Recombinant Haemophilus influenzae Uncharacterized protein HI_0148.1 (HI_0148.1)

What is the structural composition of HI_0148.1 and what does its sequence suggest about potential function?

HI_0148.1 is a small uncharacterized protein consisting of 68 amino acids with the sequence: mLFIPPPLLCLFIAIAMYFLPKIASYSVHFSVIVFVISLSFLIALSSVMQSLYVKPPLILVTLKAQQN . The protein is from Haemophilus influenzae strain ATCC 51907/DSM 11121/KW20/Rd .

Sequence analysis reveals several notable features:

• High proportion of hydrophobic residues (L, I, F, V, A)

• Multiple proline residues, particularly at the N-terminus

• Cluster of polar residues near the C-terminus

The abundance of hydrophobic residues suggests HI_0148.1 may be membrane-associated, potentially functioning as a small transmembrane protein or membrane-associated signaling peptide. When analyzing uncharacterized proteins like HI_0148.1, researchers should perform comprehensive bioinformatic analyses including hydropathy plots, secondary structure predictions, and homology searches to identify functional domains or motifs.

How should researchers approach expression and purification of recombinant HI_0148.1?

Expression and purification of small hydrophobic proteins like HI_0148.1 requires careful optimization. The commercially available recombinant protein is supplied in Tris-based buffer with 50% glycerol, optimized for stability . For laboratory expression, consider:

For purification, implement a multi-step approach:

• Initial capture using affinity chromatography (based on chosen tag)

• Intermediate purification using ion exchange chromatography

• Polishing step using size exclusion chromatography

• Consider detergent solubilization if membrane-associated

Due to the small size of HI_0148.1, special attention should be paid to protein loss during concentration and dialysis steps. Using spin concentrators with appropriate molecular weight cut-offs (3-5 kDa) is recommended.

What techniques should be employed for detection and quantification of HI_0148.1 in experimental systems?

For proteins with limited characterization like HI_0148.1, researchers should implement multiple complementary detection methods:

• Immunodetection approaches:

  • Western blotting using tag-specific antibodies for recombinant protein

  • Generation of custom antibodies against synthetic peptides derived from HI_0148.1 sequence

  • ELISA-based quantification, similar to the commercially available ELISA kit

• Mass spectrometry-based detection:

  • Targeted MS approaches like selected reaction monitoring (SRM)

  • Data-independent acquisition (DIA) methods

  • Peptide mass fingerprinting after trypsin digestion

• Nucleic acid-based detection of expression:

  • qPCR to measure transcript levels

  • RNA-seq for comprehensive transcriptomic analysis

  • Northern blotting for confirmation of transcript size

When developing detection methods, researchers should consider the challenges posed by the small size of HI_0148.1 and its potentially membrane-associated nature. Extraction protocols should be optimized to ensure complete recovery from membrane fractions.

How can researchers determine the potential role of HI_0148.1 in H. influenzae pathogenesis?

Investigating the role of HI_0148.1 in pathogenesis requires a multi-faceted approach, especially considering that H. influenzae remains a significant public health burden with increasing multi-drug resistance . Researchers should consider:

• Gene knockout/knockdown studies:

  • CRISPR-Cas9 gene editing to create HI_0148.1 mutants

  • Antisense RNA approaches for transient knockdown

  • Assessment of virulence in infection models

• Comparative genomics:

  • Analysis of HI_0148.1 presence/absence across clinical isolates

  • Sequence variation in invasive vs. non-invasive strains

  • Population structure analysis similar to that performed in recent Thai cohort studies

• Transcriptomic/proteomic profiling:

  • Expression changes during infection processes

  • Differential expression between non-typeable (NT) and encapsulated strains

  • Response to antibiotic pressure and stress conditions

• Host-pathogen interaction studies:

  • Protein-protein interaction screens with host factors

  • Cell adhesion and invasion assays with wild-type vs. mutant strains

  • Immune response triggered by purified recombinant HI_0148.1

The absence of lineage enrichment among disease samples in recent population studies suggests that invasive disease capability may not be restricted to specific H. influenzae subpopulations . Therefore, functional studies of proteins like HI_0148.1 should be conducted across diverse strain backgrounds.

What post-translational modifications might occur on HI_0148.1 and how can they be identified?

Post-translational modifications (PTMs) can significantly impact protein function and may be relevant for HI_0148.1. Drawing from techniques used to study PTMs like hydroxylation , researchers can employ:

• Mass spectrometry-based approaches:

  • High-resolution MS/MS for identification of mass shifts

  • Electron transfer dissociation (ETD) or electron capture dissociation (ECD) for labile modifications

  • Enrichment strategies for specific PTMs prior to MS analysis

• PTM-specific detection methods:

  • Phospho-specific staining (if phosphorylation is suspected)

  • Glycosylation detection using lectins or specific dyes

  • Ubiquitination detection using specific antibodies

• In silico prediction and verification:

  • Computational prediction of potential modification sites

  • Site-directed mutagenesis of predicted sites

  • Functional assessment of PTM-deficient mutants

For hydroxylation specifically, researchers can adapt methods used to detect HIF hydroxylation , such as:

• CO2 capture assays to detect hydroxylation-associated decarboxylation

• VHL capture assays (if hydroxylation affects protein-protein interactions)

• Pharmacological substrate-trapping using inhibitors like DMOG

How can protein-protein interaction networks involving HI_0148.1 be comprehensively mapped?

Mapping protein-protein interactions (PPIs) for an uncharacterized protein like HI_0148.1 requires multiple complementary approaches:

• Affinity-based methods:

  • Tandem affinity purification (TAP-tag) of HI_0148.1 followed by MS

  • BioID or TurboID proximity labeling to identify neighboring proteins

  • Pull-down assays using recombinant HI_0148.1 as bait

• Library screening approaches:

  • Yeast two-hybrid screening against H. influenzae or host proteomes

  • Phage display libraries to identify peptide binders

  • Protein arrays containing human proteins to identify host targets

• In situ methods:

  • Fluorescence resonance energy transfer (FRET)

  • Bimolecular fluorescence complementation (BiFC)

  • Proximity ligation assay (PLA) for detecting interactions in tissue

• Computational prediction:

  • Interolog mapping based on homologs with known interactions

  • Structural docking simulations

  • Co-expression network analysis

When studying membrane-associated proteins like HI_0148.1, special consideration should be given to detergent selection for extraction and maintaining native membrane environments during interaction studies. Split-membrane yeast two-hybrid systems or mammalian membrane two-hybrid approaches may be more suitable than conventional methods.

What structural biology techniques are most appropriate for determining the 3D structure of HI_0148.1?

For small membrane-associated proteins like HI_0148.1, multiple structural biology approaches should be considered:

• Solution NMR spectroscopy:

  • Particularly suitable for small proteins (<20 kDa)

  • Can be performed in membrane-mimetic environments (micelles, bicelles)

  • Provides dynamic information alongside structure

• X-ray crystallography:

  • Requires successful crystallization, which can be challenging for membrane proteins

  • Consider lipidic cubic phase (LCP) crystallization

  • May require fusion partners to aid crystallization

• Cryo-electron microscopy:

  • Traditionally challenging for small proteins, but advances in technology make it increasingly viable

  • Consider scaffold approaches or incorporation into nanodiscs

  • Particle averaging from multiple images to enhance resolution

• Computational structure prediction:

  • AlphaFold2 or RoseTTAFold can provide initial structural models

  • Molecular dynamics simulations to assess stability in membrane environments

  • Refinement using limited experimental data (chemical shifts, crosslinking)

For the 68-amino acid HI_0148.1, solution NMR may be the most practical approach, particularly if the protein can be isotopically labeled during recombinant expression. The resulting structural data would provide crucial insights into potential functional domains and interaction surfaces.

What approaches can determine if HI_0148.1 is essential for H. influenzae viability and growth?

Determining essentiality requires systematic genetic and phenotypic analysis:

• Genetic disruption methods:

  • Targeted gene deletion attempts (failure may indicate essentiality)

  • Conditional expression systems (tet-on/off)

  • CRISPRi for partial knockdown to assess dose-dependent effects

• Transposon mutagenesis approaches:

  • Tn-seq to identify essential genes under various conditions

  • TraDIS (Transposon Directed Insertion-site Sequencing)

  • Comparison of insertion patterns in HI_0148.1 vs. known essential and non-essential genes

• Complementation studies:

  • Heterologous complementation to rescue deletion phenotypes

  • Domain complementation to identify essential regions

  • Cross-species complementation with homologs

• Growth phenotype analysis:

  • Monitoring growth curves under various conditions

  • Competition assays between wild-type and mutant strains

  • Stress response profiling (antibiotic, oxidative, pH, temperature)

Given the rising concern about multi-drug resistant H. influenzae lineages , determining whether HI_0148.1 is essential could have significant implications for identifying new drug targets. If essential, researchers should conduct targeted small-molecule screens against the protein.

What are the optimal experimental conditions for studying potential enzymatic activities of HI_0148.1?

Despite being uncharacterized, HI_0148.1 may possess enzymatic activities that can be systematically investigated:

• Activity screening approaches:

  • Generic substrate panels (protease, kinase, phosphatase, etc.)

  • Metabolite profiling in presence/absence of recombinant protein

  • Co-factor dependency tests (metal ions, nucleotides, etc.)

• Assay optimization considerations:

  • Buffer composition screening (pH, ionic strength, detergents)

  • Temperature and time course analysis

  • Substrate concentration optimization

• Detection methods:

  • Spectrophotometric/fluorometric continuous assays

  • HPLC/MS-based product detection

  • Radiometric assays for high sensitivity

• Control experiments:

  • Heat-inactivated protein controls

  • Site-directed mutants of predicted catalytic residues

  • Inhibitor studies if activity is detected

When working with membrane-associated proteins like HI_0148.1, consider incorporating the protein into liposomes or nanodiscs to maintain a native-like environment that may be required for activity. Additionally, test for potential enzymatic functions in both detergent-solubilized and membrane-reconstituted forms.

How can researchers investigate the potential role of hydroxylation in regulating HI_0148.1 function?

While not specifically documented for HI_0148.1, protein hydroxylation is an important post-translational modification that could affect its function. Using techniques established for studying HIF hydroxylation , researchers can:

• Detection of hydroxylation:

  • Mass spectrometry approaches optimized for hydroxylated residues

  • CO2 capture assays to detect hydroxylation-dependent decarboxylation

  • In silico prediction of potential hydroxylation sites

• Manipulation of hydroxylation status:

  • Pharmacological inhibition using compounds like DMOG

  • Genetic manipulation of putative hydroxylases

  • Site-directed mutagenesis of predicted hydroxylation sites

• Functional consequences assessment:

  • Protein stability studies (pulse-chase, cycloheximide chase)

  • Protein-protein interaction changes upon hydroxylation

  • Subcellular localization effects

• Identification of responsible hydroxylases:

  • Co-immunoprecipitation with known hydroxylases

  • Substrate-trapping approaches using catalytically inactive hydroxylases

  • In vitro hydroxylation assays with recombinant enzymes

How might HI_0148.1 contribute to antibiotic resistance mechanisms in H. influenzae?

With increasing reports of multi-drug resistant (MDR) H. influenzae lineages , investigating the potential role of uncharacterized proteins like HI_0148.1 in resistance mechanisms is crucial:

• Expression analysis approaches:

  • Comparative transcriptomics between resistant and susceptible strains

  • Induction studies under antibiotic pressure

  • Single-cell expression analysis during antibiotic exposure

• Genetic manipulation studies:

  • Overexpression of HI_0148.1 and assessment of MIC changes

  • Knockout/knockdown and evaluation of antibiotic susceptibility

  • Site-directed mutagenesis to identify critical residues

• Protein-antibiotic interaction studies:

  • Direct binding assays between purified HI_0148.1 and antibiotics

  • Structural studies of potential drug-protein complexes

  • Competitive binding assays with known antibiotic targets

• Clinical correlation studies:

  • Sequence variations in HI_0148.1 across clinical isolates with different resistance profiles

  • Meta-analysis of genomic data from MDR lineages identified in global studies

  • Prospective monitoring of HI_0148.1 mutations in longitudinal surveillance

Given the membrane-associated nature of HI_0148.1, particular attention should be paid to its potential role in membrane permeability, drug efflux, or modification of cell envelope components that might contribute to resistance phenotypes.

What experimental approaches can determine if HI_0148.1 is a viable vaccine target against non-typeable H. influenzae?

With limitations of current Hib vaccines in protecting against non-typeable H. influenzae , novel vaccine targets are needed:

• Antigenicity assessment:

  • Epitope mapping using synthetic peptide arrays

  • B-cell epitope prediction and validation

  • T-cell epitope identification through MHC binding assays

• Conservation analysis:

  • Sequence conservation across clinical isolates

  • Population structure analysis to ensure broad coverage

  • Assessment of selective pressure on the protein

• Accessibility studies:

  • Surface exposure confirmation through protease shaving

  • Antibody binding to intact bacteria

  • Immunofluorescence or electron microscopy visualization

• Immunization studies:

  • Animal immunization with recombinant HI_0148.1

  • Evaluation of protective antibody responses

  • Challenge studies to assess vaccine efficacy

• Cross-protection potential:

  • Cross-reactivity testing against diverse clinical isolates

  • Opsonophagocytic killing assays with immune sera

  • Complement-mediated killing assessment

If HI_0148.1 proves to be surface-exposed and conserved across non-typeable strains, it could represent a valuable target for next-generation vaccines aimed at broader protection against H. influenzae infections.