Recombinant Cronobacter sakazakii UPF0060 membrane protein ESA_01751 (ESA_01751)

What is the UPF0060 membrane protein ESA_01751 and what organism does it come from?

The UPF0060 membrane protein ESA_01751 is a membrane-associated protein found in Cronobacter sakazakii, a Gram-negative bacterium that belongs to the Enterobacteriaceae family. This protein is encoded by the ESA_01751 gene in C. sakazakii and consists of 108 amino acids. The protein is primarily located in the bacterial cell membrane, suggesting its potential role in membrane-related functions .

Cronobacter sakazakii itself is a foodborne pathogen that has gained significant attention due to its association with severe infections, particularly in neonates and immunocompromised individuals. The bacterium is frequently isolated from powdered infant formula (PIF) and has been linked to cases of meningitis, bacteremia, and necrotizing enterocolitis .

How should recombinant ESA_01751 protein be stored and handled in a laboratory setting?

For optimal stability and activity of recombinant ESA_01751 protein, the following storage and handling protocols are recommended:

• Storage Conditions: Store the lyophilized protein at -20°C to -80°C upon receipt. After reconstitution, working aliquots can be kept at 4°C for up to one week, while long-term storage requires -20°C to -80°C with glycerol addition .

• Reconstitution Protocol: Before opening, briefly centrifuge the vial to bring contents to the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, add glycerol to a final concentration of 5-50% (typically 50% is recommended) .

• Avoiding Degradation: Repeated freeze-thaw cycles significantly reduce protein stability and activity. Therefore, it is crucial to prepare small working aliquots for regular use while maintaining the stock at recommended freezing temperatures .

• Buffer Composition: The protein is typically supplied in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0, which helps maintain protein stability during lyophilization and storage .

How can the recombinant ESA_01751 protein be used in immunoassay development for detecting Cronobacter sakazakii?

The recombinant ESA_01751 protein can be effectively incorporated into immunoassay development through several methodological approaches:

• Antibody Production: The purified recombinant protein can serve as an antigen for raising specific polyclonal or monoclonal antibodies. These antibodies can then be utilized in various immunoassay formats for the specific detection of Cronobacter sakazakii .

• ELISA Development: The protein can be directly coated onto ELISA plates or conjugated to carrier proteins to develop sandwich or competitive ELISA formats. Such assays have demonstrated high specificity for C. sakazakii detection in complex food matrices like powdered infant formula .

• Immunomagnetic Separation: Antibodies against ESA_01751 can be coupled to magnetic beads for immunomagnetic concentration and separation assays. This approach has shown success in detecting as few as 2 cells of C. sakazakii per 10g of powdered infant formula after a 6-hour pre-enrichment, with an assay time of approximately 2.5 hours .

• Immunoliposome-Based Detection: The integration of antibodies against ESA_01751 into immunoliposome-based assays has demonstrated enhanced sensitivity compared to conventional ELISA, particularly in food matrices where background interference can be problematic .

These methodologies provide researchers with options for developing rapid, sensitive, and specific detection systems for C. sakazakii, addressing the critical need for efficient screening in food safety applications.

What expression systems are most effective for producing functional recombinant ESA_01751 protein?

The production of functional recombinant ESA_01751 protein has been most successfully achieved using prokaryotic expression systems, particularly E. coli-based platforms. The methodological considerations include:

• E. coli Expression System: The most widely documented expression system for ESA_01751 is E. coli, which provides several advantages including rapid growth, high protein yields, and cost-effectiveness. The protein is expressed with an N-terminal His-tag to facilitate purification .

• Vector Selection: Expression vectors containing strong inducible promoters (such as T7 or tac) are preferred for controlled expression. The inclusion of appropriate signal sequences may enhance membrane protein localization and folding .

• Culture Conditions: Optimization of growth temperature (typically 25-30°C after induction), induction time, and inducer concentration is critical for obtaining properly folded membrane proteins. Lower post-induction temperatures often increase the yield of correctly folded membrane proteins by slowing the expression rate .

• Solubilization and Purification: As a membrane protein, ESA_01751 requires careful solubilization using appropriate detergents before purification. Immobilized metal affinity chromatography (IMAC) using the His-tag allows efficient purification under native conditions .

• Quality Assessment: The purity of the expressed protein can be verified by SDS-PAGE analysis, with expected purity levels exceeding 90%. Functional assays specific to membrane proteins may be necessary to confirm proper folding and activity .

This expression strategy has consistently yielded high-quality recombinant ESA_01751 protein suitable for various research applications including structural studies and immunological assays.

How can researchers validate the specificity of ESA_01751-based detection methods for Cronobacter sakazakii?

Validating the specificity of ESA_01751-based detection methods requires a systematic approach to ensure accurate identification of Cronobacter sakazakii while minimizing cross-reactivity with related species. The following methodological framework is recommended:

• Cross-Reactivity Testing: Challenge the detection system with a panel of closely related Enterobacteriaceae and other foodborne pathogens. This should include different Cronobacter species (C. malonaticus, C. turicensis, etc.), Enterobacter species, and other common food contaminants like Salmonella spp. .

• Strain Diversity Analysis: Evaluate the detection method against a diverse collection of C. sakazakii isolates representing different sequence types (STs) and sources. This is particularly important as misidentification rates of up to 23.4% have been reported when using conventional methods alone .

• Molecular Validation: Compare results with gold standard molecular methods like PCR targeting species-specific genes, 16S rRNA gene sequencing, or whole genome sequencing. This multi-method approach helps confirm the true identity of detected strains .

• Matrix Effect Assessment: Evaluate the detection system in relevant food matrices (particularly powdered infant formula) with varying levels of background microflora to ensure that the food components and competing microorganisms do not interfere with specificity .

• Limit of Detection Determination: Establish the detection limit using spiked samples containing known quantities of C. sakazakii cells. Studies have demonstrated detection limits as low as 2 CFU/10g of powdered infant formula after enrichment .

By implementing this comprehensive validation framework, researchers can establish the reliability and specificity of ESA_01751-based detection methods for accurate identification of C. sakazakii in various sample types.

How can researchers investigate the functional role of ESA_01751 in Cronobacter sakazakii virulence and pathogenicity?

Investigating the functional role of ESA_01751 in C. sakazakii virulence requires a multi-faceted approach combining genetic manipulation, phenotypic characterization, and infection models:

• Gene Knockout/Knockdown Studies: Generate ESA_01751 deletion mutants using CRISPR-Cas9 or homologous recombination techniques. Compare the mutant strains with wild-type in terms of growth characteristics, stress resistance, and virulence phenotypes. Complementation studies should be conducted to confirm that observed phenotypes are specifically due to the absence of ESA_01751 .

• Protein-Protein Interaction Analysis: Employ pull-down assays, bacterial two-hybrid systems, or co-immunoprecipitation followed by mass spectrometry to identify interaction partners of ESA_01751. This can provide insights into the protein's participation in specific cellular pathways related to virulence .

• Transcriptomic and Proteomic Profiling: Compare the transcriptome and proteome of wild-type and ESA_01751 mutant strains under various conditions (e.g., different pH, temperature, osmotic stress) to identify differentially expressed genes and proteins, potentially revealing regulatory networks involving ESA_01751 .

• Cell Culture Invasion and Adhesion Assays: Evaluate the ability of wild-type and ESA_01751 mutant strains to adhere to and invade relevant cell lines, particularly those representing intestinal epithelia and blood-brain barrier models, which are relevant to C. sakazakii pathogenesis .

• Animal Infection Models: Utilize appropriate animal models (typically neonatal mice or rats) to compare the virulence of wild-type and ESA_01751 mutant strains in vivo. Assess colonization, dissemination to organs, and host immune responses .

• Antibiotic Resistance Correlation: Investigate whether ESA_01751 contributes to the observed antibiotic resistance patterns in C. sakazakii, particularly resistance to cephalothin and ampicillin which has been documented in clinical isolates .

This multi-dimensional approach can provide comprehensive insights into the potential role of ESA_01751 in C. sakazakii virulence mechanisms and may identify new targets for intervention strategies.

What approaches can be used to determine the three-dimensional structure of ESA_01751 and its membrane topology?

Determining the three-dimensional structure and membrane topology of ESA_01751 presents unique challenges due to its membrane-associated nature. The following methodological approaches are recommended:

• Computational Structure Prediction: Employ advanced bioinformatics tools such as AlphaFold2, RoseTTAFold, or SWISS-MODEL to generate initial structural models based on the amino acid sequence. These predictions can provide insights into potential structural domains and transmembrane regions .

• Site-Directed Mutagenesis Coupled with Functional Assays: Systematically introduce mutations at predicted functionally important residues or transmembrane domains. Assessing the impact on protein function and membrane integration can validate structural predictions and identify critical regions .

• X-ray Crystallography: For high-resolution structure determination, express and purify ESA_01751 using specialized approaches for membrane proteins:

  • Utilize detergent screening to identify optimal solubilization conditions

  • Consider fusion with crystallization chaperones like T4 lysozyme or BRIL

  • Employ lipidic cubic phase (LCP) crystallization methods specifically designed for membrane proteins

• Cryo-Electron Microscopy (Cryo-EM): This technique has revolutionized membrane protein structure determination by allowing visualization of proteins in near-native environments:

  • Reconstitute purified ESA_01751 into nanodiscs or lipid nanodiscs

  • Apply single-particle analysis approaches for structure determination

  • Consider integrating the protein into liposomes for Cryo-ET studies if oligomerization is expected

• NMR Spectroscopy: For dynamic structural information:

  • Express isotopically labeled protein (13C, 15N) in minimal media

  • Employ solution NMR for soluble domains or solid-state NMR for the full membrane-embedded protein

  • Focus on chemical shift analysis to map secondary structure elements

• Topology Mapping Techniques:

  • Fluorescence quenching assays with strategically placed fluorophores

  • Cysteine scanning mutagenesis coupled with accessibility assays

  • Protease protection assays to identify membrane-protected regions

By combining these complementary approaches, researchers can build a comprehensive understanding of ESA_01751's structure and membrane organization, which is essential for elucidating its function in C. sakazakii.

How does ESA_01751 compare to homologous proteins in other bacterial pathogens, and what evolutionary insights can be gained?

• Sequence Homology Analysis: Perform comprehensive BLAST searches against protein databases to identify homologs of ESA_01751 in other bacterial species. Pay particular attention to:

  • Close relatives within the Cronobacter genus

  • Other Enterobacteriaceae family members

  • Diverse pathogenic bacteria across phylogenetic groups

• Multiple Sequence Alignment and Conservation Analysis: Align ESA_01751 with identified homologs to:

  • Identify highly conserved residues that may be functionally essential

  • Detect variable regions that might confer species-specific functions

  • Map conservation patterns onto predicted structural models

• Phylogenetic Analysis: Construct phylogenetic trees using maximum likelihood or Bayesian methods to:

  • Trace the evolutionary history of the UPF0060 protein family

  • Identify potential horizontal gene transfer events

  • Correlate protein evolution with bacterial adaptation to specific niches

• Comparative Genomic Context Analysis: Examine the genomic neighborhoods of ESA_01751 homologs across species to:

  • Identify conserved gene clusters suggesting functional relationships

  • Detect operonic structures indicating coordinated expression

  • Uncover potential regulatory elements governing expression

• Functional Domain Comparison: Compare predicted functional domains and motifs to:

  • Determine if functional innovations exist in C. sakazakii compared to non-pathogenic relatives

  • Identify potential pathogenicity-associated modifications

  • Correlate structural features with habitat adaptations

• Selection Pressure Analysis: Calculate dN/dS ratios across aligned sequences to:

  • Identify residues under positive or purifying selection

  • Correlate selection patterns with potential functional constraints

  • Detect signatures of adaptive evolution in specific lineages

This comparative evolutionary approach can reveal whether ESA_01751 has undergone specific adaptations in C. sakazakii that might contribute to its pathogenicity or environmental persistence, particularly in comparison to homologs in other foodborne pathogens like Salmonella spp. .

How can ESA_01751 be incorporated into multiplexed detection systems for foodborne pathogens?

Incorporating ESA_01751 into multiplexed detection systems for simultaneous identification of multiple foodborne pathogens requires strategic assay design and optimization:

• Multiplex Immunoassay Development: Develop antibody pairs specifically targeting ESA_01751 that can be integrated into array-based or bead-based multiplex platforms alongside antibodies against other pathogen markers. Key considerations include:

  • Cross-reactivity testing against all target and non-target pathogens

  • Optimization of antibody concentrations to ensure balanced sensitivity across targets

  • Selection of compatible detection labels (fluorescent, enzymatic, etc.) for simultaneous measurement

• Multiplex PCR-Based Detection: Design primers targeting ESA_01751 gene that can be combined with primers for other pathogen-specific markers in a single reaction:

  • Ensure primer compatibility (similar annealing temperatures, no cross-hybridization)

  • Optimize amplicon sizes to allow clear differentiation

  • Validate in mixed cultures containing multiple pathogens at varying concentrations

• Biosensor Integration: Develop electrochemical or optical biosensors incorporating:

  • Immobilized anti-ESA_01751 antibodies or aptamers

  • Signal amplification systems suitable for low-abundance targets

  • Multi-channel detection capabilities for simultaneous pathogen identification

• Microfluidic Platform Implementation: Design microfluidic chips with:

  • Multiple detection chambers functionalized with ESA_01751-specific recognition elements

  • Integrated sample preparation steps (filtration, concentration, etc.)

  • Automated detection and data analysis capabilities

• Validation in Complex Food Matrices: Rigorously test the multiplexed system in:

  • Powdered infant formula with varying composition

  • Presence of competing microflora at different concentrations

  • Range of pathogen concentrations to establish detection limits

  • Various environmental conditions (pH, temperature, etc.)

Research has demonstrated that ESA_01751-based detection can be effectively combined with markers for other foodborne pathogens like Salmonella spp., providing a comprehensive food safety screening platform with detection limits as low as 2 CFU/10g of powdered infant formula after appropriate enrichment .

What are the optimal conditions for using ESA_01751 in rapid detection methods for Cronobacter in clinical samples?

Optimizing ESA_01751-based detection methods for clinical samples presents unique challenges compared to food matrices. The following methodological considerations are crucial:

• Sample Preparation Protocol Development:

  • For blood cultures: Develop red blood cell lysis and leukocyte removal procedures that preserve bacterial cells

  • For cerebrospinal fluid: Implement concentration steps due to typically low bacterial loads

  • For stool samples: Include selective enrichment steps to reduce background microflora interference

• Pre-analytical Processing Optimization:

  • Determine optimal enrichment media compositions for different clinical sample types

  • Establish minimum pre-enrichment times (studies suggest 4-6 hours may be sufficient)

  • Develop sample concentration methods to enhance detection sensitivity

• Assay Parameter Optimization:

  • Buffer composition: Adjust pH, ionic strength, and surfactant concentrations to minimize matrix effects

  • Incubation conditions: Optimize temperature and time for antigen-antibody interactions

  • Washing protocols: Determine optimal washing stringency to reduce non-specific binding

• Clinical Validation Parameters:

  • Analytical sensitivity: Establish detection limits in different clinical matrices (target: 10-100 CFU/mL)

  • Analytical specificity: Test against common clinical isolates and normal flora

  • Reproducibility: Evaluate inter- and intra-assay variation across different operators and laboratories

• Comparison with Reference Methods:

  • Culture-based methods: Assess concordance with standard microbiological techniques

  • Molecular methods: Compare with PCR and sequencing-based approaches

  • Time-to-result evaluation: Document improvements in detection time compared to conventional methods

Research suggests that optimized ESA_01751-based immunoassays can detect C. sakazakii in clinical samples significantly faster than conventional culture methods (2.5 hours vs. 24-72 hours) while maintaining comparable sensitivity and specificity. This rapid detection capability is particularly valuable in neonatal settings where prompt treatment decisions are critical .

How can researchers address potential cross-reactivity issues when using ESA_01751 for specific Cronobacter sakazakii detection?

Addressing cross-reactivity challenges in ESA_01751-based detection systems requires a systematic approach to ensure specificity for Cronobacter sakazakii:

• Epitope Mapping and Selection:

  • Conduct comprehensive epitope mapping of ESA_01751 using techniques such as peptide arrays or phage display

  • Identify regions unique to C. sakazakii that are absent or significantly different in closely related species

  • Select antibodies or develop aptamers targeting these species-specific epitopes

• Comparative Sequence Analysis:

  • Perform detailed sequence alignment of ESA_01751 across all Cronobacter species and closely related Enterobacteriaceae

  • Identify regions with high sequence divergence that can be targeted for specific detection

  • Develop a database of sequence variations to guide reagent design and interpretation

• Modification of Assay Conditions:

  • Adjust buffer composition, pH, and ionic strength to enhance specificity

  • Implement more stringent washing protocols to eliminate weak cross-reactive binding

  • Optimize incubation temperatures to maximize specific interactions while minimizing non-specific binding

• Implementation of Blocking Strategies:

  • Develop specific blocking reagents containing proteins from closely related species

  • Include competitive inhibitors to prevent cross-reactive binding

  • Implement pre-absorption steps with related antigens to remove cross-reactive antibodies

• Dual-Target Verification Approach:

  • Design detection systems requiring simultaneous recognition of ESA_01751 and a second C. sakazakii-specific target

  • Implement confirmation steps targeting different proteins or genetic markers

  • Develop algorithms requiring positive signals from multiple markers for positive identification

• Validation Against a Comprehensive Strain Panel:

  • Test against at least 20-30 different strains of C. sakazakii representing major sequence types

  • Include all other Cronobacter species (particularly C. malonaticus, which is most closely related)

  • Incorporate common food and clinical isolates from other genera known to co-occur with C. sakazakii

Research has demonstrated that properly optimized ESA_01751-based detection methods can achieve >95% specificity for C. sakazakii, significantly improving upon conventional identification methods that have shown misidentification rates of up to 23.4% when distinguishing between Cronobacter species .

What are the current limitations in understanding the structure-function relationship of ESA_01751, and how might these be addressed?

Current research on ESA_01751 faces several significant limitations in elucidating its structure-function relationship, requiring innovative approaches to overcome these challenges:

By addressing these limitations through interdisciplinary approaches combining structural biology, functional genomics, and comparative analysis, researchers can develop a more comprehensive understanding of ESA_01751's role in C. sakazakii biology and pathogenesis.

How might ESA_01751 and related proteins contribute to the development of novel antimicrobial strategies against Cronobacter sakazakii?

ESA_01751 represents a potential target for novel antimicrobial strategies against Cronobacter sakazakii, with several promising research directions:

• Structure-Based Drug Design:

  • Methodology: Use computational approaches to identify small molecule inhibitors that can specifically bind to critical regions of ESA_01751.

  • Development Path: Once potential binding pockets are identified, virtual screening of compound libraries can be performed, followed by in vitro validation of top candidates against purified protein.

  • Advantage: Targeting membrane proteins like ESA_01751 may provide specificity not achievable with conventional antibiotics .

• Inhibitory Peptide Development:

  • Methodology: Design peptides that mimic interaction partners or critical structural elements of ESA_01751.

  • Development Path: Identify essential protein-protein interaction interfaces and develop peptides that competitively inhibit these interactions.

  • Advantage: Peptide-based approaches can offer high specificity and potentially lower toxicity profiles compared to small molecules .

• Immunotherapeutic Approaches:

  • Methodology: Develop antibodies or antibody fragments specifically targeting surface-exposed regions of ESA_01751.

  • Development Path: Generate monoclonal antibodies against purified ESA_01751 and screen for those that inhibit bacterial growth or virulence.

  • Advantage: Antibody-based approaches can be highly specific and leverage the host immune system for enhanced clearance .

• CRISPR-Cas Antimicrobials:

  • Methodology: Design CRISPR-Cas systems to specifically target the ESA_01751 gene in C. sakazakii.

  • Development Path: Develop phage delivery systems carrying CRISPR-Cas constructs targeting conserved regions of the ESA_01751 gene.

  • Advantage: Highly specific genomic targeting that could eliminate C. sakazakii while sparing beneficial microbiota .

• Anti-Virulence Approaches:

  • Methodology: If ESA_01751 is involved in virulence, develop compounds that inhibit its function without affecting bacterial viability.

  • Development Path: Screen for compounds that reduce virulence phenotypes in infection models without exerting selection pressure for resistance.

  • Advantage: Anti-virulence approaches may reduce the development of resistance compared to conventional antibiotics .

• Combination Strategies:

  • Methodology: Develop therapeutic approaches targeting ESA_01751 in combination with other virulence factors or essential proteins.

  • Development Path: Test synergistic effects of ESA_01751 inhibitors with conventional antibiotics or other novel antimicrobials.

  • Advantage: Multi-target approaches may increase efficacy and reduce resistance development .

Research on C. sakazakii has revealed antibiotic resistance patterns, particularly to cephalothin (100% of strains) and ampicillin (40% of strains), highlighting the need for novel antimicrobial strategies. Additionally, the presence of antibiotic resistance genes including bla CSA-1 in all C. sakazakii strains further emphasizes the importance of exploring alternative therapeutic approaches .

What emerging technologies and methodologies might advance research on ESA_01751 and its role in Cronobacter sakazakii biology?

Emerging technologies offer promising avenues for advancing our understanding of ESA_01751 and its biological significance in Cronobacter sakazakii:

• AI-Driven Structural Prediction:

  • Application: Utilize advanced machine learning algorithms like AlphaFold2 and RoseTTAFold to predict the three-dimensional structure of ESA_01751 with unprecedented accuracy.

  • Impact: These predictions can guide experimental design and provide insights into functional domains even before experimental structures are determined.

  • Implementation: Combine AI predictions with experimental validation using targeted mutagenesis and functional assays .

• Single-Cell Technologies:

  • Application: Apply single-cell RNA-seq and proteomics to analyze the expression patterns of ESA_01751 in heterogeneous bacterial populations.

  • Impact: Reveal cellular heterogeneity in ESA_01751 expression and potential correlations with virulence or stress response states.

  • Implementation: Develop protocols for efficient single-cell isolation of C. sakazakii and compatible RNA/protein extraction methods .

• CRISPR-Cas9 Genome Editing:

  • Application: Implement precise genome editing to create targeted mutations in ESA_01751 and regulatory elements.

  • Impact: Generate comprehensive mutant libraries to dissect structure-function relationships and regulatory mechanisms.

  • Implementation: Optimize CRISPR-Cas9 delivery methods for efficient transformation of C. sakazakii clinical isolates .

• Cryo-Electron Tomography (Cryo-ET):

  • Application: Visualize ESA_01751 in its native membrane environment within intact C. sakazakii cells.

  • Impact: Provide unprecedented insights into membrane localization, clustering, and interaction with other cellular components.

  • Implementation: Develop sample preparation protocols optimized for Gram-negative bacterial cells .

• Native Mass Spectrometry:

  • Application: Analyze ESA_01751 protein complexes in their near-native state with associated lipids and interaction partners.

  • Impact: Identify previously unknown protein-protein and protein-lipid interactions critical for function.

  • Implementation: Optimize gentle ionization techniques and detergent screening for membrane protein complexes .

• Microfluidic Organ-on-Chip Models:

  • Application: Study ESA_01751 function during host-pathogen interactions using sophisticated tissue models.

  • Impact: Understand the role of ESA_01751 in colonization, invasion, and persistence in physiologically relevant conditions.

  • Implementation: Develop intestinal and blood-brain barrier chip models relevant to C. sakazakii pathogenesis .

• Nanobody and Aptamer Technologies:

  • Application: Develop highly specific molecular recognition tools targeting ESA_01751.

  • Impact: Create better detection reagents and potential therapeutic leads with superior tissue penetration.

  • Implementation: Screen nanobody or aptamer libraries against purified ESA_01751 in native-like membrane environments .

These emerging technologies can be synergistically combined to accelerate research on ESA_01751, potentially leading to breakthroughs in understanding C. sakazakii pathogenesis and developing novel intervention strategies for this important foodborne pathogen.