Recombinant Haemophilus influenzae ATP-dependent zinc metalloprotease FtsH (ftsH), partial

What is ATP-dependent zinc metalloprotease FtsH and what is its function in Haemophilus influenzae?

FtsH is an ATP- and Zn²⁺-dependent metalloprotease with a molecular mass of approximately 70 kDa that belongs to the AAA family (ATPases associated with a variety of cellular activities) . In bacteria like H. influenzae, FtsH is anchored to the cytoplasmic membrane via two transmembrane regions, with its short amino terminus and longer carboxy terminus exposed to the cytoplasm . The protein combines both protease and chaperone functions, serving a crucial role in protein quality control by degrading unstable proteins including regulatory proteins and improperly assembled membrane proteins .

The dual functionality of FtsH as both a protease and a chaperone—characteristics of proteins designated as "charonins"—makes it particularly important for maintaining cellular homeostasis in H. influenzae, a common inhabitant of the upper respiratory tract that can cause serious infections .

What domains characterize the functional architecture of H. influenzae FtsH?

The H. influenzae FtsH protein exhibits an architecture similar to that found in other bacterial species, with four key domains:

This arrangement allows FtsH to identify, unfold, and degrade target proteins in an ATP-dependent manner . The zinc-binding domain contains the HEXXH motif characteristic of metalloproteases and is essential for proteolytic activity, while the AAA module provides the energy for substrate unfolding through ATP hydrolysis .

What experimental designs are most appropriate for studying substrate specificity of H. influenzae FtsH?

To study substrate specificity of H. influenzae FtsH, fractional factorial experimental designs can be particularly valuable when multiple factors influence substrate recognition and processing. These designs allow researchers to examine multiple variables with fewer experimental runs compared to full factorial designs .

For example, a 2³ factorial design could examine three key factors affecting substrate specificity (e.g., temperature, pH, and substrate concentration) at two levels each, while managing experimental resources efficiently . The mathematical model for analyzing such designs follows:

$\hat{\tau}^*(k) = \frac{1}{2^{2(K-p-1)}} \sum_{j=1}^{J'} g^*_{kj} \bar{Y}^{obs}(z^*_j)$

Where K is the number of factors, p is the fraction of the full factorial design, and g*kj defines the contrast for estimating the kth factor effect .

When evaluating interactions between multiple factors affecting FtsH activity, it's important to consider that the variance under fractional factorial designs equals:

$Var (\hat{\tau}^*(k)) = \frac{1}{2^{2(K-p-1)}} \frac{(J')^2}{n} S^2 = \frac{4}{n}S^2$

This approach allows researchers to systematically explore the complex substrate recognition mechanisms of H. influenzae FtsH while optimizing experimental resources .

What methods are most effective for expressing and purifying recombinant H. influenzae FtsH?

Based on successful approaches with other bacterial proteins, recombinant expression of H. influenzae FtsH typically employs E. coli as the expression host with His-tag purification strategies. The presence of transmembrane domains in native FtsH creates challenges that can be addressed through several strategies:

• Construct Design: Replace the N-terminal lipid modification signal sequence with one for protein secretion to improve solubility and yield, similar to techniques used for other H. influenzae proteins .

• Expression Conditions:

  • Temperature: 25-30°C (reduced temperature improves folding)

  • Induction: 0.1-0.5 mM IPTG

  • Media: Enriched media supplemented with zinc

• Purification Protocol:

  Step	Buffer Composition	Purpose
  Lysis	Tris/PBS-based buffer (pH 8.0) with protease inhibitors	Cell disruption
  IMAC	Above + 20-300 mM imidazole gradient	Capture
  Size exclusion	Tris/PBS with 6% trehalose	Polishing
  Storage	Final product in Tris/PBS with 6% trehalose (pH 8.0)	Stabilization

• Storage and Handling: Aliquot and store at -20°C/-80°C to avoid repeated freeze-thaw cycles, with 5-50% glycerol addition recommended for long-term stability .

How can researchers differentiate between the chaperone and protease activities of H. influenzae FtsH?

Differentiating between the dual functions of FtsH requires carefully designed assays that can isolate each activity:

• Protease Activity Assays:

  • Fluorogenic peptide substrates with FRET (Förster Resonance Energy Transfer) pairs

  • SDS-PAGE analysis of substrate degradation kinetics

  • ATP-dependence assays (proteolysis requires ATP)

• Chaperone Activity Assays:

  • Protein aggregation prevention assays using model substrates

  • Thermal stability assays of client proteins

  • Co-immunoprecipitation of non-degraded bound substrates

• Mutational Analysis:
  Creating point mutations in the zinc-binding HEXXH motif can abolish proteolytic activity while potentially preserving chaperone function, allowing researchers to isolate and measure chaperone activity independently .

• Comparative Substrate Analysis:
  Some FtsH substrates, such as denatured alkaline phosphatase in E. coli, associate with FtsH without being degraded, providing a model system for studying chaperone function specifically .

What role does FtsH play in H. influenzae pathogenicity and stress response?

While the specific role of FtsH in H. influenzae pathogenicity is not fully characterized in the provided materials, research with related bacteria suggests FtsH likely plays important roles in:

• Stress Adaptation: FtsH likely regulates heat shock response in H. influenzae by controlling sigma factor levels, similar to its role in E. coli where it degrades sigma 32 .

• Membrane Protein Quality Control: By eliminating uncomplexed or misfolded membrane proteins, FtsH maintains membrane integrity during infection and environmental stress .

• Virulence Regulation: FtsH may modulate expression of virulence factors in H. influenzae, which is known to cause serious infections of mucosal surfaces .

• Host-Pathogen Interactions: H. influenzae, as a common inhabitant of the upper respiratory tract, requires precise regulation of surface proteins during colonization and invasion, a process likely influenced by FtsH activity .

What are the challenges in studying partial versus full-length recombinant H. influenzae FtsH?

Working with partial versus full-length FtsH constructs presents distinct challenges and opportunities:

• Membrane Domain Challenges:

  • Full-length FtsH contains transmembrane domains that reduce solubility

  • Partial constructs lacking transmembrane regions show improved solubility but may not reflect native conformational states

  • Activity assays must account for the absence of membrane anchoring in partial constructs

• Oligomerization Effects:
  Native FtsH functions as a hexameric complex; partial constructs may show altered oligomerization properties affecting both structure and function .

• Domain Interactions:
  Partial constructs may disrupt interdomain communication critical for coordinating ATP hydrolysis with proteolytic activity.

• Experimental Design Considerations:
  When comparing partial and full-length constructs, incomplete factorial designs may be necessary when certain combinations cannot be tested due to technical limitations, requiring careful statistical analysis as shown in the equation below :

  $Var(\hat{\dot{\tau}}(k)) = \frac{1}{2^{2(K-1)}} \sum_{j=1}^{J} \frac{\dot{g}_{kj}^2}{n_j} S^2(z_j) - \frac{1}{n} S^2_k$

How can researchers accurately identify and differentiate H. influenzae from related species when studying FtsH?

Accurate species identification is crucial as H. influenzae, H. parainfluenzae, and H. haemolyticus differ considerably in pathogenicity yet are frequently misidentified . When studying FtsH from these species, researchers should:

• Employ Multiple Identification Methods:

  • Fluorescence in situ hybridization (FISH) with newly designed DNA probes (85% accuracy, 0% misidentifications)

  • MALDI-TOF-MS with formic acid extraction (88% accuracy, 7% misidentifications)

• Database Considerations:
  Misidentifications in MALDI-TOF-MS systems can be resolved by adding suitable reference spectra to the system's database .

• Confirmation Strategy:

  Method	Accuracy	Misidentification Rate	Uninterpretable Results
  FISH	85%	0%	15%
  Shimadzu MALDI-TOF-MS	70%	7%	23%
  Bruker MALDI-TOF-MS with formic acid extraction	88%	7%	5%

• Verification Approach:
  Apply alternative tests in case of ambiguous results, especially for isolates from seriously ill patients, as no analyzed diagnostic procedure was free of errors .

What experimental approaches can identify novel substrates of H. influenzae FtsH?

Identifying novel substrates of H. influenzae FtsH requires systematic approaches:

• Proteomics-Based Methods:

  • Comparative proteomics between wild-type and FtsH-deficient strains

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture) to quantify protein turnover rates

  • Proximity-dependent biotin identification (BioID) with FtsH as the bait protein

• Biochemical Approaches:

  • In vitro degradation assays with recombinant FtsH and candidate substrates

  • Co-immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screening using catalytically inactive FtsH mutants

• Genetic Screens:

  • Suppressor mutations that rescue FtsH deletion phenotypes

  • Synthetic lethal screens to identify genetic interactions

• Experimental Design Considerations:
  Given the complexity of substrate recognition, fractional factorial designs as described in search result 3 are particularly useful for identifying factors that influence substrate selection .

What insights can structural studies of recombinant H. influenzae FtsH provide for antimicrobial development?

Structural studies of H. influenzae FtsH can accelerate antimicrobial development through several avenues:

• Active Site Targeting:
  The zinc-binding domain containing the HEXXH motif represents a potential target for small-molecule inhibitors .

• Allosteric Site Identification:
  Structural studies can reveal non-catalytic sites that regulate FtsH activity, offering additional targets for drug development.

• Species-Specific Features:
  Identifying structural differences between H. influenzae FtsH and human cellular proteases can guide the development of selective inhibitors.

• Structure-Based Drug Design:
  Crystal structures of H. influenzae FtsH bound to substrates or inhibitors can facilitate rational design of antimicrobial compounds through computational methods like molecular docking and virtual screening.

• Evolutionary Conservation Analysis: Comparison of FtsH structures across bacterial species can identify conserved regions essential for function, representing potentially broad-spectrum antimicrobial targets.