosphoproteomics analysis of wild-type versus ΔSAR1684 strains can reveal alterations in signaling networks, potentially positioning SAR1684 within specific regulatory pathways. Similarly, secretome analysis may identify extracellular proteins whose secretion depends on SAR1684 function, particularly important if SAR1684 influences virulence factor expression or secretion.

Integration of these proteomic datasets with transcriptomics data strengthens functional predictions by identifying concordant changes at both RNA and protein levels. Network analysis algorithms can then position SAR1684 within functional modules based on correlation patterns across multiple datasets. This integrated approach parallels successful studies of other S. aureus proteins involved in virulence regulation and immune evasion mechanisms .

How does SAR1684 compare with other uncharacterized proteins (UPFs) in S. aureus MRSA252?

The comparative analysis of SAR1684 with other uncharacterized proteins in S. aureus MRSA252 provides important context for understanding its potential significance. The table below summarizes key characteristics of selected UPF proteins from S. aureus MRSA252, enabling researchers to identify patterns and prioritize targets for functional investigation .

This comparative analysis reveals several notable patterns. SAR1684 is among the more highly conserved UPF proteins, suggesting functional importance across S. aureus lineages. Its predicted cytoplasmic localization differentiates it from membrane-associated or secreted UPFs that might directly interact with host factors. The constitutive expression pattern of SAR1684 contrasts with stress-induced or growth phase-dependent UPFs, potentially indicating a housekeeping function rather than a specialized stress response role.

The mixed α/β secondary structure prediction for SAR1684 parallels structural features observed in bacterial proteins involved in nucleotide binding or enzymatic functions. While this prediction alone cannot determine function, it narrows the range of potential biochemical activities compared to predominantly α-helical proteins often involved in protein-protein interactions or predominantly β-sheet proteins frequently associated with transport functions.

Researchers investigating SAR1684 should consider these comparative characteristics when designing experiments and interpreting results. The high conservation suggests that functional insights gained from laboratory strains will likely translate to clinical isolates, while the constitutive expression pattern indicates that standard laboratory growth conditions should be sufficient for initial characterization studies without requiring specialized induction conditions.

What experimental evidence exists for SAR1684's role in S. aureus biology compared to characterized virulence factors?

The current experimental evidence for SAR1684's biological role remains limited compared to well-characterized S. aureus virulence factors. The table below contrasts the available evidence for SAR1684 with established virulence determinants, highlighting the substantial knowledge gap that represents an opportunity for novel discoveries .

This comparative analysis highlights the extensive characterization gap between SAR1684 and established virulence factors like SpA. While SpA has been extensively characterized structurally, functionally, and immunologically—with clear roles in immune evasion through B cell superantigen activity and antibody neutralization —SAR1684 remains at the prediction stage across all categories.

The lack of experimental data for SAR1684 should not be interpreted as indicating functional insignificance. Many bacterial proteins with important roles remain uncharacterized due to research prioritization rather than biological importance. The absence of phenotypic data from SAR1684 knockout studies represents a particularly critical knowledge gap, as such studies have been instrumental in establishing the significance of proteins like SpA, where knockout strains showed increased susceptibility to phagocytosis and reduced abscess formation .

For researchers investigating SAR1684, this comparison emphasizes the value of foundational experiments that have already proven informative for characterized virulence factors. Gene deletion studies, structural determination efforts, and protein interaction screening represent high-priority approaches likely to yield meaningful insights. The extensive methodological framework established through SpA research—including modified versions like SpA KKAA for vaccine development —provides valuable templates for similar investigations of SAR1684.