Recombinant Staphylococcus aureus UPF0754 membrane protein SAUSA300_1796 (SAUSA300_1796)

What is Recombinant Staphylococcus aureus UPF0754 membrane protein SAUSA300_1796?

Recombinant Staphylococcus aureus UPF0754 membrane protein SAUSA300_1796 is a membrane-associated protein expressed in Staphylococcus aureus strain USA300. This protein is encoded by the SAUSA300_1796 gene, with the UniProt accession number Q2FFP9. The full-length protein consists of 374 amino acids and is part of the UPF0754 protein family, a group of proteins with currently uncharacterized functions. The protein is predominantly hydrophobic with multiple transmembrane domains, suggesting its integration within the bacterial cell membrane .

What are the optimal storage conditions for this recombinant protein?

For optimal preservation of protein integrity and activity, store the recombinant UPF0754 membrane protein at -20°C. For extended storage periods, it is recommended to conserve the protein at -20°C or -80°C. The protein is typically supplied in a Tris-based buffer containing 50% glycerol, optimized specifically for this protein's stability. Importantly, repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of activity. For short-term use, working aliquots can be stored at 4°C for up to one week . This approach minimizes protein degradation while maintaining accessibility for ongoing experiments.

What expression systems can be used to produce this protein?

The choice of expression system should be guided by the intended application, with consideration for required protein authenticity, yield, and experimental timeline. For structural studies requiring large quantities of protein, E. coli systems might be preferable, while functional studies may benefit from insect or mammalian cell expression to maintain native protein conformation and activity.

How does the UPF0754 membrane protein relate to S. aureus pathogenicity and persistence?

The UPF0754 membrane protein may play a significant role in S. aureus pathogenicity, particularly in the context of intracellular persistence within host cells. S. aureus USA300, which expresses this membrane protein, demonstrates remarkable ability to survive and persist inside macrophages, even establishing small colony variants (SCVs) under various stress conditions . Research indicates that S. aureus USA300 can be internalized by RAW 264.7 murine macrophages and form SCVs as an adaptation mechanism.

Evidence suggests that during internalization, S. aureus undergoes significant changes in gene expression, particularly in genes related to adhesion, virulence, and metabolism. While direct experimental evidence specifically linking UPF0754 to these processes is limited in the available literature, as a membrane protein, it potentially participates in bacterial adaptation to the intracellular environment. The protein's structure suggests possible roles in signal transduction, transport, or membrane integrity maintenance during phagocytosis and intracellular residence .

What methodologies can be used to study the role of UPF0754 in small colony variant formation?

Several sophisticated methodologies can be employed to investigate the potential role of UPF0754 in small colony variant (SCV) formation:

• Targeted Gene Disruption: Using techniques such as CRISPR-Cas9 or transposon mutagenesis to create UPF0754 knockout strains, followed by assessment of SCV formation capabilities under stress conditions.

• Macrophage Infection Models: Experimental protocols using RAW 264.7 murine macrophages can be implemented, as demonstrated in previous studies. These macrophages can be infected with wild-type and UPF0754-deficient S. aureus strains, with subsequent quantification of intracellular survival and SCV formation over time .

• Comparative Genomics: Performing whole genome sequencing of wild-type S. aureus and its SCV derivatives to identify mutations potentially involving UPF0754 or interacting partners. This approach has previously identified significant mutations in genes such as aroK, purR, and intergenic regions that contribute to the SCV phenotype .

• Transcriptomics Analysis: Quantitative PCR (qPCR) or RNA-seq can be used to analyze expression changes in UPF0754 and related genes during SCV formation. Previous studies have shown differential expression of genes involved in transcriptional regulation (sarA, agrA), virulence (psmA, hla, hld), and metabolism (pdhA, fumC, uhpt) in SCVs compared to wild-type strains .

How can researchers assess the membrane localization and topology of UPF0754?

Determining the precise membrane localization and topology of UPF0754 requires a multi-faceted experimental approach:

What is the relationship between UPF0754 and phagosomal maturation in macrophages?

S. aureus has the ability to subvert normal phagosomal maturation in macrophages, establishing an intracellular niche. Research indicates that S. aureus USA300 strain JE2 phagosomes progress to a late phagosome state but fail to mature to a phagolysosome state and do not acidify appropriately . While the specific role of UPF0754 in this process remains to be fully elucidated, several experimental approaches can be used to investigate its potential involvement:

• Fluorescent pH Indicators: Using pH-sensitive fluorescent probes to monitor phagosomal acidification in macrophages infected with wild-type versus UPF0754-deficient S. aureus strains.

• Immunofluorescence Colocalization Studies: Employing antibodies against UPF0754 and various phagosomal markers (e.g., LAMP-1, Rab7) to determine whether the protein localizes to the phagosomal membrane during infection.

• High-throughput Microscopy Screening: Implementing automated microscopy protocols to screen libraries of bacterial mutants, including those affecting UPF0754, for alterations in phagosomal acidification patterns .

• Transcriptional Response Analysis: Analyzing host cell transcriptional responses to infection with wild-type versus UPF0754-deficient strains to identify differences in phagosome-related gene expression.

Understanding the relationship between UPF0754 and phagosomal maturation could provide valuable insights into S. aureus intracellular persistence mechanisms and potentially reveal new therapeutic targets.

How should researchers design experiments to study UPF0754 functional interactions?

Designing robust experiments to study UPF0754 functional interactions requires a comprehensive approach:

• Bacterial Two-Hybrid Assays: Implement bacterial two-hybrid systems to screen for potential protein-protein interactions involving UPF0754. This method can identify both bacterial and potentially host interaction partners.

• Co-Immunoprecipitation with Tagged Variants: Express epitope-tagged UPF0754 variants in S. aureus, followed by immunoprecipitation and mass spectrometry to identify proteins that physically interact with UPF0754 under different environmental conditions.

• Cross-linking Studies: Perform in vivo cross-linking followed by mass spectrometry (XL-MS) to capture transient interactions that might occur during infection or stress responses.

• Synthetic Genetic Arrays: Create a library of S. aureus strains with paired mutations (UPF0754 plus another gene) to identify genetic interactions through growth phenotypes, particularly under conditions that mimic the intracellular environment.

• Differential RNA-seq Analysis: Compare transcriptional profiles between wild-type and UPF0754 mutant strains under various conditions to identify genes whose expression is influenced by UPF0754, suggesting functional relationships.

• Protein Structure Prediction and Docking: Utilize computational approaches to predict the three-dimensional structure of UPF0754 and perform in silico docking studies with potential interaction partners identified through experimental methods.

These complementary approaches can provide a comprehensive understanding of UPF0754's functional interactions within the bacterial cell and potentially with host components during infection.

What are the challenges in purifying functional recombinant UPF0754 and how can they be addressed?

Purification of functional membrane proteins presents several technical challenges, and UPF0754 is no exception. Researchers should consider the following challenges and strategies:

• Solubilization Optimization:

  • Challenge: Membrane proteins require detergents for solubilization, but harsh detergents can denature the protein.

  • Solution: Screen a panel of mild detergents (e.g., DDM, LDAO, CHAPS) at different concentrations to identify conditions that maximize protein extraction while preserving structure and function.

• Expression System Selection:

  • Challenge: Traditional E. coli systems may not properly fold complex membrane proteins.

  • Solution: Consider alternative expression systems such as insect cells for proper post-translational modifications and protein folding . For initial screening, E. coli and yeast systems offer higher yields but potentially at the cost of protein quality.

• Protein Stability:

  • Challenge: UPF0754 may be unstable once removed from its native membrane environment.

  • Solution: Incorporate stabilizing agents in buffers (glycerol, specific lipids) and consider reconstitution into nanodiscs or liposomes to provide a membrane-like environment.

• Verification of Functionality:

  • Challenge: Assessing whether the purified protein retains its native function.

  • Solution: Develop functional assays based on predicted activities (e.g., transport assays if it's a transporter, binding assays if it's a receptor). In the absence of known function, assess structural integrity through circular dichroism or thermal shift assays.

• Protein Yield:

  • Challenge: Membrane proteins often express at lower levels than soluble proteins.

  • Solution: Optimize expression conditions (temperature, induction time, media composition) and consider fusion tags that enhance expression and solubility.

• Storage Conditions:

  • Challenge: Maintaining protein stability during storage.

  • Solution: Store aliquots at -20°C or -80°C in buffer containing 50% glycerol to prevent freeze-thaw damage, and avoid repeated freezing and thawing cycles .

How can researchers effectively analyze the role of UPF0754 in S. aureus intracellular survival?

To comprehensively analyze UPF0754's role in S. aureus intracellular survival, researchers should implement a multi-dimensional experimental approach:

• Genetic Manipulation:

  • Generate clean deletion mutants and complemented strains of UPF0754

  • Create point mutations in specific domains to identify critical functional regions

  • Develop inducible expression systems to control protein levels during infection

• Cellular Infection Models:

  • Utilize RAW 264.7 murine macrophages as established infection models

  • Expand to primary human macrophages and other relevant cell types

  • Implement time-course analyses of bacterial survival with wild-type versus mutant strains

• Microscopy Techniques:

  • Apply high-throughput microscopy screening methods to assess phagosomal acidification

  • Use fluorescence microscopy with pH-sensitive dyes to track bacterial compartmentalization

  • Implement super-resolution microscopy to visualize UPF0754 localization during infection

• Transcriptional Profiling:

  • Perform RNA-seq on both bacteria and host cells during infection

  • Compare expression patterns between wild-type and UPF0754-deficient strains

  • Focus on genes involved in stress response, virulence, and metabolic adaptation

• Small Colony Variant Analysis:

  • Assess the frequency of SCV formation in UPF0754 mutants versus wild-type

  • Characterize the stability of SCVs derived from different genetic backgrounds

  • Analyze genomic changes in SCVs using whole genome sequencing approaches

• Metabolic Studies:

  • Investigate changes in bacterial metabolism during intracellular residence

  • Examine the potential role of UPF0754 in nutrient acquisition or stress adaptation

  • Consider how membrane protein function might influence metabolic pathways critical for survival

This comprehensive approach integrates multiple techniques to elucidate UPF0754's specific contributions to intracellular survival and persistence mechanisms.

How should researchers interpret changes in UPF0754 expression during infection?

Interpreting changes in UPF0754 expression during infection requires careful consideration of multiple factors:

• Temporal Expression Patterns: Changes in UPF0754 expression should be analyzed across different time points during infection. Early upregulation might suggest a role in initial adaptation to the intracellular environment, while later expression changes could indicate involvement in persistence mechanisms. Similar to the observed expression patterns in other genes during S. aureus SCV formation, temporal dynamics can provide insights into the protein's functional role .

• Correlation with Environmental Triggers: Researchers should correlate expression changes with specific environmental conditions (pH changes, oxidative stress, nutrient limitation) to identify potential regulatory mechanisms. S. aureus gene expression is known to adapt significantly to stressors encountered within macrophages .

• Relationship to Other Virulence Factors: Analyze whether UPF0754 expression correlates with known virulence factors such as those regulated by the agr system or sarA. Previous studies have shown significant upregulation of uhpt and sdrE genes in SCVs, suggesting metabolic adaptation patterns that might also involve membrane proteins .

• Host Cell Response Correlation: Cross-reference UPF0754 expression changes with host cell responses to identify potential host-pathogen interaction patterns. This could reveal whether the protein participates in immune evasion mechanisms or adaptation to host defense strategies.

• Strain Variability Considerations: Compare expression patterns across different S. aureus strains to determine whether UPF0754 expression changes represent strain-specific or conserved responses to intracellular environments.

• Regulatory Network Analysis: Place UPF0754 expression changes within the context of global regulatory networks, particularly those involving transcription factors known to regulate membrane proteins and stress responses.

When interpreting expression data, researchers should recognize that correlation does not necessarily imply causation, and functional studies are essential to confirm the biological significance of observed expression changes.

What statistical approaches are most appropriate for analyzing UPF0754 functional studies?

Selecting appropriate statistical approaches for UPF0754 functional studies ensures robust, reproducible, and meaningful data interpretation:

For SCV formation studies specifically, researchers should consider using generalized linear models with appropriate error distributions (e.g., binomial for proportion data). When analyzing genomic data, such as the mutations identified in SCVs (as seen in previous studies with mutations in aroK and purR genes), appropriate corrections for multiple testing should be applied to control false discovery rates .

Sample size calculations should be performed prior to experiments, considering the expected effect size based on preliminary data or similar studies with other membrane proteins in S. aureus. All statistical analyses should be transparently reported, including all data transformations and handling of outliers.

How can contradictory findings about UPF0754 function be reconciled in the literature?

Reconciling contradictory findings about UPF0754 function requires a systematic approach to identify sources of variability and establish consensus:

• Methodological Differences Analysis: Create a comprehensive table comparing experimental methods across studies, including:

  • Bacterial strains and genetic backgrounds

  • Expression systems and purification methods

  • Assay conditions (pH, temperature, buffer composition)

  • Cell types used for infection studies

  • Measurement techniques and endpoints

• Context-Dependent Function Assessment: Consider whether UPF0754 might have different functions depending on:

  • Growth phase or metabolic state of bacteria

  • Environmental conditions during experiments

  • Presence of specific stress factors

  • Interactions with strain-specific factors

• Technical Validation Approaches:

  • Cross-laboratory validation using standardized protocols

  • Developing consensus assays for UPF0754 function

  • Implementing more sensitive or specific detection methods

  • Using multiple complementary techniques to assess function

• Biological Variability Considerations:

  • Evaluate whether contradictions reflect true biological variability

  • Consider post-translational modifications that might differ between studies

  • Assess natural strain variations in UPF0754 sequence and expression

• Integrated Data Analysis:

  • Perform meta-analyses where appropriate

  • Develop computational models that might reconcile seemingly contradictory data

  • Consider how different aspects of UPF0754 function might be revealed under different experimental conditions

• Collaborative Resolution Strategies:

  • Establish research consortia to systematically address contradictions

  • Develop shared resources (antibodies, constructs, protocols)

  • Create public databases of experimental conditions and results

By systematically analyzing sources of variability and implementing rigorous validation approaches, researchers can reconcile contradictory findings and develop a more comprehensive understanding of UPF0754 function in S. aureus biology.

What are the most promising approaches for determining the function of UPF0754?

Several innovative approaches show particular promise for elucidating the function of UPF0754:

• Integrated Structural Biology: Combining X-ray crystallography, cryo-electron microscopy, and NMR spectroscopy to determine the three-dimensional structure of UPF0754, providing insights into its potential molecular function. The amino acid sequence suggests multiple transmembrane domains that could form channels, transporters, or signaling complexes .

• Chemical Genetics Approaches: Using small molecule libraries to identify compounds that specifically affect UPF0754 function, then characterizing the resulting phenotypes to infer the protein's role. This approach is particularly valuable for proteins of unknown function.

• Comparative Genomics and Evolutionary Analysis: Examining the conservation and co-evolution of UPF0754 across bacterial species, particularly focusing on patterns of conservation that might suggest functional domains or interacting partners.

• High-resolution In vivo Imaging: Developing fluorescent reporters to visualize UPF0754 dynamics during infection processes, potentially revealing spatial and temporal patterns associated with specific cellular events.

• CRISPR Interference (CRISPRi) Time-course Studies: Implementing inducible CRISPRi systems to downregulate UPF0754 expression at specific time points during infection, helping to distinguish between roles in initial invasion versus persistence.

• Proteomic Profiling: Conducting comparative proteomics between wild-type and UPF0754-deficient strains under various conditions, particularly focusing on membrane protein complexes that might be affected by UPF0754 absence.

• Metabolic Flux Analysis: Examining how UPF0754 affects cellular metabolism, particularly in the context of adaptation to the intracellular environment where S. aureus is known to undergo significant metabolic reprogramming .

These approaches, particularly when used in combination, offer powerful means to move beyond correlative observations and establish causal relationships between UPF0754 and specific cellular processes in S. aureus.

How might UPF0754 be exploited as a potential therapeutic target?

UPF0754's potential as a therapeutic target merits systematic exploration, given S. aureus's significant clinical impact and the protein's possible role in bacterial persistence:

• Target Validation Strategies:

  • Demonstrate that UPF0754 inhibition reduces bacterial survival in infection models

  • Establish whether UPF0754 is essential for virulence or persistence in vivo

  • Confirm expression of UPF0754 during human infections through clinical samples

• Drug Discovery Approaches:

  • Develop high-throughput screening assays for UPF0754 inhibitors

  • Design structure-based inhibitors once protein structure is determined

  • Consider antisense or CRISPR-based therapeutics to downregulate gene expression

  • Explore antibody-based approaches if portions of the protein are surface-exposed

• Delivery Strategies for Intracellular Targeting:

  • Develop nanoparticle formulations to deliver inhibitors to infected macrophages

  • Explore cell-penetrating peptides as delivery vehicles

  • Consider prodrug approaches that activate in the phagosomal environment

• Resistance Mitigation Strategies:

  • Identify potential resistance mechanisms through laboratory evolution studies

  • Design combination therapies targeting multiple persistence factors

  • Explore adjuvant approaches that might sensitize bacteria to existing antibiotics

• Translational Considerations:

  • Assess inhibitor specificity against human proteins to minimize off-target effects

  • Develop appropriate animal models that recapitulate intracellular persistence

  • Consider pharmacokinetic requirements for reaching intracellular bacteria

What interdisciplinary approaches could accelerate UPF0754 research?

Accelerating UPF0754 research requires innovative interdisciplinary approaches that integrate diverse expertise and methodologies:

• Computational Biology and Artificial Intelligence Integration:

  • Implement machine learning algorithms to predict protein function based on sequence features

  • Develop computational models of membrane protein dynamics in bacterial membranes

  • Use network analysis tools to place UPF0754 within global S. aureus interaction networks

• Synthetic Biology Approaches:

  • Engineer synthetic systems to isolate and study UPF0754 function in simplified contexts

  • Develop orthogonal expression systems to probe function without native regulatory influences

  • Create biosensors that report on UPF0754 activity in real-time during infection

• Systems Biology Integration:

  • Combine transcriptomics, proteomics, and metabolomics to develop comprehensive models of UPF0754's role

  • Map system-wide changes resulting from UPF0754 manipulation

  • Identify emergent properties not apparent from isolated studies

• Advanced Imaging Technologies:

  • Implement super-resolution microscopy to visualize UPF0754 localization and dynamics

  • Use correlative light and electron microscopy to connect protein localization with ultrastructural features

  • Develop live-cell imaging approaches to track UPF0754 during infection processes

• Immunology-Microbiology Interface:

  • Investigate how UPF0754 affects host-pathogen interactions at the molecular level

  • Examine potential recognition of UPF0754 by host immune receptors

  • Study how UPF0754 might influence immune cell function and signaling

• Collaborative Research Networks:

  • Establish international consortia focusing specifically on UPF0754 and related proteins

  • Develop shared resources, protocols, and databases to accelerate research

  • Implement multi-laboratory validation studies to enhance reproducibility

• Clinical Microbiology Connections:

  • Analyze UPF0754 expression in clinical isolates from various infection types

  • Correlate genetic variations in UPF0754 with clinical outcomes

  • Collect infection samples to validate laboratory findings in authentic clinical contexts

These interdisciplinary approaches can overcome traditional research silos, accelerating progress in understanding UPF0754's function and potentially revealing novel therapeutic opportunities.