Recombinant Haemophilus influenzae UPF0114 protein HI_0507 (HI_0507)

What expression systems are recommended for producing recombinant HI_0507?

The recommended expression system for HI_0507 is E. coli with an N-terminal His-tag. This approach has been validated to produce full-length protein (amino acids 1-183) with purity exceeding 90% as confirmed by SDS-PAGE analysis. The expression construct should include:

• A strong inducible promoter (T7 or tac)

• N-terminal His-tag for purification

• Appropriate signal sequence if membrane localization is desired

For optimal expression, consider testing multiple E. coli strains (BL21(DE3), Rosetta, or Origami) as membrane proteins often present expression challenges. Induction parameters should be optimized at lower temperatures (16-25°C) to enhance proper folding .

What purification and storage protocols should be followed for HI_0507?

Purification Protocol:

• Harvest E. coli cells and lyse using appropriate buffer systems containing mild detergents

• Purify using IMAC (Immobilized Metal Affinity Chromatography) leveraging the His-tag

• Further purify via size exclusion chromatography if higher purity is required

• Confirm purity via SDS-PAGE (>90% purity is achievable)

Storage Requirements:

• Store lyophilized protein at -20°C/-80°C upon receipt

• For reconstituted protein, aliquot to avoid freeze-thaw cycles

• Short-term storage (up to one week): 4°C in working buffer

• Long-term storage: Add 5-50% glycerol (optimal: 50%) and store at -20°C/-80°C

• Use Tris/PBS-based buffer with 6% Trehalose, pH 8.0 for storage

How should researchers reconstitute and prepare HI_0507 for experimental use?

For optimal reconstitution of lyophilized HI_0507:

• Briefly centrifuge the vial before opening to collect contents at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• For long-term storage preparations, add glycerol to a final concentration of 5-50% (recommended: 50%)

• Aliquot into single-use volumes to prevent repeated freeze-thaw cycles

• Validate protein integrity via SDS-PAGE before experimental use

When designing experiments, consider that HI_0507 is a membrane protein, which may require specific buffer conditions to maintain proper folding and functionality. For functional assays, perform initial validation experiments to determine optimal protein concentration ranges .

What analytical methods are appropriate for characterizing HI_0507?

When performing SDS-PAGE analysis, which is the most commonly reported method for HI_0507, ensure complete denaturation of the sample and use appropriate molecular weight markers. The expected band should appear at approximately 20-22 kDa (accounting for the His-tag) .

How does HI_0507 relate to other virulence factors in H. influenzae?

While direct evidence for HI_0507's role in virulence is still emerging, comparative analysis with other H. influenzae proteins provides context:

• Protein H (PH): A surface-exposed lipoprotein found in encapsulated H. influenzae type b (Hib) and type f (Hif) that binds factor H, a major regulator of the alternative pathway of complement activation. This interaction is crucial for bacterial resistance against complement activation and pathogenesis .

• HMW1 Adhesin: A key factor in intracellular invasion by H. influenzae, with transformation of the hmw1 operon resulting in ~1,000-fold increased invasion into airway epithelial cells. This protein facilitates bacterial self-aggregation and adherence to airway cells .

Given that HI_0507 is also a membrane-associated protein, researchers should investigate potential interactions with these better-characterized virulence factors, particularly in the context of host-pathogen interactions .

What genetic manipulation strategies can be employed to study HI_0507 function in H. influenzae?

For in-depth functional analysis of HI_0507, consider these genetic approaches:

• Gene Deletion/Knockout: Create lph (gene encoding for HI_0507) deletion mutants using homologous recombination or CRISPR-Cas9 systems. Compare phenotypes with wild-type strains to identify functional roles, similar to approaches used for studying Protein H .

• Transformed Recombinant Enrichment Profiling (TREP): This technique could be particularly valuable, as demonstrated with HMW1. TREP uses natural transformation to generate complex pools of recombinants, followed by phenotypic selection and deep sequencing to identify genetic variations responsible for specific phenotypes .

• Allelic Replacement: Replace the native hi_0507 gene with tagged or mutated versions to study protein domains and their functions.

• Complementation Studies: Reintroduce the wild-type gene in knockout strains to confirm phenotype restoration.

These approaches would help establish the specific role of HI_0507 in H. influenzae biology and potential contributions to pathogenesis .

How can researchers investigate potential interactions between HI_0507 and host factors?

To explore potential host-pathogen interactions:

• Protein-Protein Interaction Studies:

  • Pull-down assays using His-tagged HI_0507

  • Co-immunoprecipitation with potential host targets

  • Surface plasmon resonance to measure binding kinetics

  • Bacterial two-hybrid systems

• Cell Culture Models:

  • Infection assays using respiratory epithelial cell lines

  • Quantification of adhesion, invasion, and intracellular survival

  • Immunofluorescence to track protein localization during infection

  • Flow cytometry to measure host cell responses

• Comparative Analysis:

  • Study HI_0507 alongside known virulence factors like Protein H and HMW1

  • Investigate if HI_0507 contributes to complement evasion similar to Protein H

  • Assess if HI_0507 affects bacterial aggregation or adhesion similar to HMW1

These methodologies would provide insights into whether HI_0507 participates in host-pathogen interactions that contribute to H. influenzae colonization or pathogenesis.

What bioinformatic approaches can predict structure-function relationships for HI_0507?

For computational analysis of HI_0507:

• Sequence-Based Analysis:

  • Multiple sequence alignment with homologs across bacterial species

  • Identification of conserved domains and motifs

  • Prediction of post-translational modifications

  • Signal peptide and transmembrane domain prediction

• Structural Prediction:

  • Ab initio modeling using tools like AlphaFold2 or RoseTTAFold

  • Molecular dynamics simulations to study protein flexibility

  • Protein-protein docking with potential interaction partners

  • Identification of potential ligand binding pockets

• Evolutionary Analysis:

  • Phylogenetic trees to understand evolutionary relationships

  • Analysis of selection pressure on specific residues

  • Identification of co-evolving residues suggesting functional importance

These computational approaches can guide experimental design by highlighting regions of potential functional importance and generating testable hypotheses about HI_0507's role in H. influenzae biology .

How does the structure and function of HI_0507 compare with other characterized H. influenzae membrane proteins?

While detailed structural information about HI_0507 is still emerging, comparative analysis with better-characterized H. influenzae membrane proteins provides valuable context:

Researchers should consider investigating whether HI_0507 shares functional similarities with these better-characterized proteins, particularly in the context of host-pathogen interactions and immune evasion strategies .

What experimental challenges should researchers anticipate when working with HI_0507 compared to other H. influenzae proteins?

Working with membrane proteins like HI_0507 presents specific challenges:

• Solubility Issues: As a membrane protein, HI_0507 may require detergents or specialized buffer systems to maintain solubility and proper folding.

• Structural Determination: Membrane proteins typically present greater challenges for structural studies compared to soluble proteins.

• Functional Assays: Establishing appropriate functional assays may be difficult without clear understanding of the protein's role.

• Expression Optimization: Expression levels may be lower than for soluble proteins, requiring optimization of expression conditions.

By comparison, proteins like Protein H have established functional assays (Factor H binding) and HMW1 has clear phenotypic outcomes (adhesion, invasion) that facilitate their study. Researchers should develop systematic approaches to characterize HI_0507's function through comparative studies with these better-understood proteins .

What are promising research avenues for elucidating HI_0507 function in H. influenzae pathogenesis?

Future research on HI_0507 should consider these approaches:

• Systems Biology Integration:

  • Proteomics to identify interaction partners under different conditions

  • Transcriptomics to determine expression patterns during infection

  • Metabolomics to identify pathway alterations in knockout strains

• In vivo Models:

  • Animal models to compare virulence of wild-type vs. hi_0507 mutants

  • Competitive index assays to measure fitness contributions

  • Tissue-specific expression analysis during infection

• Structural Biology:

  • Cryo-EM or X-ray crystallography to determine 3D structure

  • Structure-guided mutagenesis to identify functional domains

  • Protein dynamics studies using hydrogen-deuterium exchange mass spectrometry

These approaches would contribute to a comprehensive understanding of HI_0507's role in H. influenzae biology and potential contributions to pathogenesis, building on established methodologies used for other virulence factors .

How might clinical isolates differ in HI_0507 sequence and expression, and what implications might this have?

Understanding HI_0507 variation across clinical isolates is crucial:

• Sequence Variation Analysis:

  • Compare hi_0507 sequences across clinical isolates from different:

    • Disease manifestations (invasive vs. non-invasive)

    • Body sites (respiratory tract, blood, CSF)

    • Patient populations (age groups, immunocompetent vs. immunocompromised)

• Expression Level Comparison:

  • Quantify HI_0507 expression in different clinical contexts

  • Identify regulatory mechanisms controlling expression

  • Correlate expression levels with virulence or antibiotic resistance

• Function Comparison:

  • Assess whether sequence variants have altered functions

  • Determine if specific variants correlate with disease severity