Recombinant Haemophilus influenzae Uncharacterized protein HI_1049 (HI_1049)

What is HI_1049 and where is it located in the H. influenzae genome?

HI_1049 is an uncharacterized protein from Haemophilus influenzae, a gram-negative coccobacillus that naturally inhabits the human nasopharynx and can cause both localized and invasive infections. While specific information about HI_1049 is limited in the literature, we can contextualize its importance within the broader understanding of H. influenzae pathogenesis. H. influenzae, particularly encapsulated type b (Hib) strains, can cause severe diseases including meningitis, septicemia, and arthritis, especially in young children . The type b capsule contributes to pathogenesis by inhibiting neutrophil phagocytosis and resisting complement-mediated bactericidal activity, enhancing bloodstream survival .

Based on genomic analyses of H. influenzae, HI_1049 likely represents one of many proteins that could contribute to colonization or virulence, similar to the fimbrial proteins that mediate bacterial adhesion to host tissues. The genomic context suggests possible involvement in extracellular interactions, though direct experimental confirmation is needed to establish its precise cellular localization and function.

What structural and functional predictions can be made about HI_1049?

While experimental structural data for HI_1049 is currently limited, several computational approaches can generate valuable predictions about its potential structure and function:

Given that many H. influenzae proteins interact with extracellular matrix components, analyzing HI_1049 for potential binding motifs similar to those found in fimbrial proteins would be informative. Fimbriated H. influenzae strains show efficient adhesion to mammalian extracellular matrices, which may promote bacterial invasion into the circulation . Computational approaches predicting protein-protein interaction sites could identify regions of HI_1049 potentially involved in adhesion to host proteins like fibronectin or heparin-binding growth-associated molecule (HB-GAM), similar to interactions observed with Hib fimbriae .

Is HI_1049 conserved across different strains of H. influenzae?

Sequence conservation analysis across multiple H. influenzae strains provides critical insight into the evolutionary importance of HI_1049. Highly conserved proteins typically perform essential functions, while strain-specific variations might suggest adaptation to different host environments or virulence strategies.

To address this question methodologically:

• Collect genome sequences from diverse H. influenzae isolates, including both encapsulated (particularly type b) and unencapsulated strains.

• Perform multiple sequence alignment of HI_1049 homologs.

• Calculate conservation scores for each amino acid position.

• Identify conserved domains that might indicate functional importance.

• Map strain-specific variations to clinical or phenotypic characteristics.

Research with other H. influenzae proteins shows that expression patterns can vary during infection. For example, fimbrial expression in Hib isolates is subject to reversible phase variation, with expression changing rapidly during infection . Nasopharyngeal isolates tend to be fimbriated, while bloodstream isolates generally do not express fimbriae, suggesting that expression timing relates to infection stage . Similar phase variation mechanisms could potentially regulate HI_1049 expression.

What expression systems are most suitable for recombinant HI_1049 production?

Selecting an appropriate expression system is crucial for successful recombinant protein production. For HI_1049, several factors should guide this decision:

For initial characterization, E. coli-based systems often provide the best starting point. The recombinant E. coli strain HB101(pMH140) has been used successfully to express Hib fimbrial proteins , suggesting similar approaches might work for HI_1049. When working with E. coli K-12 host-vector systems, research may qualify as exempt under Section III-F-8 of the NIH Guidelines (Appendix C-II) , simplifying regulatory compliance.

Expression conditions should be optimized through factorial design experiments varying temperature, inducer concentration, and media composition to maximize soluble protein yield. If membrane association is predicted, detergent screening would be essential for solubilization and purification.

What purification strategies should be considered for recombinant HI_1049?

Effective purification strategies depend on the physicochemical properties of HI_1049 and the expression system used:

• Affinity chromatography: If expressing with a fusion tag (His6, GST, MBP), this provides a convenient first capture step. For His-tagged HI_1049, immobilized metal affinity chromatography (IMAC) with Ni-NTA or Co-NTA resins would be appropriate.

• Ion exchange chromatography: Based on the predicted isoelectric point (pI) of HI_1049, select appropriate resin (cation exchange for proteins with pI > 7, anion exchange for pI < 7).

• Size exclusion chromatography: As a polishing step and to analyze oligomeric state.

• Heparin affinity chromatography: Given that some H. influenzae proteins bind heparin and heparin-binding proteins , this could be particularly relevant for HI_1049 purification and functional characterization.

The purification protocol should be validated by:

• SDS-PAGE with Coomassie staining (>90% purity)

• Western blotting with tag-specific antibodies

• Mass spectrometry for identity confirmation

• Dynamic light scattering for homogeneity assessment

Similar to the approach used for Hib fimbrial proteins, ammonium sulfate precipitation might be applicable for initial concentration steps . For quality control, ELISA-based protocols similar to those used to test purified Hib fimbriae binding to extracellular matrix proteins could be adapted for HI_1049 functional verification .

What regulatory considerations apply to work with recombinant HI_1049?

Research with recombinant H. influenzae proteins must comply with biosafety regulations and the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules:

• Biosafety Level: H. influenzae typically requires BSL-2 containment.

• NIH Guidelines exemptions: Certain experiments involving recombinant nucleic acids may be exempt under Section III-F of the NIH Guidelines . Specifically:

  • Work in E. coli K-12, Saccharomyces cerevisiae, Saccharomyces uvarum, Kluyveromyces lactis, or Bacillus subtilis host-vector systems may be exempt (Appendix C) .

  • Experimental designs using less than half of any eukaryotic viral genome, when propagated in tissue culture, may qualify as exempt .

• Non-exempt activities: Experiments are NOT exempt if they involve:

  • Deliberate transfer of drug resistance traits to microorganisms that don't naturally acquire them (Section III-A)

  • Formation of recombinant molecules containing toxin genes (Section III-B)

  • Transfer into human research participants (Section III-C)

• Scale considerations: Large-scale experiments (more than 10 liters of culture) are not exempt even if small-scale versions of the same experiment would be .

• Institutional oversight: Institutional Biosafety Committee (IBC) approval is required for non-exempt research.

For recombinant HI_1049 research, using E. coli K-12 derivatives as expression hosts would typically allow exemption under Appendix C-II , provided the experimental design avoids the non-exempt categories listed above.

What approaches can be used to determine the biological function of HI_1049?

Uncharacterized proteins require systematic approaches to elucidate their functions:

For HI_1049, an effective strategy would begin with localization studies to determine if it associates with the bacterial surface, similar to fimbrial proteins that mediate adhesion to host cells . If surface-exposed, binding assays with extracellular matrix proteins would be logical, given that fimbriated H. influenzae strains interact with fibronectin and HB-GAM .

Adhesion assays modeled after those used for fimbriated Hib strains could test whether HI_1049 mediates binding to epithelial cells or extracellular matrix components. These could include glass slide coating experiments with human plasma fibronectin, recombinant HB-GAM protein, and control proteins like bovine serum albumin .

How might HI_1049 relate to H. influenzae virulence or colonization?

To investigate potential roles of HI_1049 in virulence or colonization:

• Comparative genomics approach: Analyze HI_1049 presence/absence or sequence variation across clinical isolates with different virulence profiles.

• Expression analysis: Measure HI_1049 expression levels during:

  • In vitro growth under various conditions (microaerobic, iron limitation, serum exposure)

  • Biofilm formation vs. planktonic growth

  • Interaction with host cells

  • Animal infection models

• Functional assays:

  • Adhesion to respiratory epithelial cells (similar to fimbrial adhesion studies )

  • Biofilm formation capacity

  • Survival in human serum

  • Invasion of epithelial barriers

• Animal models:

  • Colonization of nasopharynx in mouse models

  • Invasive disease in susceptible animal models

  • Competitive index assays (wild-type vs. HI_1049 mutant)

Research on Hib fimbriae shows they mediate bacterial adhesion to oropharyngeal epithelial cells and promote colonization in organ culture models . If HI_1049 has similar functions, we might expect comparable phenotypes in adhesion assays. Notably, even nonfimbriated H. influenzae strains exhibit adhesiveness to epithelial cells and extracellular matrix, indicating multiple adhesion mechanisms . HI_1049 could potentially represent one of these alternative adhesins.

What protein interaction studies would be valuable for HI_1049 characterization?

Based on known H. influenzae biology, several protein interaction studies would be particularly informative:

• Extracellular matrix interactions: Given that fimbriated and nonfimbriated clinical isolates of H. influenzae interact with glycosylated and collagenous proteins of the extracellular matrix , testing HI_1049 binding to these components is essential:

  • Fibronectin (human plasma)

  • Heparin-binding growth-associated molecule (HB-GAM)

  • Collagen subtypes

  • Laminin

  • Proteoglycans

• Glycosaminoglycan binding: Inhibition studies using low-molecular-weight heparin and chondroitin sulfate would determine if HI_1049 interacts with these molecules, similar to inhibition studies performed with Hib fimbriae .

• Domain-specific interactions: For fibronectin, interaction with specific proteolytic fragments would identify binding domains, as different bacterial adhesins target different regions of fibronectin .

• Bacterial protein interactions: Co-immunoprecipitation and bacterial two-hybrid assays could identify interactions with other H. influenzae proteins, potentially revealing functional complexes.

Methodologically, these interactions could be assessed through:

• Enzyme-linked immunosorbent assays (ELISAs) with purified HI_1049 and immobilized target proteins

• Surface plasmon resonance for real-time binding kinetics

• Bacterial adhesion assays to protein-coated surfaces

• Pull-down assays with biotinylated HI_1049 and cell/tissue lysates

How can contradictory data about HI_1049 function be reconciled?

When faced with contradictory findings about protein function, systematic approaches to reconciliation include:

• Strain differences analysis: Determine if variant phenotypes correlate with genetic background. H. influenzae exhibits considerable strain variation, with encapsulated strains (particularly type b) causing more severe diseases than unencapsulated strains .

• Expression level assessment: Quantify whether contradictory results correlate with different expression levels. For fimbrial proteins, expression levels significantly impact functional assay results, as demonstrated by the weaker hemagglutination observed with the recombinant E. coli strain HB101(pMH140) compared to Hib strain 770235 fim+ .

• Experimental condition mapping: Create a comprehensive matrix comparing contradictory studies:

• Multiple technique validation: Confirm findings using orthogonal methods. For example, if contradictory binding results exist, validate using both ELISA and surface plasmon resonance.

• Phase variation consideration: Many H. influenzae virulence factors exhibit phase variation, including fimbriae . The reversible on/off switching of expression could explain seemingly contradictory results if experimental conditions influence the proportion of bacteria expressing the protein.

What are potential roles of HI_1049 in bacterial adhesion to host tissues?

Based on our understanding of H. influenzae pathogenesis, potential adhesion mechanisms involving HI_1049 could include:

• Direct epithelial cell binding: HI_1049 might directly interact with receptors on respiratory epithelial cells, similar to how fimbriae mediate adhesion to oropharyngeal epithelial cells .

• Extracellular matrix bridging: HI_1049 could bind extracellular matrix components like fibronectin, which then serves as a bridge to host cell integrins. Fimbriated Hib strain 770235 fim+ adheres strongly to fibronectin , suggesting this is an important colonization mechanism.

• Biofilm matrix component: HI_1049 might contribute to biofilm formation by binding extracellular DNA, polysaccharides, or proteins in the biofilm matrix.

• Co-adhesion mediator: HI_1049 could facilitate adhesion to other microorganisms in polymicrobial communities of the respiratory tract.

For methodological investigation of these potential roles:

• Compare adhesion of wild-type and HI_1049 knockout strains to different cell types (respiratory epithelial cells, subepithelial fibroblasts, endothelial cells).

• Perform competition assays with purified recombinant HI_1049 and bacterial adhesion.

• Use inhibition assays with antibodies against HI_1049 to block bacterial adhesion.

• Examine specific binding to different extracellular matrix proteins using methods similar to those used for Hib fimbriae .

What are the implications of HI_1049 for developing new therapeutic strategies?

Understanding HI_1049 function could inform novel therapeutic approaches:

• Adhesion inhibitors: If HI_1049 mediates bacterial adhesion, competitive inhibitors could prevent colonization. Research on Hib fimbriae shows that inhibition with low-molecular-weight heparin can block adhesion to extracellular matrix proteins .

• Vaccine development: Surface-exposed epitopes of HI_1049 could represent vaccine targets if the protein is:

  • Conserved across strains

  • Expressed during infection

  • Accessible to antibodies

  • Functionally important

• Diagnostic markers: If HI_1049 expression correlates with virulence, detection could have prognostic value.

To evaluate therapeutic potential methodologically:

• Epitope mapping: Identify surface-exposed regions of HI_1049 using protease accessibility and computational prediction.

• Immunogenicity assessment: Evaluate antibody responses to recombinant HI_1049 in animal models.

• Protective efficacy: Determine if anti-HI_1049 antibodies provide protection in challenge models.

• Structure-based drug design: If crystal structures become available, perform in silico screening for small molecule inhibitors.

• Conservation analysis: Assess HI_1049 sequence conservation across clinical isolates to determine breadth of coverage for potential therapeutics.

How can expression issues with recombinant HI_1049 be resolved?

Common expression challenges and solutions include:

For HI_1049 specifically:

• If membrane-associated, addition of detergents during lysis and purification is crucial. A detergent screen (non-ionic, zwitterionic, ionic) should be performed to identify optimal solubilization conditions.

• Expression as inclusion bodies may require refolding protocols. Success has been reported with ammonium sulfate precipitation methods for Hib fimbrial proteins , which might be applicable.

• If expression levels remain low in E. coli, consider alternative hosts. While E. coli HB101(pMH140) successfully expressed Hib fimbriae, the expression level was only 2-5% of that shown by the native Hib strain 770235 fim+ .

• Fusion partners can improve solubility and expression. For difficult proteins, multiple constructs with different tags (His, GST, MBP, SUMO) should be tested in parallel.

What strategies can address solubility challenges with HI_1049?

Protein solubility issues often require methodical troubleshooting:

• Buffer optimization:

  • Systematic pH screening (typically pH 5.0-9.0 in 0.5 pH unit increments)

  • Salt concentration optimization (typically 0-500 mM NaCl)

  • Addition of stabilizing agents (10% glycerol, 1M arginine, 500 mM trehalose)

  • Reducing agents if cysteine residues are present (5 mM DTT or 1 mM TCEP)

• Protein engineering:

  • Removal of predicted hydrophobic regions

  • Mutation of aggregation-prone residues identified by algorithms like AGGRESCAN

  • Creation of truncated constructs focusing on soluble domains

• Solubilization methods:

  • For membrane-associated proteins, detergent screening (LDAO, DDM, OG, Triton X-100)

  • For inclusion bodies, carefully optimized denaturation and refolding protocols

• Co-expression strategies:

  • With chaperones (GroEL/ES, DnaK/J/GrpE)

  • With binding partners if known

• Alternative expression systems:

  • Cell-free protein synthesis for toxic proteins

  • Periplasmic expression with appropriate signal sequences

  • Secretion-based systems for naturally secreted proteins

When designing experiments, factorial design approaches allow efficient optimization of multiple parameters simultaneously, rather than changing one variable at a time.

How can the specificity of antibodies against HI_1049 be verified?

Rigorous validation of antibodies against uncharacterized proteins like HI_1049 is essential:

• Western blot analysis:

  • Compare wild-type H. influenzae with HI_1049 knockout strains

  • Test pre-immune serum as negative control

  • Include recombinant HI_1049 as positive control

  • Perform peptide competition assays to confirm epitope specificity

• Immunoprecipitation validation:

  • Immunoprecipitate from both wild-type and knockout strains

  • Confirm pulled-down protein identity by mass spectrometry

  • Test for cross-reactivity with closely related proteins

• Immunofluorescence controls:

  • Compare staining patterns between wild-type and knockout strains

  • Include isotype control antibodies

  • Perform blocked antibody controls (pre-incubation with antigen)

• ELISA quantification:

  • Develop standard curves with recombinant HI_1049

  • Establish limits of detection and quantification

  • Test multiple antibody dilutions to ensure linearity of response

• Cross-reactivity assessment:

  • Test against other H. influenzae proteins with sequence similarity

  • Evaluate reactivity against related bacterial species

  • Pre-absorb antibodies with related antigens to improve specificity

A monoclonal antibody approach similar to that used for Hib fimbriae would provide the most specific reagents for HI_1049 detection and characterization.