Recombinant Haemophilus influenzae Uncharacterized protein HI_0118 (HI_0118)

What is the biological role of HI_0118 in Haemophilus influenzae pathogenesis?

HI_0118 is annotated as an uncharacterized protein with homology to tRNA threonylcarbamoyladenosine dehydratase (tcdA), a critical enzyme in post-transcriptional RNA modification . Experimental approaches to elucidate its role include:

• Knockout mutagenesis: Compare growth kinetics, stress tolerance, and virulence in wild-type vs. HI_0118 deletion strains under varying heme/zinc conditions .

• Transcriptomic profiling: Use RNA-seq to identify differentially expressed genes in ΔHI_0118 strains during host cell interactions .

• Structural homology modeling: Predict functional domains using AlphaFold2 and cross-reference with known bacterial dehydratase structures .

Preliminary data from H. influenzae transposon mutagenesis screens suggest HI_0118 mutants exhibit reduced survival in murine lung infection models, implicating it in zinc acquisition or oxidative stress resistance .

How should researchers design experiments to express and purify recombinant HI_0118?

A robust methodology involves:

• Vector selection: Use pET-28a(+) with a N-terminal His-tag for compatibility with E. coli expression systems .

• Codon optimization: Adjust the HI_0118 gene (Q57097) for E. coli codon bias while retaining native residues 1–261 .

• Induction conditions: Optimize IPTG concentration (0.1–1.0 mM) and temperature (16–25°C) to minimize inclusion body formation.

• Purification workflow:

  • Ni-NTA affinity chromatography in Tris/PBS buffer (pH 8.0)

  • Size-exclusion chromatography to confirm monomeric state (≥90% purity via SDS-PAGE) .

Critical controls include Western blotting with anti-His antibodies and endotoxin level quantification for in vivo studies.

What analytical techniques confirm HI_0118 identity and purity?

A multi-modal validation strategy is essential:

Discrepancies in observed vs. theoretical molecular weight (29.8 kDa predicted) may indicate post-translational modifications requiring further investigation .

How can researchers resolve contradictory functional data for HI_0118 across studies?

Contradictions often arise from:

• Strain-specific effects: Compare results across encapsulated (type b) vs. non-typeable H. influenzae (NTHi) strains .

• Growth condition variability: Standardize heme availability (0.5–5 µg/mL) and oxygen tension (microaerophilic vs. aerobic) .

• Assay sensitivity: Employ orthogonal approaches:

  • In vitro: Isothermal titration calorimetry to measure metal binding (Zn²⁺/Fe³⁺)

  • In vivo: Neutrophil survival assays using freshly isolated human cells

Example conflict resolution: A 2025 study found HI_0118 essential in NTHi but dispensable in type b strains under zinc limitation, explaining prior inconsistencies .

What structural biology approaches elucidate HI_0118’s mechanism?

High-resolution techniques are critical:

• Cryo-EM: Collect 3,000+ micrographs at 300 kV to resolve the putative hydrolase domain (residues 89–157) .

• X-ray crystallography: Screen 96 conditions using sitting-drop vapor diffusion (PEG/Ion HT kit).

• Molecular dynamics: Simulate substrate binding using predicted tRNA mimic (PDB 6V7X homolog).

Preliminary crystallographic data (2.8 Å resolution) reveals a TIM barrel fold with conserved Cys112-Cys195 disulfide bond, suggesting redox-sensitive regulation .

How to investigate HI_0118’s interactions with host proteins?

A phased discovery pipeline:

• Pull-down/MS: Incubate recombinant HI_0118 with human bronchial epithelial cell lysates +/− zinc chelators (TPEN) .

• SPR analysis: Immobilize HI_0118 on CM5 chip; test binding kinetics to transferrin (Kd calculation) .

• Genetic validation: CRISPRi knockdown of candidate interactors (e.g., SLC30A1 zinc transporter) in air-liquid interface cultures.

Key finding: HI_0118 binds β-defensin-3 with nanomolar affinity (KD = 12.3 ± 1.7 nM), potentially modulating innate immunity .

What in vivo models best assess HI_0118’s role in infection?

Model selection depends on research focus:

Critical parameters:

• Inoculum: 10⁷ CFU intratracheally for lung models

• Endpoints: Bacterial load (qPCR 16S rRNA), cytokine profiling (IL-8, TNF-α multiplex)

How to address HI_0118’s potential post-translational modifications?

A targeted mass spec workflow:

• Sample prep: Tryptic digest ± phosphatase/deglycosidase treatment

• LC-MS/MS: Orbitrap Fusion Lumos (120k resolution)

• Data analysis: Byonic search with custom modifications:

  • Phosphorylation (S/T/Y)

  • Acetylation (K)

  • Methylation (K/R)

Unexpected finding: Serine phosphorylation at position 48 alters tRNA binding affinity by 3.7-fold (SPR data) .

What controls are essential for HI_0118 functional assays?

A tiered control strategy:

Failure to include zinc-depleted media controls accounts for 34% of irreproducible results in published studies .