Recombinant Haemophilus influenzae Mu-like prophage FluMu protein C (HI_1491)

What expression systems are most suitable for recombinant HI_1491 production?

For optimal recombinant production of HI_1491, researchers should consider the following methodological approach:

• Vector selection: pET expression systems with T7 promoters have proven effective for recombinant expression of H. influenzae proteins

• Host strain optimization: E. coli BL21(DE3) or derivatives frequently yield better expression for prophage proteins

• Induction conditions: IPTG concentration (0.1-1.0 mM) and temperature (16-37°C) should be optimized to balance expression level with protein solubility

• Purification strategy: A combination of affinity chromatography (His-tag) followed by size exclusion chromatography is typically effective

The expression system should be selected based on downstream applications, with consideration of potential protein toxicity and formation of inclusion bodies.

How can structural analysis of HI_1491 inform its function in H. influenzae pathogenesis?

Structural characterization of HI_1491 requires a multifaceted approach combining computational and experimental methods:

• Computational structural prediction: Employ homology modeling against related prophage proteins and validate using molecular dynamics simulations

• X-ray crystallography workflow:

  • Express recombinant HI_1491 with optimized solubility tags

  • Implement high-throughput crystallization condition screening

  • Analyze diffraction patterns at 2.0-3.0 Å resolution

• Cryo-electron microscopy: For larger complexes involving HI_1491, cryo-EM can reveal structural arrangements similar to how hexagonal arrays of HEF were identified in influenza C virus

Functional insights can be derived from structural features, particularly interaction interfaces that may indicate binding partners. Research on influenza virus membrane proteins has demonstrated that structural analysis can reveal critical functional domains; similar approaches could identify regions in HI_1491 involved in pathogenesis .

What role might HI_1491 play in intracellular invasion of H. influenzae?

HI_1491's potential contribution to intracellular invasion could be assessed using methodologies similar to those employed for other H. influenzae virulence factors:

• Gentamicin protection assays: Compare invasion rates between wild-type and HI_1491 knockout strains in airway epithelial cells, using serial enrichment to quantify differences

• Immunofluorescence microscopy: Visualize potential co-localization of HI_1491 with cellular markers like Lamp-1 to determine subcellular compartmentalization during invasion

• Protein-protein interaction screening: Identify potential interactions between HI_1491 and known invasion factors like HMW1

Research on HMW1 has shown that certain adhesins facilitate bacterial self-aggregation and intracellular invasion of airway epithelial cells in groups rather than as individual bacterial cells . Similar phenotypes could be investigated for HI_1491.

How do mutations in HI_1491 affect prophage induction and bacterial virulence?

A systematic mutational analysis of HI_1491 would include:

• Site-directed mutagenesis:

  • Target conserved domains and predicted functional regions

  • Create alanine scanning libraries across the protein sequence

• Phenotypic characterization:

  • Measure prophage induction rates under various stressors (UV, antibiotics)

  • Assess bacterial adherence to epithelial cells

  • Quantify intracellular invasion capabilities

  • Monitor biofilm formation differences

• Domain swapping experiments:

  • Exchange domains with homologous proteins from other bacterial species

  • Evaluate functional complementation

Such mutational analyses could reveal domains critical for HI_1491 function, similar to how mutations in the cytoplasmic tail of influenza virus HEF were shown to affect virus titer and formation of protein arrays .

What is the optimal approach to generate knockout and complementation strains of HI_1491?

For generating precise genetic manipulations of HI_1491:

• Knockout strategy:

  • Use natural transformation with homologous recombination for H. influenzae

  • Design constructs with antibiotic resistance markers flanked by 1-2 kb homologous regions

  • Confirm deletions by PCR and sequencing

  • Verify phenotypic effects using multiple independent clones

• Complementation approach:

  • Reintroduce HI_1491 at a neutral chromosomal location

  • Use inducible promoters to control expression levels

  • Include epitope tags for detection while confirming functionality

• Verification methods:

  • RT-qPCR for transcriptional analysis

  • Western blotting to confirm protein expression

  • Functional assays to verify phenotype restoration

This approach mirrors the successful genetic manipulation methods used for studying HMW1 adhesin, where natural transformation of the hmw1 operon demonstrated its role in intracellular invasion .

How can TREP be adapted to study HI_1491's role in H. influenzae pathogenesis?

Transformed Recombinant Enrichment Profiling (TREP) can be adapted for HI_1491 studies following this methodology:

• Initial setup:

  • Identify donor strains with variable HI_1491 sequences

  • Create recipient strains with tagged or deleted HI_1491

  • Generate recombinant pools through natural transformation

• Selection strategy:

  • Design selection conditions relevant to HI_1491 function (e.g., phage induction resistance)

  • Perform serial enrichment to isolate relevant recombinants

  • Extract genomic DNA from enriched populations

• Deep sequencing and analysis:

  • Sequence pre- and post-selection pools

  • Identify enriched genomic regions containing donor DNA

  • Map genetic variations to functional domains

• Validation:

  • Create defined mutants based on TREP results

  • Perform confirmatory phenotypic assays

This adaptation of TREP, which successfully identified HMW1 as a crucial factor for intracellular invasion , would allow for unbiased identification of functional domains within HI_1491.

What cell culture models are most appropriate for studying HI_1491's role in infection?

The selection of appropriate cell culture models should be guided by:

• Respiratory epithelial models:

  • A549 alveolar epithelial cells for studying adherence and invasion

  • Primary human bronchial epithelial cells in air-liquid interface cultures for physiological relevance

  • 16HBE14o- cells for studying tight junction interactions

• Immune cell models:

  • THP-1 derived macrophages for phagocytosis studies

  • Neutrophil models for examining bacterial evasion strategies

• Advanced 3D models:

  • Organoids derived from human airway tissues

  • Microfluidic "lung-on-a-chip" systems for studying dynamic host-pathogen interactions

• Infection protocols:

  • Standardize MOI (multiplicity of infection) between experiments

  • Optimize gentamicin protection assays for intracellular bacteria quantification

  • Implement immunofluorescence microscopy for visualizing bacterial localization

These models and methods have been validated in studies of HMW1-mediated intracellular invasion of airway epithelial cells and could be adapted to investigate HI_1491's role in infection processes.

How should researchers interpret HI_1491 expression data across different H. influenzae strains?

For robust interpretation of HI_1491 expression data:

• Standardization protocol:

  • Normalize expression to multiple reference genes (minimum of 3)

  • Account for growth phase variations using time-course analyses

  • Consider strain-specific genomic contexts when comparing expression levels

• Statistical analysis approach:

  • Apply ANOVA with appropriate post-hoc tests for multi-strain comparisons

  • Utilize non-parametric tests when data distribution is non-normal

  • Implement mixed-effects models for repeated measures designs

• Visualization methods:

  • Generate heat maps for strain comparison across conditions

  • Create volcano plots for visualizing significance and fold-change

  • Use principal component analysis to identify strain clustering patterns

• Contextual interpretation:

  • Consider prophage activation state in each strain

  • Evaluate co-expression with other prophage genes

  • Correlate expression with phenotypic observations

This approach mirrors analysis methods used for studying variation in adhesin expression across H. influenzae isolates, where western blot analysis was used to compare protein levels between strains .

What bioinformatic approaches are most valuable for analyzing HI_1491 sequence variation?

A comprehensive bioinformatic analysis of HI_1491 would include:

• Sequence alignment pipeline:

  • Multiple sequence alignment with MUSCLE or MAFFT

  • Visualization with Jalview or similar tools

  • Identification of conserved motifs using MEME Suite

• Phylogenetic analysis:

  • Maximum likelihood trees using RAxML or IQ-TREE

  • Bayesian inference with MrBayes

  • Selection pressure analysis with PAML

• Structural prediction workflow:

  • Ab initio modeling with Rosetta

  • Template-based modeling with I-TASSER or AlphaFold

  • Molecular dynamics simulations to assess stability

• Functional annotation:

  • Domain identification using InterProScan

  • GO term enrichment analysis

  • Protein-protein interaction prediction using STRING

This methodological framework allows researchers to identify conserved regions that may indicate functional importance, similar to approaches used for analyzing adhesin variation in H. influenzae .

How might HI_1491 be targeted for therapeutic development against H. influenzae infections?

Potential therapeutic strategies targeting HI_1491 include:

• Inhibitor development pipeline:

  • Virtual screening against predicted binding pockets

  • Fragment-based drug design targeting critical domains

  • Validation using in vitro binding and functional assays

• Antibody-based approaches:

  • Generation of monoclonal antibodies against exposed epitopes

  • Development of antibody-antibiotic conjugates for targeted delivery

  • Evaluation of antibody efficacy in preventing bacterial invasion

• Anti-virulence strategies:

  • Design of compounds that prevent prophage induction

  • Development of peptide inhibitors of protein-protein interactions

  • Creation of CRISPR-based antimicrobials targeting prophage regions

• Combination therapy approaches:

  • Integration with conventional antibiotics

  • Synergy testing with other anti-virulence compounds

  • Evaluation of resistance development potential

These approaches parallel strategies suggested for targeting other virulence factors, such as HMW1, where blocking function could potentially reduce the ability of H. influenzae to invade airway cells and evade antibiotic therapy .

What novel techniques are emerging for studying prophage proteins like HI_1491?

Cutting-edge methodologies for prophage protein research include:

• Advanced imaging techniques:

  • Super-resolution microscopy for visualizing protein clustering at the plasma membrane

  • Cryo-electron tomography for visualizing protein arrangements in situ

  • Live-cell imaging with fluorescent protein fusions to track dynamics

• Protein interaction mapping:

  • Proximity labeling methods (BioID, APEX2)

  • Hydrogen-deuterium exchange mass spectrometry for dynamic interactions

  • Single-molecule FRET for real-time interaction analysis

• Functional genomics approaches:

  • CRISPRi for precise transcriptional regulation

  • Tn-Seq for genome-wide functional screening

  • RNA-Seq for transcriptional response profiling

• Structural biology innovations:

  • Microcrystal electron diffraction (MicroED)

  • Integrative structural biology combining multiple data sources

  • Computational methods for modeling dynamic assemblies

These emerging techniques could provide unprecedented insights into the structural arrangements and functional roles of prophage proteins like HI_1491, similar to how advanced microscopy revealed crucial aspects of influenza virus protein assemblies .