Recombinant Staphylococcus aureus UPF0754 membrane protein USA300HOU_1839 (USA300HOU_1839)

What is UPF0754 membrane protein and why is it significant in Staphylococcus aureus research?

UPF0754 membrane proteins represent a family of bacterial membrane proteins with conserved structures across multiple Staphylococcus species. In S. aureus, these proteins are embedded in the cellular membrane and may play roles in cellular processes that contribute to the pathogen's virulence and survival. The USA300HOU_1839 variant is specifically found in the USA300 strain, which is a predominant community-associated methicillin-resistant S. aureus (CA-MRSA) lineage. Understanding these membrane proteins provides insights into potential therapeutic targets and bacterial physiology .

How does the amino acid sequence of USA300HOU_1839 compare with other S. aureus UPF0754 proteins?

The USA300HOU_1839 protein shares significant sequence homology with other S. aureus UPF0754 variants, such as SaurJH1_1933, SAHV_1831, and SAS1767. Based on available data, these proteins typically contain approximately 374 amino acids with characteristic hydrophobic transmembrane domains. The amino acid sequence of the related SaurJH1_1933 begins with "MNALFIIIIFMIVVGAIIGGIT..." and contains multiple hydrophobic regions consistent with its membrane-spanning function . Sequence alignment analysis typically reveals conserved domains that may be critical for protein function across different S. aureus strains.

What is the predicted membrane topology of USA300HOU_1839?

Based on analysis of related UPF0754 membrane proteins, USA300HOU_1839 likely contains multiple transmembrane domains. The sequence pattern suggests an N-terminal region followed by alternating hydrophobic (membrane-spanning) and hydrophilic (loop) regions. Computational prediction models indicate approximately 5-7 transmembrane helices with both the N-terminal and C-terminal regions potentially located on opposite sides of the membrane. This topology is consistent with the amino acid composition observed in the SaurJH1_1933 variant, which contains segments with high hydrophobicity index values suitable for membrane integration .

What are the optimal conditions for inducing expression of USA300HOU_1839 in E. coli?

For optimal expression in E. coli, consider the following protocol:

• Select an appropriate expression vector containing a T7 or similar strong promoter.

• Transform the construct into an expression strain optimized for membrane proteins (e.g., C41(DE3) or C43(DE3)).

• Grow cultures at 37°C until OD600 reaches 0.6-0.8.

• Reduce temperature to 18-20°C before induction.

• Induce with 0.1-0.5 mM IPTG.

• Continue expression for 16-20 hours at reduced temperature.

This approach minimizes inclusion body formation and improves the yield of correctly folded membrane protein. For membrane proteins like USA300HOU_1839, lower induction temperatures significantly improve proper folding and membrane integration .

What purification strategies yield the highest purity of USA300HOU_1839?

Purification of USA300HOU_1839 typically requires a multi-step approach:

• Membrane fraction isolation via differential centrifugation.

• Solubilization using appropriate detergents (e.g., DDM, LDAO, or Triton X-100).

• Affinity chromatography using the protein's tag (commonly His-tag).

• Size exclusion chromatography for further purification.

This process typically yields preparations with ≥85% purity as determined by SDS-PAGE analysis . For higher purity applications, additional ion exchange chromatography may be incorporated. The choice of detergent is critical for maintaining protein stability and activity throughout the purification process.

How can USA300HOU_1839 be incorporated into liposomes for functional studies?

For functional reconstitution into liposomes, researchers should follow this methodological approach:

• Prepare lipid mixture (typically POPC/POPE at 3:1 ratio) dissolved in chloroform.

• Evaporate solvent under nitrogen gas and form lipid film.

• Hydrate lipid film with buffer containing purified USA300HOU_1839 protein.

• Subject the mixture to freeze-thaw cycles (5-10 times).

• Extrude through polycarbonate membranes (100-400 nm pore size).

• Remove non-incorporated protein via density gradient centrifugation.

This protocol allows for controlled incorporation of the membrane protein into lipid bilayers, creating proteoliposomes suitable for functional assays including ion transport, ligand binding, or interaction studies with other membrane components .

What methods are recommended for studying protein-protein interactions involving USA300HOU_1839?

To investigate protein-protein interactions involving USA300HOU_1839, several complementary approaches are recommended:

• Co-immunoprecipitation (Co-IP) using antibodies against USA300HOU_1839 or potential interacting partners.

• Proximity labeling techniques such as BioID or APEX2 fusion constructs.

• Bimolecular fluorescence complementation (BiFC) for in vivo interaction validation.

• Surface plasmon resonance (SPR) or microscale thermophoresis (MST) for quantitative interaction analysis.

• Cross-linking mass spectrometry (XL-MS) to map interaction interfaces.

These methods provide different levels of information, from validating interactions to determining binding kinetics and identifying specific interaction domains. For membrane proteins like USA300HOU_1839, detergent selection and concentration are critical parameters that must be optimized to maintain native protein conformation while enabling detection of specific interactions .

What approaches are most suitable for determining the structure of USA300HOU_1839?

Determining the structure of membrane proteins like USA300HOU_1839 presents unique challenges. The most suitable approaches include:

• X-ray crystallography, which requires production of well-diffracting crystals:

  • Utilize lipidic cubic phase (LCP) crystallization.

  • Screen multiple detergents and additives.

  • Consider fusion proteins (e.g., T4 lysozyme) to enhance crystallization.

• Cryo-electron microscopy (cryo-EM):

  • Particularly useful for larger membrane protein complexes.

  • Can visualize protein in near-native lipid environments using nanodiscs.

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Suitable for smaller domains or regions of the protein.

  • Requires isotopic labeling (15N, 13C, 2H).

• Computational modeling approaches:

  • Homology modeling based on related structures.

  • Molecular dynamics simulations to predict dynamic properties.

Each method has specific advantages and limitations for membrane protein structural analysis, and often a combination of approaches yields the most comprehensive structural information .

How can researchers assess the proper folding and stability of recombinant USA300HOU_1839?

Assessment of proper folding and stability for USA300HOU_1839 should include multiple complementary techniques:

• Circular dichroism (CD) spectroscopy to evaluate secondary structure content.

• Fluorescence spectroscopy to monitor tertiary structure properties.

• Thermal shift assays to determine protein stability under various conditions.

• Limited proteolysis to assess compact, folded domains.

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to evaluate oligomeric state and homogeneity.

For membrane proteins specifically, additional methods include:

• Tryptophan fluorescence quenching to assess membrane integration.

• Detergent screening by differential scanning fluorimetry to identify stabilizing conditions.

Proper folding is typically indicated by monodisperse elution profiles, expected secondary structure content, and resistance to proteolytic degradation. Purity should be confirmed by SDS-PAGE, with expected levels ≥85% for most research applications .

What approaches can reveal the physiological function of USA300HOU_1839 in S. aureus?

To elucidate the physiological function of USA300HOU_1839, a systematic approach combining genetic, biochemical, and physiological methods is recommended:

• Gene knockout or knockdown studies:

  • CRISPR-Cas9 or antisense RNA approaches to reduce expression

  • Phenotypic analysis of resulting mutants

• Complementation experiments:

  • Expression of wild-type protein in knockout strains

  • Site-directed mutagenesis to identify critical residues

• Transcriptomic analysis:

  • RNA-seq to identify co-regulated genes

  • Comparison of expression profiles between wild-type and mutant strains

• Metabolomic profiling:

  • Identification of altered metabolic pathways in mutants

  • Isotope labeling to track specific metabolic processes

• Interaction studies:

  • Pull-down assays to identify protein partners

  • Metabolite binding assays to identify potential substrates

These approaches should be conducted in physiologically relevant conditions, such as under various stresses that S. aureus encounters during infection processes .

How does USA300HOU_1839 compare to UPF0754 proteins in other bacterial species?

UPF0754 membrane proteins are found across multiple bacterial species, with notable examples in Bacillus subtilis (yheB), Bacillus pumilus (BPUM_0927), Bacillus cereus (BCAH820_0954), and Anoxybacillus flavithermus (Aflv_2299). Comparative analysis reveals:

*Estimated sequence identity ranges based on typical conservation patterns within protein families .

This evolutionary conservation suggests functional importance, with the highest conservation observed within Staphylococcus species and more divergence in other genera, while maintaining similar membrane topology and predicted structural features.

How can USA300HOU_1839 be utilized in vaccine development against S. aureus infections?

The potential of USA300HOU_1839 as a vaccine component can be evaluated through the following research approaches:

• Antigenicity assessment:

  • Epitope mapping to identify immunogenic regions

  • Analysis of conservation across clinical isolates

  • Evaluation of surface exposure in intact bacteria

• Immunization studies:

  • Recombinant protein formulations with appropriate adjuvants

  • Design of fusion proteins combining multiple antigens

  • Testing protein-conjugate formulations similar to successful approaches used with capsular polysaccharides

• Challenge models:

  • Evaluation of protection in various infection models

  • Assessment of correlates of protection (antibody titers, cellular responses)

  • Analysis of bacterial clearance mechanisms

Recent failures in S. aureus vaccine development suggest that single-antigen approaches may be insufficient. A multi-component strategy incorporating UPF0754 membrane proteins alongside other antigens could provide broader protection. Particularly promising is the "designer" glycoconjugate approach where bacterial proteins (like USA300HOU_1839) could be conjugated to capsular polysaccharides from the same organism, potentially increasing immunogenicity compared to traditional conjugates using carrier proteins from unrelated bacteria .

What are the key considerations for developing antibodies against USA300HOU_1839?

Development of high-quality antibodies against USA300HOU_1839 requires special considerations due to its membrane-embedded nature:

• Antigen preparation strategies:

  • Use of peptide antigens from predicted extracellular loops

  • Purification of full-length protein in detergent micelles

  • Preparation of proteoliposomes displaying native conformation

• Immunization protocols:

  • Multiple boost strategies to enhance response against weakly immunogenic epitopes

  • Use of specialized adjuvants suited for membrane protein antigens

  • Prime-boost strategies combining different antigen formats

• Screening methodologies:

  • ELISA using properly folded protein in detergent micelles

  • Flow cytometry against intact bacterial cells

  • Immunofluorescence microscopy to confirm surface localization

• Validation approaches:

  • Western blotting against native and denatured forms

  • Immunoprecipitation under native conditions

  • Functional blocking assays if the protein has known activity

The resulting antibodies can serve multiple research purposes, including localization studies, functional inhibition experiments, and potentially therapeutic applications if the antibodies show opsonizing or neutralizing activity .

What statistical approaches are recommended for analyzing USA300HOU_1839 expression data?

When analyzing expression data for USA300HOU_1839, consider these statistical approaches:

• For qPCR expression analysis:

  • Use 2^-ΔΔCt method for relative quantification

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

  • Implement mixed-effects models for time-course experiments

• For protein expression quantification:

  • Employ densitometry analysis with proper normalization

  • Use non-parametric tests when assumptions of normality cannot be met

  • Apply multiple comparison corrections (e.g., Bonferroni, FDR) when testing many conditions

• For experimental design:

  • Implement power analysis to determine appropriate sample sizes

  • Use randomized block designs to control for batch effects

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

Data should be organized in clear tables following scientific reporting standards, with independent variables (e.g., growth conditions, strain types) in the left column and dependent variables (e.g., expression levels) with corresponding trials in subsequent columns .

How should researchers design experiments to evaluate the effects of environmental conditions on USA300HOU_1839 expression?

To properly evaluate environmental effects on USA300HOU_1839 expression, implement the following experimental design approach:

For each condition:

• Establish time course measurements at 0, 1, 2, 4, 8, and 24 hours

• Include minimum three biological replicates per condition

• Implement factorial designs to detect interaction effects

• Use appropriate statistical analysis (two-way ANOVA, mixed models)

This systematic approach allows for comprehensive characterization of how USA300HOU_1839 expression responds to environmental cues, potentially revealing insights into its physiological role .