Recombinant Staphylococcus aureus UPF0316 protein USA300HOU_1913 (USA300HOU_1913)

What is UPF0316 protein USA300HOU_1913 and what are its key characteristics?

UPF0316 protein USA300HOU_1913 is an uncharacterized protein from Staphylococcus aureus strain USA300. The protein consists of 200 amino acids and belongs to the UPF (Uncharacterized Protein Family) 0316 family. The full amino acid sequence is: MSFVTENPWLMVLTIFIINICYVTFLTMRTILTLKGYRYIAASVSFLEVLVYIVGLGLVMSNLDHIQNIIAYAFGFSIGIIVGMKIEEKLALGYTVVNVTSAEYELDLPNELRNLGYGVTHYAAFGRDGSRMVMQILTPRKYERKLMDTIKNLDPKAFIIAYEPRNIHGGFWTKGIRRRKLKDYEPEELESVVEHEIQSK . Based on sequence analysis, the protein likely contains transmembrane domains, suggesting it could be a membrane-associated protein, though its precise function remains to be fully elucidated.

When recombinantly produced, the protein is typically expressed with an N-terminal His-tag to facilitate purification and is expressed in E. coli expression systems for research applications . The recombinant version maintains the full-length sequence to preserve potential functional domains.

How should recombinant USA300HOU_1913 protein be handled and stored for optimal stability?

For optimal stability and activity maintenance, recombinant USA300HOU_1913 protein requires specific handling and storage protocols. The lyophilized protein should be briefly centrifuged prior to opening to ensure the material is at the bottom of the vial. Reconstitution should be performed in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL .

For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being standard practice) and aliquot the protein to avoid repeated freeze-thaw cycles, which can significantly compromise protein integrity . The aliquoted protein should be stored at -20°C or preferably -80°C for extended shelf life.

Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be strictly avoided as this can lead to protein degradation and loss of activity . The storage buffer typically consists of a Tris/PBS-based solution with 6% trehalose at pH 8.0, which helps maintain protein stability.

What expression systems are most effective for producing functional recombinant USA300HOU_1913?

Several expression systems can be utilized for producing recombinant USA300HOU_1913, each with distinct advantages depending on research requirements. For standard applications, E. coli expression systems are most commonly employed due to their cost-effectiveness and high yield . The protein is typically expressed with an N-terminal His-tag to facilitate purification through affinity chromatography.

For studies requiring post-translational modifications or improved protein folding, alternative expression systems might be considered:

What methodological approaches can elucidate the function of this uncharacterized protein?

Elucidating the function of uncharacterized proteins like USA300HOU_1913 requires a multi-faceted experimental approach. Based on the UPF family characteristics, several methodologies are particularly valuable:

• Comparative Genomics and Phylogenetic Analysis: Identification of conserved domains across bacterial species can provide initial functional hints. Analysis should include comparison with characterized UPF proteins such as UPF1, UPF2, and UPF3, which are known to function in nonsense-mediated mRNA decay pathways and interact with release factors .

• Protein-Protein Interaction Studies: Techniques such as co-immunoprecipitation, yeast two-hybrid assays, or proximity-dependent biotin identification (BioID) can identify binding partners. Based on other UPF proteins, potential binding partners might include translation termination factors or components of RNA decay machinery .

• Gene Knockout/Knockdown Studies: Creating deletion mutants in S. aureus followed by phenotypic characterization can reveal functional roles. Analysis should focus on growth characteristics, antibiotic susceptibility, virulence in infection models, and potential nonsense suppression phenotypes as observed with other UPF proteins .

• Transcriptomics and Proteomics: RNA-seq and mass spectrometry analysis comparing wild-type and knockout strains can identify affected pathways. Special attention should be paid to changes in nonsense-containing transcripts and proteins involved in translation termination.

• Structural Biology: X-ray crystallography or cryo-EM studies can provide structural insights. The membrane-associated nature of USA300HOU_1913 may require specific solubilization techniques using appropriate detergents or nanodiscs.

These approaches should be implemented sequentially, starting with bioinformatic analyses to guide subsequent experimental designs for efficient functional characterization.

How can site-directed mutagenesis be optimally designed to study functional domains of USA300HOU_1913?

Site-directed mutagenesis represents a powerful approach for identifying functional domains within USA300HOU_1913. Based on sequence analysis and structural predictions, a systematic mutagenesis strategy should target:

• Transmembrane Domains: The protein sequence suggests membrane association (residues approximately 10-32, 45-67, and 75-97). Alanine scanning of these regions can disrupt membrane integration. Conservative (e.g., Leu to Ile) and non-conservative (e.g., Leu to Asp) substitutions should be compared to distinguish structural versus functional roles.

• Conserved Motifs: The sequence "YTVVNVTSAEYE" (residues 112-123) represents a potentially conserved motif. Mutations should target invariant residues first, particularly charged or polar amino acids that often participate in functional interactions.

• C-terminal Region: The C-terminal sequence "AFIIAYEPRNIHGGFWT" (residues 169-185) contains a potential active site based on conservation patterns. Alanine substitutions of each residue can identify critical positions.

Mutants should be generated using overlap extension PCR with the following protocol:

• Design complementary primers containing the desired mutation

• Perform two separate PCR reactions to generate overlapping fragments

• Combine fragments in a third PCR to generate the full mutant construct

• Clone into expression vector with N-terminal His-tag

• Verify by sequencing before expression in E. coli

Each mutant protein should be purified and subjected to functional assays comparing activity to wild-type protein. If USA300HOU_1913 shares functional similarities with other UPF proteins, nonsense suppression assays would be particularly informative .

What are the optimal conditions for crystallizing USA300HOU_1913 for structural determination?

Crystallizing membrane-associated proteins like USA300HOU_1913 presents significant challenges that require specialized approaches. Based on successful crystallization of similar bacterial membrane proteins, the following optimized protocol is recommended:

• Protein Preparation:

  • Express with removable His-tag (TEV protease cleavage site)

  • Solubilize using mild detergents: initial screening with n-Dodecyl β-D-maltoside (DDM), n-Decyl-β-D-Maltopyranoside (DM), and LDAO

  • Purify to >95% homogeneity using sequential chromatography (IMAC, followed by size exclusion)

  • Concentrate to 5-15 mg/mL in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, with detergent at 2× CMC

• Crystallization Screening:

  • Implement sparse matrix screens specifically designed for membrane proteins

  • Use sitting drop vapor diffusion at both 4°C and 18°C

  • Test protein:reservoir ratios of 1:1, 1:2, and 2:1

  • Include additives such as lipids (particularly E. coli polar lipids)

• Alternative Approaches:

  • Lipidic cubic phase (LCP) crystallization

  • Bicelle-based crystallization

  • Co-crystallization with antibody fragments (Fab or nanobody)

• Optimization Strategy:

  • Fine-grid screens around initial hits

  • Additive screens including divalent cations (particularly Mg2+)

  • Seeding techniques to improve crystal quality

If crystallization proves challenging, alternative structural biology approaches such as cryo-electron microscopy or NMR (for specific domains) should be considered. The presence of multiple transmembrane domains will necessitate careful detergent selection to maintain native protein conformation while allowing crystal contacts to form.

How does the expression profile of USA300HOU_1913 change under different stress conditions?

Understanding the expression profile of USA300HOU_1913 under various stress conditions can provide valuable insights into its physiological role. A comprehensive approach should employ both transcriptomic and proteomic analyses under conditions relevant to S. aureus pathogenesis:

• Antibiotic Stress Response:

  • Expose S. aureus cultures to sub-inhibitory concentrations of various antibiotic classes

  • Monitor USA300HOU_1913 expression using RT-qPCR and Western blotting

  • Compare with known stress response genes as controls

  • Special attention should be paid to vancomycin and daptomycin exposure, given S. aureus adaptation mechanisms to these antibiotics

• Environmental Stress Conditions:

  • Test temperature shifts (30°C, 37°C, 42°C)

  • Osmotic stress (varying NaCl concentrations)

  • pH stress (pH 5.5, 7.0, 8.5)

  • Oxidative stress (hydrogen peroxide exposure)

  • Nutrient limitation (carbon, nitrogen, phosphate)

• Host-Relevant Conditions:

  • Growth in serum or serum-supplemented media

  • Exposure to neutrophil antimicrobial peptides

  • Biofilm versus planktonic growth conditions

  • Intracellular survival conditions (after phagocytosis)

• Data Analysis Strategy:

  • Normalize expression against multiple reference genes (gyrB, rpoB)

  • Use hierarchical clustering to identify co-regulated genes

  • Perform pathway enrichment analysis for co-regulated gene clusters

  • Compare expression patterns with known UPF family members

This comprehensive profiling approach can reveal conditions where USA300HOU_1913 is significantly up- or down-regulated, providing clues to its physiological role in stress adaptation or virulence regulation.

How does USA300HOU_1913 compare structurally and functionally to similar proteins in other bacterial species?

Comparative analysis of USA300HOU_1913 with homologous proteins reveals important evolutionary and potentially functional relationships. Sequence homology searches indicate several key comparisons:

• S. aureus Strain Variations:
  The USA300HOU_1913 protein shares significant homology with NWMN_1849 from S. aureus strain Newman, another UPF0316 family protein of identical length (200 amino acids) . The high sequence conservation (>95% identity) suggests critical functional importance. Minor amino acid variations between strains may correlate with strain-specific virulence or adaptation characteristics.

• Cross-Species Comparisons:
  Homologs exist in other Staphylococcus species and more distantly in other Gram-positive bacteria. Sequence conservation patterns reveal:

  • Highly conserved transmembrane domains suggesting structural importance

  • Variable loop regions that may confer species-specific functions

  • Conservation of the C-terminal region (residues 169-185) across Staphylococcal species

• Functional Domain Analysis:
  While direct functional data on USA300HOU_1913 is limited, comparison with other UPF proteins suggests potential roles in:

  • Translation termination processes, similar to other UPF family members

  • Membrane transport or signaling, based on predicted transmembrane topology

  • Potential mRNA surveillance mechanisms, given the role of other UPF proteins in nonsense-mediated decay

This comparative analysis suggests that USA300HOU_1913 likely performs a conserved function in Staphylococcal species, potentially related to translation quality control or membrane-associated processes. The high conservation across pathogenic strains may indicate importance for bacterial fitness or virulence.

What is the relationship between USA300HOU_1913 and known virulence factors in S. aureus?

• Genomic Context Analysis:
  Examining genes adjacent to USA300HOU_1913 in the S. aureus genome can reveal functional relationships through operonic organization or co-regulation patterns. Analysis should focus on whether nearby genes have established roles in virulence or antibiotic resistance.

• Expression Correlation Studies:
  RNA-seq data analysis across infection models and stress conditions can identify whether USA300HOU_1913 expression correlates with known virulence factors. Of particular interest would be correlation with:

  • Toxin production genes (e.g., alpha-toxin, Panton-Valentine leukocidin)

  • Immune evasion factors

  • Biofilm formation components

  • Regulators like agr, sarA, or alternative sigma factors

• Pathogenicity Analysis of Deletion Mutants:
  Constructing USA300HOU_1913 deletion mutants and testing them in various infection models can directly assess virulence contributions. Key assays should include:

  • Mammalian cell adhesion and invasion capacity

  • Immune cell survival (particularly neutrophil interactions)

  • Animal infection models (skin abscess, systemic infection)

  • Biofilm formation capacity

• Antibiotic Resistance Connections:
  Given the adaptation mechanisms S. aureus employs against antibiotics like vancomycin and daptomycin , USA300HOU_1913 should be investigated for potential contributions to resistance development through:

  • Expression analysis in resistant versus sensitive strains

  • Impact of gene deletion on minimum inhibitory concentrations

  • Potential membrane-associated functions that might alter cell envelope properties

These investigative approaches can establish whether USA300HOU_1913 directly contributes to S. aureus virulence or indirectly supports pathogenesis through basic cellular functions that promote bacterial fitness during infection.

What are the key quality control parameters for validating recombinant USA300HOU_1913 preparation?

Rigorous quality control is essential for ensuring reliable research outcomes when working with recombinant USA300HOU_1913. The following comprehensive validation protocol is recommended:

• Purity Assessment:

  • SDS-PAGE analysis (target: >90% purity)

  • Silver staining for detection of low-abundance contaminants

  • Western blot using anti-His antibody to confirm identity

  • Mass spectrometry to verify molecular weight and detect potential truncations or modifications

• Structural Integrity:

  • Circular dichroism spectroscopy to assess secondary structure

  • Thermal shift assays to determine stability and proper folding

  • Limited proteolysis to verify domain organization

  • Dynamic light scattering to evaluate homogeneity and aggregation state

• Functional Verification:

  • Binding assays with potential interaction partners (if known)

  • Activity assays based on predicted function

  • For membrane-associated proteins, reconstitution into liposomes or nanodiscs to verify membrane integration

• Endotoxin Testing:

  • LAL (Limulus Amebocyte Lysate) test to measure endotoxin levels

  • Target: <1 EU/mg protein for cell-based assays

• Storage Stability Assessment:

  • Regular testing of aliquots stored under recommended conditions

  • Accelerated stability testing at elevated temperatures

  • Freeze-thaw stability testing to determine maximum number of cycles

A detailed record of each batch's quality parameters should be maintained in laboratory information management systems to ensure experimental reproducibility and facilitate troubleshooting of unexpected results.

What bioinformatic approaches are most effective for predicting potential functions of USA300HOU_1913?

Given the uncharacterized nature of USA300HOU_1913, sophisticated bioinformatic analyses represent crucial first steps toward functional elucidation. A comprehensive bioinformatic workflow should include:

• Sequence-Based Analyses:

  • Position-Specific Scoring Matrix (PSSM) searches using PSI-BLAST to identify distant homologs

  • Multiple sequence alignment of homologs to identify conserved residues

  • Motif identification using MEME, PROSITE, and other pattern recognition tools

  • Disorder prediction to identify flexible regions potentially involved in protein-protein interactions

• Structural Prediction:

  • Transmembrane topology prediction using TMHMM, Phobius, and MEMSAT

  • Secondary structure prediction using PSIPRED and JPred

  • Tertiary structure modeling using AlphaFold2 or RoseTTAFold

  • Molecular dynamics simulations to evaluate structural stability and potential conformational changes

• Functional Inference:

  • Gene neighborhood analysis across multiple bacterial genomes

  • Gene fusion detection to identify potential functional associations

  • Co-expression network analysis using publicly available transcriptomic data

  • Protein-protein interaction prediction based on structure and sequence

• Specialized Analyses for UPF Family Proteins:

  • Based on the established roles of UPF proteins in translation termination and nonsense-mediated decay , specific searches for:

    • RNA-binding motifs

    • Translation factor interaction domains

    • Similarities to known surveillance complex components

• Integration of Multiple Lines of Evidence:

  • Weighted scoring system combining predictions from multiple tools

  • Bayesian integration of diverse data types

  • Machine learning approaches trained on proteins with known functions

These bioinformatic analyses should be iteratively refined as experimental data becomes available, creating a virtuous cycle of prediction and validation that efficiently narrows the functional possibilities.

What emerging technologies show promise for characterizing membrane-associated proteins like USA300HOU_1913?

Several cutting-edge technologies are transforming our ability to characterize challenging membrane-associated proteins like USA300HOU_1913:

• Cryo-Electron Microscopy Advances:

  • Single-particle analysis for membrane proteins in nanodiscs or amphipols

  • Microcrystal electron diffraction (MicroED) for small, well-ordered crystals

  • Tomography with subtomogram averaging for in situ structural determination

  • These approaches overcome traditional crystallization barriers and can achieve near-atomic resolution

• Integrative Structural Biology:

  • Combining lower-resolution techniques (SAXS, cryo-EM) with computational modeling

  • Cross-linking mass spectrometry to identify domain arrangements and protein-protein interfaces

  • Hydrogen-deuterium exchange mass spectrometry for dynamics and ligand-binding studies

  • These integrated approaches provide complementary structural information when single techniques are insufficient

• Advanced Functional Genomics:

  • CRISPR interference for precise transcriptional regulation

  • Dual RNA-seq for simultaneous host-pathogen transcriptomics during infection

  • Transposon sequencing (Tn-seq) for high-throughput identification of genetic interactions

  • These approaches can reveal phenotypes and genetic relationships not apparent in simple knockout studies

• Single-Molecule Technologies:

  • Single-molecule FRET to study protein dynamics and conformational changes

  • Nanopore recording for membrane protein conductance measurements

  • Super-resolution microscopy for in vivo localization and trafficking studies

  • These techniques provide dynamic information lost in ensemble measurements

• AI-Driven Approaches:

  • AlphaFold2 and RoseTTAFold for accurate structure prediction

  • Machine learning for function prediction from sequence and structure

  • Molecular dynamics with AI-accelerated sampling techniques

  • These computational tools can guide experimental design and provide insights when experimental data is limited

These emerging technologies, particularly when used in combination, offer unprecedented opportunities to overcome the traditional challenges associated with membrane protein characterization, potentially accelerating our understanding of USA300HOU_1913's structure and function.

How might USA300HOU_1913 contribute to novel therapeutic approaches against S. aureus infections?

Understanding USA300HOU_1913's function could open several promising avenues for therapeutic development against S. aureus infections, particularly given the critical need for new approaches to combat antibiotic-resistant strains:

• Target Validation Considerations:

  • If USA300HOU_1913 proves essential for bacterial viability or virulence, it could represent a novel drug target

  • High conservation across S. aureus strains would suggest broad-spectrum activity

  • Limited homology to human proteins would minimize off-target effects

  • Membrane localization could provide accessibility to drug molecules without requiring cellular entry

• Potential Therapeutic Strategies:

  • If involved in translation termination like other UPF proteins :

    • Small molecule inhibitors could disrupt protein synthesis

    • Peptide mimetics could interfere with binding partners

  • If involved in membrane functions:

    • Compounds disrupting protein-lipid interactions

    • Pore-forming agents targeting specific membrane domains

• Vaccine Development Potential:

  • If exposed regions are identified, epitope mapping could guide subunit vaccine design

  • Surface-exposed conserved domains would be priority candidates

  • Potential for multivalent vaccines combining with established S. aureus antigens

• Combination Therapy Approaches:

  • Synergistic effects with existing antibiotics should be investigated

  • Particularly promising would be combinations with vancomycin or daptomycin, given S. aureus adaptation mechanisms to these drugs

  • Inhibitors might sensitize resistant strains to conventional antibiotics

• Diagnostic Applications:

  • Antibodies against USA300HOU_1913 could facilitate rapid diagnostic tests

  • Expression patterns during infection might serve as biomarkers

  • Point-of-care tests could guide appropriate antibiotic selection

The development of any therapeutic approach would require comprehensive understanding of USA300HOU_1913's function, structure, and role in pathogenesis. Given the membrane association and potential involvement in fundamental cellular processes, inhibitors could potentially disrupt multiple aspects of S. aureus physiology simultaneously, reducing the likelihood of resistance development.