Recombinant Staphylococcus aureus Uncharacterized lipoprotein SAS0073 (SAS0073)

What is the predicted structure and function of SAS0073 lipoprotein in S. aureus?

SAS0073 is an uncharacterized lipoprotein from Staphylococcus aureus that likely follows the typical structural patterns of bacterial lipoproteins. Like other S. aureus lipoproteins, SAS0073 likely contains an N-terminal signal sequence with a conserved C-terminal lipobox motif that is recognized by the processing machinery. The structural analysis would involve identifying the conserved cysteine residue in the lipobox that serves as the site for lipid attachment. Functionally, while its specific role remains uncharacterized, bacterial lipoproteins generally serve diverse functions including nutrient acquisition, cell signaling, and host-pathogen interactions. As with characterized S. aureus lipoproteins such as FhuD2, Csa1A, and SpA, SAS0073 may potentially contribute to bacterial virulence or survival mechanisms .

How is SAS0073 processed and localized within the S. aureus cell?

The processing of SAS0073 likely follows the canonical lipoprotein maturation pathway observed in S. aureus. After Sec-dependent secretion across the cytoplasmic membrane, the lipoprotein precursor undergoes sequential processing by conserved enzymes. First, the phosphatidylglycerol prolipoprotein diacylglyceryl transferase (Lgt) attaches a diacylglyceryl group to the conserved cysteine in the lipobox. Subsequently, the signal peptidase II (LspA) cleaves the signal peptide, resulting in the mature lipoprotein anchored to the membrane via its lipid moiety . This processing pathway is essential for proper localization and function of lipoproteins in S. aureus. Mutations in either lgt or lspA genes would disrupt proper processing and membrane anchoring of SAS0073, potentially affecting its functional capabilities .

What expression systems are most suitable for producing recombinant SAS0073?

For recombinant expression of SAS0073, several expression systems could be employed depending on research objectives. E. coli expression systems are commonly used due to their high yield and ease of genetic manipulation. When expressing SAS0073 in E. coli, fusion to the lpp leader sequence has been shown to effectively direct lipoproteins to the outer membrane vesicles (OMVs), with expression levels potentially reaching 10-20% of total OMV proteins . Alternatively, yeast, baculovirus, or mammalian cell expression systems might be considered for specific applications requiring eukaryotic post-translational modifications . For vaccine development purposes, the E. coli BL21(DE3)ΔompA strain has demonstrated efficient incorporation of lipidated S. aureus antigens into OMVs, suggesting it as a viable platform for SAS0073 expression .

What strategies can be employed to determine the immunogenic potential of SAS0073?

Determining the immunogenic potential of SAS0073 requires a multi-faceted approach. Initially, bioinformatic analysis to identify potential B-cell and T-cell epitopes within the protein sequence would guide experimental design. Subsequently, recombinant SAS0073 should be expressed, preferably as a lipidated form to maintain native conformation and adjuvant properties. The purified protein could then be used in immunization studies in mice to evaluate antibody titers, as demonstrated with other S. aureus lipoproteins where doses as low as 0.2 μg/dose elicited high, saturating antigen-specific antibody responses . Flow cytometry and ELISA assays would quantify humoral immune responses, while ELISpot assays would assess T-cell responses. Protection studies involving challenge with virulent S. aureus strains would ultimately determine protective efficacy. Additionally, incorporation of SAS0073 into bacterial outer membrane vesicles (OMVs) could enhance its immunogenicity due to the adjuvant properties of OMVs, potentially achieving 5-20% incorporation efficiency of total OMV proteins .

How can researchers engineer SAS0073 for optimal incorporation into outer membrane vesicles (OMVs)?

Engineering SAS0073 for optimal incorporation into OMVs involves several critical steps. First, the coding sequence of SAS0073 should be fused immediately downstream from, and in frame with, the "lipobox" cysteine of an E. coli lipoprotein leader sequence such as lpp. This fusion construct should be cloned into a suitable expression vector such as pET21. For optimal expression and reduced reactogenicity, the E. coli BL21(DE3)ΔompAΔmsbBΔpagP strain has demonstrated excellent results with other S. aureus antigens . When expressed in this system, lipidated proteins not only accumulate efficiently in OMVs (10-20% of total OMV proteins) but also modify the lipid A structure, reducing TLR4 agonist activity by approximately 100-fold compared to empty OMVs, thus improving the safety profile for vaccine applications . Expression conditions should be optimized regarding induction time, temperature, and IPTG concentration to maximize protein yield while ensuring proper incorporation into OMVs.

What are the key considerations when designing knockout studies to determine SAS0073 function?

Designing knockout studies to determine SAS0073 function requires careful consideration of multiple factors. First, create a precise gene deletion using allelic replacement techniques rather than insertional inactivation to avoid polar effects on adjacent genes. The pKOR1 system, which utilizes lambda recombination and counter-selection with antisense secY, has proven effective for creating markerless deletions in S. aureus. When evaluating the ΔSAS0073 mutant, perform comprehensive phenotypic characterization including growth kinetics in various media, biofilm formation capacity, resistance to antimicrobial peptides, oxidative stress response, and virulence in infection models. Additionally, consider creating complemented strains where SAS0073 is reintroduced on a plasmid under both native and inducible promoters to confirm phenotype restoration. For more nuanced understanding, site-directed mutagenesis of specific domains within SAS0073 would help identify functional regions. Finally, perform transcriptomic and proteomic analyses comparing wild-type and mutant strains to identify dysregulated pathways that might reveal SAS0073's functional networks .

How should researchers design experiments to investigate potential signaling functions of SAS0073-derived peptides?

To investigate potential signaling functions of SAS0073-derived peptides, researchers should implement a comprehensive experimental design. Begin by examining if SAS0073 undergoes processing by the Eep metalloprotease and subsequent export via the EcsAB transporter, as these components are essential for linear peptide processing and secretion in S. aureus . Create isogenic mutants lacking eep and ecsAB genes to determine their impact on SAS0073 processing. Next, employ high-resolution mass spectrometry to identify specific peptide fragments derived from SAS0073 in culture supernatants of wild-type S. aureus compared to these mutants. Once identified, synthesize these peptides chemically and test their ability to induce transcriptional changes in both producer and recipient bacterial cells using RNA-seq or targeted qRT-PCR. Additionally, examine if the peptides mediate aggregation or other phenotypic changes in S. aureus or other bacterial species, similar to the interspecies signaling observed with staph-cAM373 . Finally, develop reporter systems with promoters potentially regulated by these peptides fused to fluorescent proteins to visualize signaling events in real-time.

What approaches can be used to identify potential interaction partners of SAS0073?

Identifying potential interaction partners of SAS0073 requires multiple complementary approaches. Begin with co-immunoprecipitation (Co-IP) studies using anti-SAS0073 antibodies to pull down protein complexes from S. aureus lysates, followed by mass spectrometry to identify bound proteins. For membrane-associated interactions, employ crosslinking with membrane-permeable agents like formaldehyde or DSP (dithiobis[succinimidyl propionate]) prior to Co-IP to stabilize transient interactions. Bacterial two-hybrid systems can screen for direct protein-protein interactions, while split-GFP complementation assays could visualize interactions in vivo. For a global approach, perform APEX2 proximity labeling by fusing SAS0073 to an engineered ascorbate peroxidase, which biotinylates nearby proteins upon activation with hydrogen peroxide. Additionally, surface plasmon resonance (SPR) or microscale thermophoresis can determine binding kinetics with candidate partners. Finally, structural studies using X-ray crystallography or cryo-EM of SAS0073 in complex with identified partners would provide definitive evidence of interaction surfaces and mechanisms .

How can researchers effectively evaluate the impact of SAS0073 on S. aureus pathogenesis in vivo?

Evaluating SAS0073's impact on S. aureus pathogenesis requires robust in vivo experimental designs. Create isogenic deletion mutants (ΔSAS0073) and complemented strains in clinically relevant S. aureus backgrounds such as USA300 LAC. Employ multiple infection models that recapitulate different aspects of staphylococcal disease: skin and soft tissue infection (SSTI) models to assess abscess formation and bacterial burden; systemic infection models to evaluate bacterial dissemination to organs; and specialized models like osteomyelitis, endocarditis, or pneumonia depending on hypothesized function. When conducting these experiments, monitor both bacterial metrics (CFU recovery, dissemination patterns) and host response parameters (neutrophil recruitment, cytokine profiles, tissue damage). Implement longitudinal imaging using bioluminescent or fluorescent S. aureus strains to track infection progression non-invasively. Additionally, assess the ΔSAS0073 mutant's ability to colonize mucosal surfaces or skin in carrier models. For vaccine potential assessment, immunize mice with recombinant SAS0073 and evaluate protection against subsequent challenge with various S. aureus strains, as demonstrated successfully with other S. aureus antigens that provided complete protection in mouse challenge models .

What statistical methods are most appropriate for analyzing antibody responses to SAS0073 in immunization studies?

When analyzing antibody responses to SAS0073 in immunization studies, several statistical approaches should be employed for robust data interpretation. For comparing antibody titers between experimental groups (e.g., different vaccine formulations or doses), use one-way ANOVA followed by Tukey's or Dunnett's post-hoc tests for multiple comparisons. For non-normally distributed data, apply non-parametric alternatives such as Kruskal-Wallis with Dunn's post-test. When examining antibody responses over time, implement repeated measures ANOVA or mixed-effects models to account for within-subject correlations. Correlation analyses (Pearson's or Spearman's) should be used to examine relationships between antibody titers and protection outcomes. Power calculations should be performed prior to experimentation, typically aiming for 80-90% power to detect meaningful differences in antibody responses, with group sizes of at least 8-10 animals per condition. Additionally, consider analyzing antibody subclasses and functional antibody assays (e.g., opsonophagocytic activity) to gain insights into the quality of immune responses beyond simple titers. This comprehensive statistical approach aligns with methodologies used in successful S. aureus antigen studies where high, saturating antigen-specific antibody titers were achieved with as little as 0.2 μg/dose of lipidated antigens .

How should researchers interpret conflicting results between in vitro and in vivo studies of SAS0073 function?

When faced with conflicting results between in vitro and in vivo studies of SAS0073 function, researchers should implement a systematic interpretation framework. First, explicitly acknowledge the discrepancies rather than selectively reporting aligned findings. Consider fundamental differences in experimental conditions—in vitro systems lack the complex host environment including immune factors, tissue architecture, and physiological stresses that might influence SAS0073 expression or function. Verify that the in vitro conditions appropriately model relevant aspects of the in vivo environment; for instance, if studying SAS0073's role during infection, in vitro media should mimic host nutritional conditions. Examine temporal dynamics, as discrepancies might reflect differences in observation timepoints rather than true functional conflicts. Perform dose-response studies across both systems to identify potential threshold effects. Additionally, consider strain-specific effects, as genetic background can influence lipoprotein expression and function in S. aureus. Employ complementary approaches such as ex vivo experiments using infected host cells or tissues to bridge the gap between systems. Finally, use systems biology approaches including transcriptomics or proteomics to identify regulatory networks affecting SAS0073 expression differently between settings .

What approaches should be used to analyze mass spectrometry data for identifying SAS0073-derived peptides?

Analysis of mass spectrometry data for identifying SAS0073-derived peptides requires a sophisticated computational pipeline. Begin with high-resolution LC-MS/MS data acquisition from culture supernatants of wild-type S. aureus and appropriate mutants (ΔSAS0073, Δeep, ΔecsAB). For data processing, implement database searching using engines such as Mascot, SEQUEST, or MaxQuant against a custom database containing the SAS0073 sequence and known processing intermediates. Set search parameters to accommodate post-translational modifications including lipidation at cysteine residues and signal peptide removal. Apply false discovery rate (FDR) thresholds of 1% at both peptide and protein levels using target-decoy approaches. For quantitative comparisons between strains, employ label-free quantification methods such as normalized spectral counting or MS1 intensity-based approaches. Verify identified peptides through comparison with synthetic standards matching predicted SAS0073-derived sequences. Additionally, implement de novo sequencing algorithms to identify novel processed forms not predicted by database searches. For detecting small linear peptides specifically, optimize extraction methods using hydrophobic resins and implement specialized LC gradients for small peptide resolution. This approach parallels successful identification of processed linear peptides from S. aureus observed in previous studies .

What are the potential applications of SAS0073 in vaccine development against S. aureus infections?

SAS0073 represents a promising candidate for S. aureus vaccine development based on several advantageous properties of lipoproteins. As a lipoprotein, SAS0073 likely possesses intrinsic adjuvant properties through TLR2 activation, potentially enhancing immune responses without requiring additional adjuvants. For optimal vaccine formulation, SAS0073 could be expressed as a lipidated protein and incorporated into outer membrane vesicles (OMVs), which have demonstrated remarkable efficacy with other S. aureus antigens. Using the E. coli BL21(DE3)ΔompAΔmsbBΔpagP expression system would allow high-level incorporation of SAS0073 into OMVs (potentially 5-20% of total OMV proteins) while simultaneously reducing reactogenicity through modified lipid A structure . This approach has shown remarkable success with five other S. aureus antigens, which when combined provided complete protection against S. aureus challenge in mouse models. The lipidation strategy appears to be universally applicable to diverse antigens, suggesting it would work effectively with SAS0073. For clinical development, SAS0073 could be combined with other protective antigens to create a multivalent vaccine targeting multiple virulence factors simultaneously, addressing the historical challenge of single-antigen vaccine failures against this complex pathogen .

How might researchers investigate the potential role of SAS0073 in interspecies bacterial communication?

Investigating SAS0073's potential role in interspecies bacterial communication requires a systematic experimental approach. First, determine if SAS0073 undergoes processing to generate linear peptides by the membrane metalloprotease Eep and the EcsAB transporter, critical components identified in the processing and secretion of signaling peptides in S. aureus . Generate isogenic mutants lacking eep and ecsAB genes and analyze supernatants using high-resolution mass spectrometry to identify potential SAS0073-derived peptides present in wild-type but absent in mutant strains. Once candidate peptides are identified, synthesize them chemically and test their ability to induce aggregation or gene expression changes in other bacterial species, particularly those sharing niches with S. aureus such as Enterococcus faecalis. Develop reporter strains of recipient bacteria with promoters of interest fused to fluorescent proteins to visualize responses to SAS0073-derived peptides. Additionally, investigate if these peptides can mediate horizontal gene transfer between species, similar to the staph-cAM373 peptide (AIFILAA) that promotes aggregation of E. faecalis cells harboring the pAM373 plasmid . Finally, employ transcriptomic approaches to identify the complete regulon controlled by these peptides in both producer and recipient species.

What technologies are emerging that could advance our understanding of SAS0073 function in S. aureus biology?

Emerging technologies offer unprecedented opportunities to elucidate SAS0073 function in S. aureus biology. CRISPR interference (CRISPRi) systems adapted for S. aureus enable precise temporal control of SAS0073 expression, allowing researchers to determine the immediate consequences of its depletion without genetic compensation effects associated with traditional knockouts. Single-cell RNA sequencing of heterogeneous S. aureus populations can reveal cell-to-cell variability in SAS0073 expression and correlation with specific bacterial states or subpopulations. Advanced imaging techniques including super-resolution microscopy and correlative light-electron microscopy can visualize SAS0073 localization with nanometer precision, potentially revealing microdomains within the bacterial membrane. For structural studies, cryo-electron tomography enables visualization of SAS0073 in its native membrane environment without crystallization. Metabolic labeling approaches such as bio-orthogonal non-canonical amino acid tagging (BONCAT) allow selective labeling of newly synthesized SAS0073 to track its production under various conditions. Additionally, bacterial cytological profiling using high-content imaging could identify cellular pathways disrupted by SAS0073 manipulation. Finally, interactome mapping through proximity labeling methods like TurboID would comprehensively identify proteins in close proximity to SAS0073 within living bacteria .

How does SAS0073 compare structurally and functionally to characterized S. aureus lipoproteins?

While the specific structural and functional characteristics of SAS0073 remain uncharacterized, comparison with well-studied S. aureus lipoproteins provides insight into its potential properties. Like other S. aureus lipoproteins, SAS0073 likely contains a characteristic N-terminal signal sequence with a conserved lipobox motif that directs processing by Lgt and LspA. Based on observations with characterized lipoproteins such as FhuD2, Csa1A, and SpA, which show efficient incorporation into OMVs (10-20% of total OMV proteins), SAS0073 would likely demonstrate similar behavior when expressed as a recombinant lipoprotein in appropriate systems . Functionally, it may parallel FhuD2's role in nutrient acquisition, contribute to immune evasion like SpA, or serve as an unidentified virulence factor. The expression of lipidated proteins has been shown to reduce TLR4 stimulation by modifying lipid A structure, with reductions ranging from 20 to 100-fold depending on the specific lipoprotein; SAS0073 would likely exhibit similar immunomodulatory effects .

What animal models are most appropriate for studying SAS0073 in S. aureus pathogenesis?

Selecting appropriate animal models for studying SAS0073 in S. aureus pathogenesis depends on hypothesized functions and research objectives. For systemic infection studies, the murine bacteremia model involving tail vein injection of S. aureus followed by assessment of bacterial burden in kidneys, liver, and heart provides quantitative readouts of bacterial dissemination and persistence. For localized infections, the skin abscess model created by subcutaneous injection allows evaluation of abscess formation, bacterial clearance, and local immune responses, potentially revealing SAS0073's role in tissue-specific pathogenesis. If SAS0073 is hypothesized to participate in biofilm formation, catheter-associated infection models or the tibial implant model would be most relevant. For colonization studies, the nasal colonization model would determine if SAS0073 contributes to mucosal persistence. When evaluating SAS0073 as a vaccine candidate, challenge studies following immunization with recombinant SAS0073 should employ clinically relevant infection models aligned with the target disease presentation. Importantly, studies should use both laboratory and clinical S. aureus strains, as strain background can significantly influence protein expression and function. This multi-model approach parallels successful evaluation strategies used for other S. aureus antigens that demonstrated complete protection in mouse challenge models .

What heterologous expression systems provide optimal yields of functional recombinant SAS0073?

For optimal expression of recombinant SAS0073, the E. coli BL21(DE3)ΔompA system has demonstrated exceptional results with other S. aureus lipoproteins, achieving expression levels of 10-20% of total OMV proteins for similar antigens like FhuD2, Csa1A, and SpA . This system efficiently directs lipidated proteins to the vesicular compartment, maintaining native conformation important for immunological studies. For vaccine applications specifically, the E. coli BL21(DE3)ΔompAΔmsbBΔpagP strain offers additional advantages through reduced endotoxicity while maintaining high expression yields . Expression optimization should include testing various induction temperatures (16-30°C), IPTG concentrations (0.1-1.0 mM), and induction times (4-24 hours) to maximize protein yield while ensuring proper lipidation and folding. Purification should employ gentle methods that preserve the lipid moiety, such as membrane fractionation followed by detergent solubilization and affinity chromatography with appropriate tags.