Recombinant Haemophilus influenzae Uncharacterized protein HI_0870.1 (HI_0870.1)

What is the amino acid sequence and basic properties of HI_0870.1?

HI_0870.1 is a 96-amino acid protein with the sequence: MVLYFYNKINRSFGLMILLYFFRSGGLAFRISELFLIFSIPLFALLINELSGVNRVLMFFILVYYISIVFLRRIYVHVVFGVNTFLPYKPIFDSYL . The protein has a UniProt ID of O86229 and is classified as an uncharacterized protein, meaning its specific function has not yet been definitively established through experimental verification. The sequence analysis suggests it may contain hydrophobic regions consistent with membrane association, which is typical of many bacterial proteins involved in cellular processes like transport, signaling, or structural integrity of the membrane.

The protein appears to be relatively small at only 96 amino acids, which could indicate it functions as part of a larger complex or serves as a regulatory component. Given its identification in the complete genome sequencing of Haemophilus influenzae Rd, it represents one of many potential gene products encoded in this pathogen's genomic sequence .

What expression systems are recommended for producing Recombinant HI_0870.1?

For recombinant expression of HI_0870.1, E. coli has been successfully employed as demonstrated in commercial preparations. The protein can be expressed with an N-terminal His tag, which facilitates subsequent purification steps . The expression typically involves transformation of E. coli BL21(DE3) or similar strains with plasmids containing the HI_0870.1 gene sequence optimized for bacterial expression.

Based on methodologies used for similar H. influenzae proteins, induction of protein expression can be achieved using 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) . After induction, the cells are typically grown for 3-4 hours at 37°C or overnight at a lower temperature (16-18°C) to improve protein folding. The protein may form inclusion bodies, which would require additional processing steps including solubilization with denaturing agents like urea followed by refolding procedures .

Alternative expression systems, such as yeast or mammalian cells, might be considered if the bacterial system yields poorly folded protein, though no specific data on these alternatives for HI_0870.1 is currently available.

How should Recombinant HI_0870.1 be stored for optimal stability?

According to available data, recombinant HI_0870.1 should be stored as follows:

The protein is typically provided as a lyophilized powder, which should be briefly centrifuged prior to opening to bring contents to the bottom of the vial . For optimal results, reconstituted protein should be supplemented with glycerol (recommended final concentration of 50%) before aliquoting for long-term storage to prevent damage from freeze-thaw cycles and maintain structural integrity.

What structural features can be predicted for HI_0870.1 based on sequence analysis?

Analysis of the HI_0870.1 amino acid sequence reveals several potential structural features. The sequence (MVLYFYNKINRSFGLMILLYFFRSGGLAFRISELFLIFSIPLFALLINELSGVNRVLMFFILVYYISIVFLRRIYVHVVFGVNTFLPYKPIFDSYL) contains a high proportion of hydrophobic residues (F, L, I, V, Y, M), suggesting potential membrane-spanning domains .

Using modern prediction algorithms, we can identify:

This pattern of multiple transmembrane domains is reminiscent of small membrane proteins that may function in transport, signaling, or as structural components. Similar to Haemophilus influenzae protein E (PE), which functions as an adhesin and mediates interactions with host cells, HI_0870.1 might play a role in interactions with the host environment, though this would require experimental validation .

What experimental approaches are most appropriate for characterizing the function of HI_0870.1?

Characterizing an uncharacterized protein like HI_0870.1 requires a systematic experimental approach:

• Structural Characterization:

  • X-ray crystallography or NMR spectroscopy to determine three-dimensional structure

  • Circular dichroism (CD) spectroscopy to assess secondary structure elements

  • Mass spectrometry for accurate molecular weight determination and identification of post-translational modifications

• Localization Studies:

  • Generation of antibodies against synthetic peptides from surface-exposed regions (similar to the approach used for H. influenzae PE)

  • Immunofluorescence microscopy to determine cellular localization

  • Cell fractionation followed by Western blotting to confirm membrane association

• Functional Characterization:

  • Yeast two-hybrid or pull-down assays to identify interaction partners

  • Gene knockout or knockdown studies to assess phenotypic changes

  • Binding assays with potential host factors (extracellular matrix proteins, complement factors)

  • Virulence assessment using isogenic mutants in appropriate infection models

• Expression Profiling:

  • qRT-PCR to determine expression patterns under different conditions

  • RNA-seq to identify co-regulated genes

  • Promoter analysis to understand regulation

A rigorous experimental design would include appropriate controls and replication to ensure statistical validity of results . For instance, when creating deletion mutants, complementation studies would be essential to confirm that observed phenotypes are due to the specific deletion rather than polar effects or secondary mutations.

How might HI_0870.1 contribute to bacterial pathogenesis based on knowledge of other H. influenzae proteins?

While the specific function of HI_0870.1 remains undetermined, insights can be drawn from other characterized H. influenzae proteins. For example, Protein E (PE) serves as a multifunctional adhesin involved in interactions with lung epithelial cells and host proteins such as vitronectin, laminin, and plasminogen .

Potential pathogenic roles for HI_0870.1 might include:

• Host-Pathogen Interaction: As suggested by studies on PE, HI_0870.1 might mediate binding to host cell receptors or extracellular matrix components, facilitating colonization.

• Immune Evasion: Many bacterial membrane proteins participate in mechanisms to evade host immune responses, such as binding to complement regulatory proteins.

• Nutrient Acquisition: Small membrane proteins often play roles in uptake of essential nutrients from the host environment.

• Biofilm Formation: Membrane proteins can contribute to cell-cell interactions necessary for biofilm establishment.

• Stress Response: Adaptation to host environmental stresses often involves specialized membrane proteins.

To investigate these possibilities, researchers could design experiments similar to those used for PE, which identified specific binding regions (e.g., amino acids 84-108 involved in vitronectin binding) . Synthetic peptides corresponding to different regions of HI_0870.1 could be tested for binding to host proteins or cells, and immunization studies could assess protective efficacy against H. influenzae infection.

What purification protocols are optimal for obtaining high-quality Recombinant HI_0870.1?

Based on established methods for similar H. influenzae proteins, the following purification protocol is recommended:

• Cell Lysis and Inclusion Body Preparation:

  • Sonication in an appropriate buffer (e.g., PBS with protease inhibitors)

  • Collection of inclusion bodies by centrifugation

  • Washing with 5 M urea to remove contaminants

  • Solubilization in 8 M urea for complete denaturation

• Protein Refolding:

  • Dilution method in refolding buffer (50 mM Tris-HCl, pH 7.8, 500 mM NaCl, 5 mM DTT, 0.005% Tween 20, and 2 M urea)

  • Gradual dilution with constant stirring at 4°C

  • Concentration using ultrafiltration devices

• Affinity Chromatography:

  • Nickel-NTA affinity chromatography utilizing the N-terminal His tag

  • Washing with increasing imidazole concentrations to remove non-specific binding

  • Elution with 250-500 mM imidazole

• Additional Purification Steps:

  • Size exclusion chromatography to ensure homogeneity

  • Ion exchange chromatography if necessary for removing contaminants

• Quality Control:

  • SDS-PAGE to confirm purity (>90%)

  • Western blotting to verify identity

  • Mass spectrometry for molecular weight confirmation

  • Circular dichroism to assess proper folding

This protocol should yield high-purity protein suitable for structural and functional studies. If the protein shows tendency to aggregate, addition of mild detergents or lipid-like molecules might help maintain solubility and native conformation.

How can researchers generate specific antibodies against HI_0870.1 for experimental studies?

Generation of specific antibodies against HI_0870.1 can follow established protocols used for other H. influenzae proteins:

• Peptide Design and Synthesis:

  • Analyze the amino acid sequence to identify potentially immunogenic regions

  • Select peptides from surface-exposed regions (based on structural predictions)

  • Synthesize 15-25 amino acid peptides corresponding to these regions

  • Conjugate to a carrier protein (e.g., KLH or BSA) to enhance immunogenicity

• Immunization Protocol:

  • Following the approach used for H. influenzae PE :

    • Prime mice with 50 μg peptide emulsified in complete Freund's adjuvant

    • After 4 weeks, administer booster doses (50 μg peptide with aluminum hydroxide)

    • Continue boosters for 3 consecutive weeks

    • Collect serum 1 week after the final booster

• Antibody Purification:

  • Affinity purification using the immunizing peptide coupled to a suitable matrix

  • Elution under mild conditions to preserve antibody activity

  • Neutralization and buffer exchange

• Validation:

  • ELISA against both the peptide and recombinant full-length protein

  • Western blotting against recombinant protein and H. influenzae lysates

  • Immunofluorescence to confirm binding to the bacterial surface

  • Negative controls with pre-immune serum and irrelevant proteins

• Storage:

  • Aliquot and store at -20°C or -80°C in a buffer containing a stabilizer like glycerol

These antibodies can serve multiple research purposes, including localization studies, pull-down assays to identify interaction partners, and functional blocking experiments if the protein is involved in host-pathogen interactions.

What comparative genomic approaches might reveal about HI_0870.1 conservation and evolution?

Understanding the evolutionary context of HI_0870.1 can provide valuable insights into its function and importance. Several comparative genomic approaches can be employed:

• Sequence Conservation Analysis:

  • Compare HI_0870.1 across different H. influenzae strains to determine conservation level

  • Investigate whether the gene is part of the core genome or accessory genome

  • Analyze selection pressure (dN/dS ratio) to identify if the protein is under positive, negative, or neutral selection

• Synteny Analysis:

  • Examine the genomic context of HI_0870.1 in different bacterial species

  • Identify conserved gene neighborhoods that might indicate functional relationships

  • Determine if the gene is part of an operon structure, suggesting coordinated expression with other genes

• Ortholog Identification:

  • Search for homologs in other bacterial species beyond the Pasteurellaceae family

  • Similar to findings with Haemophilus protein E, orthologs might be present in diverse species like Enterobacter cloacae and Listeria monocytogenes

  • Functional characterization of these orthologs might provide clues to HI_0870.1 function

• Structural Homology:

  • Even with low sequence similarity, structural conservation often indicates functional relationships

  • Threading approaches and fold recognition can identify proteins with similar structures

Conservation patterns across species can indicate essential functions that have been maintained through evolutionary pressure, while species-specific variations might suggest adaptations to particular niches or hosts.

What role might HI_0870.1 play in bacterial resistance mechanisms?

Given the increasing concern about antimicrobial resistance, investigating potential roles of HI_0870.1 in resistance mechanisms represents an important research direction:

• Expression Analysis Under Antibiotic Pressure:

  • Measure expression levels of HI_0870.1 in response to various antibiotics

  • Determine if the protein is upregulated during specific stress conditions related to antibiotic challenge

• Resistance Phenotype Assessment:

  • Generate knockout or overexpression strains to determine if HI_0870.1 affects susceptibility to antimicrobials

  • Test against multiple classes of antibiotics to identify specific patterns

• Membrane Integrity Studies:

  • As a potential membrane protein, HI_0870.1 might influence membrane permeability or structure

  • Assess membrane potential, integrity, and antibiotic accumulation in wild-type versus mutant strains

• Interaction with Known Resistance Mechanisms:

  • Investigate potential interactions with established resistance mechanisms such as efflux pumps or modified porins

  • Determine if HI_0870.1 functions as an accessory protein in multicomponent resistance systems

This research direction has significant clinical implications, as understanding resistance mechanisms is critical for developing new therapeutic strategies against H. influenzae infections.