Recombinant Haemophilus influenzae Signal peptidase I (lepB)

What is the function of Signal Peptidase I (lepB) in Haemophilus influenzae?

Signal peptidase I (lepB) in H. influenzae serves as a specialized serine protease that cleaves N-terminal signal peptides from secreted and membrane proteins during their translocation across the cytoplasmic membrane. Similar to its E. coli homolog, H. influenzae lepB likely utilizes a Ser/Lys catalytic dyad to perform this highly specialized task of removing leader sequences of approximately 15-30 amino acids from protein precursors . This processing is essential for proper protein localization and function in the periplasm or outer membrane. Beyond its catalytic role, lepB may also possess non-catalytic functions, potentially serving as a chaperone-like protein that facilitates membrane association of certain proteins, as has been demonstrated with colicins in E. coli .

How can I clone and express recombinant H. influenzae lepB for research purposes?

The cloning and expression of recombinant H. influenzae lepB can be approached using strategies similar to those employed for E. coli lepB. Based on established protocols, the H. influenzae lepB gene can be PCR-amplified and inserted into an appropriate expression vector such as pBAD or pET systems . For optimal expression, consider the following methodology:

• Design primers that include appropriate restriction sites (e.g., NcoI, BamHI) and ensure in-frame cloning

• PCR-amplify the lepB gene from H. influenzae genomic DNA

• Clone the gene into an expression vector with an inducible promoter system

• Transform into an appropriate E. coli strain (e.g., TOP10, BL21)

• Express the protein using optimal inducer concentration (e.g., 0.0002-0.05% arabinose for pBAD systems or IPTG for pET systems)

• Purify using affinity chromatography if a tag has been incorporated

Since lepB is a membrane protein with two transmembrane domains and a large periplasmic domain, consider using detergent solubilization methods during purification, such as those used for E. coli lepB purification involving Ni-NTA affinity chromatography .

How can I verify the identity and activity of recombinant H. influenzae lepB?

Verification of recombinant H. influenzae lepB identity and activity can be accomplished through multiple complementary approaches:

• Western blot analysis: Using antibodies against lepB or attached epitope tags to confirm protein expression and size. This approach has been successfully used to monitor lepB expression levels in regulatable strains .

• Enzymatic activity assays: Using synthetic peptide substrates that mimic natural signal sequences. Activity can be measured by detecting cleavage products using HPLC, mass spectrometry, or fluorescence-based methods.

• Complementation assays: Testing whether the recombinant lepB can rescue growth in lepB-depleted E. coli strains. This approach utilizes the essential nature of lepB for cell viability, as demonstrated with E. coli lepB regulatable strains .

• Binding studies: If investigating non-catalytic functions, surface plasmon resonance (SPR) techniques can be employed to assess binding interactions with potential partner proteins, similar to studies of E. coli lepB interaction with colicin D .

How can I develop a regulatable expression system for H. influenzae lepB to study its essentiality?

Developing a regulatable expression system for H. influenzae lepB follows principles demonstrated with E. coli lepB. The methodology involves:

• Clone H. influenzae lepB into an inducible expression vector: Use a tightly regulated promoter system such as the arabinose-inducible pBAD vector system, which has been successfully used for lepB regulation .

• Replace the chromosomal copy of lepB: Using homologous recombination techniques:

  • Design primers that amplify an antibiotic resistance gene (e.g., kanamycin) flanked by sequences homologous to regions surrounding the H. influenzae lepB gene

  • Transform the construct into a strain already containing the inducible lepB plasmid

  • Select recombinants on media containing both the inducer and appropriate antibiotics

  • Confirm replacement by PCR using primers targeting flanking regions

• Optimize inducer concentration: Determine the minimal concentration of inducer (e.g., arabinose) required for normal growth. Experimental data with E. coli showed that 0.0002-0.0004% arabinose was sufficient for growth similar to wild-type strains after 24 hours of incubation .

• Validate the system: Monitor growth and lepB expression levels at various inducer concentrations using growth curves and Western blot analysis. This allows correlation between lepB levels and cell viability .

This system enables controlled depletion of lepB to study its essentiality and to evaluate potential inhibitors under conditions of limited target availability.

What approaches can be used to identify potential inhibitors of H. influenzae lepB?

Identification of H. influenzae lepB inhibitors can be approached through multiple strategies:

• Under-expression screening system: Using a lepB-regulatable strain grown with limited inducer, screen compounds for enhanced growth inhibition compared to wild-type strains. This approach has successfully identified penem compounds that show increased potency against E. coli with under-expressed lepB .

• In vitro enzymatic assays: Develop high-throughput screening assays using purified recombinant lepB and fluorogenic substrates.

• Structural approaches: If structural information is available for H. influenzae lepB (or can be modeled based on E. coli lepB), structure-based drug design methods can be employed.

• Cell permeability enhancement: Combine potential inhibitors with outer membrane permeabilizers like polymyxin B nonapeptide (Pbn) to enhance access to the periplasmic space, as demonstrated in studies with E. coli lepB .

A typical experimental design would include:

• Primary screening against purified enzyme

• Secondary screening in the lepB-regulatable strain

• Counter-screening against wild-type bacteria

• Mechanistic studies to confirm on-target activity

How can specific mutations in H. influenzae lepB affect its catalytic efficiency and substrate specificity?

Investigation of lepB mutations requires a systematic approach:

• Identify catalytic and substrate-binding residues: Based on homology to E. coli lepB, identify the Ser/Lys catalytic dyad and potential substrate-binding residues. E. coli lepB utilizes Ser-90 and Lys-145 for catalysis .

• Site-directed mutagenesis: Create specific mutations targeting:

  • Catalytic residues (to confirm mechanism)

  • Substrate-binding residues (to alter specificity)

  • Regions involved in non-catalytic functions, such as the C-terminal Box E which has been implicated in colicin binding in E. coli lepB

• Functional characterization:

  • Measure enzymatic parameters (kcat, KM) for wild-type and mutant enzymes

  • Test activity against various signal peptide substrates

  • Assess structural stability using thermal shift assays or circular dichroism

• In vivo complementation: Test whether mutants can complement a lepB-depleted strain, and quantify the degree of complementation at various inducer concentrations.

This approach can provide insights into the structural determinants of lepB function and potentially identify residues that might be targeted for inhibitor design.

What are the optimal conditions for expressing and purifying membrane-bound H. influenzae lepB?

Optimizing expression and purification of membrane-bound H. influenzae lepB requires special consideration:

• Expression system selection:

  • E. coli BL21(DE3) with pET vectors for high-level expression

  • Consider C43(DE3) or C41(DE3) strains specifically engineered for membrane protein expression

  • Control expression rate by using lower inducer concentrations and lower temperatures (16-25°C)

• Membrane extraction and solubilization:

  • Use mild detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin

  • Optimize detergent:protein ratio to maintain native structure and activity

  • Consider nanodisc or liposome reconstitution for functional studies

• Purification strategy:

  • Affinity chromatography using His-tag, as demonstrated for E. coli lepB

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography for final polishing

• Activity preservation:

  • Include stabilizing agents like glycerol (10-20%)

  • Maintain critical lipids in the buffer system

  • Test activity immediately after purification to ensure functionality

How can I distinguish between the catalytic and non-catalytic functions of H. influenzae lepB in experimental systems?

Distinguishing between catalytic and non-catalytic functions requires experimental approaches that separate these activities:

• Catalytically inactive mutants: Generate mutations in the catalytic dyad (Ser/Lys) that abolish enzymatic activity but preserve protein structure. These mutants can be used to study non-catalytic functions in isolation.

• Domain-specific analysis:

  • Express and analyze the periplasmic domain separately from the transmembrane regions

  • Create chimeric proteins with domains from different species to identify species-specific functional regions

• Binding partner identification:

  • Use pull-down assays or co-immunoprecipitation to identify interacting proteins

  • Employ surface plasmon resonance (SPR) to quantify binding interactions, similar to studies that demonstrated nanomolar binding affinity between E. coli lepB and colicin D

• Functional complementation tests:

  • Test whether catalytically inactive lepB can complement specific phenotypes

  • Use domain-swapping experiments to determine which regions are required for different functions

These approaches can help delineate the multifunctional nature of lepB beyond its well-established role in signal peptide cleavage.

How can I develop a multiplex detection system that includes H. influenzae lepB for diagnostic applications?

Development of a multiplex detection system incorporating H. influenzae lepB should build upon established multiplex PCR methodologies:

• Target selection:

  • Include lepB alongside other species-specific targets like siaT (specific for H. influenzae)

  • Consider additional targets like hypD (specific for H. haemolyticus) to differentiate from non-pathogenic near-neighbors

  • Include fucP for further differentiation among H. influenzae strains

• Primer and probe design:

  • Design primers/probes specific to H. influenzae lepB sequence regions that differ from other species

  • Ensure compatible melting temperatures for multiplexing

  • Use different fluorophores for each target to enable simultaneous detection

• Optimization strategy:

  • Validate each primer pair individually before combining

  • Optimize primer concentrations to prevent competitive inhibition

  • Test with known concentrations of mixed DNA templates to establish sensitivity and specificity

• Validation approach:

  • Test across a diverse collection of Haemophilus isolates (similar to the 143 isolates used in the siaT/hypD assay validation)

  • Include near-neighbor species to confirm specificity

  • Determine limits of detection for each target

This approach builds on the successful development of the triplex assay for simultaneous detection of hypD, siaT, and fucP in Haemophilus species .

What is the impact of growth conditions on lepB expression and activity in H. influenzae?

Understanding how growth conditions affect lepB expression and activity requires systematic investigation:

• Growth phase analysis:

  • Monitor lepB expression levels at different growth phases using quantitative PCR and Western blot

  • Correlate expression with protein secretion activity

  • Compare expression patterns between standard laboratory media and host-mimicking conditions

• Environmental stress responses:

  • Assess lepB regulation under various stresses (oxidative stress, nutrient limitation, pH changes)

  • Determine whether lepB expression is altered during biofilm formation

  • Evaluate changes during host cell interaction or infection models

• Metabolic influences:

  • Investigate whether carbon source affects lepB expression and activity

  • Determine if iron limitation, which is relevant during host infection, impacts expression

• Experimental methodology:

  • Use reporter gene fusions to monitor promoter activity in real-time

  • Employ proteomic approaches to quantify lepB protein levels under various conditions

  • Measure enzymatic activity using model substrates across different growth conditions

This systematic approach can reveal condition-specific regulation of lepB that may be relevant to pathogenesis and therapeutic targeting.

How can engineered modifications of H. influenzae lepB enhance recombinant protein secretion systems?

Engineering H. influenzae lepB for enhanced recombinant protein secretion should consider both catalytic efficiency and substrate specificity modifications:

• Targeted mutagenesis approaches:

  • Modify the substrate-binding pocket to accommodate a broader range of signal sequences

  • Engineer the catalytic site to increase turnover rate

  • Adjust the membrane interaction domains to enhance stability

• Signal peptide co-engineering:

  • Design optimized signal peptides that work synergistically with modified lepB

  • Incorporate silent mutations in signal peptide mRNA to enhance translation, as demonstrated with the Usp45 signal peptide in L. lactis (which showed up to 16% increased secretion)

  • Modify amino acid composition of signal peptides, particularly increasing positive charge in the n-region, which has been shown to enhance secretion by up to 51%

• Expression system optimization:

  • Develop dual-control systems that coordinate lepB expression with recombinant protein production

  • Balance lepB levels to prevent bottlenecks in the secretion pathway

• Validation methodology:

  • Use model secreted proteins like α-amylase or nuclease to quantify secretion efficiency

  • Employ proteomics to assess global impacts on the secretome

  • Measure growth parameters to ensure cellular viability is maintained

This approach builds on successful signal peptide engineering efforts in other systems while focusing specifically on the role of lepB in the secretion process .

What are the key differences between H. influenzae lepB and its homologs in other pathogenic bacteria that could be exploited for species-specific inhibitor development?

Comparative analysis of lepB across bacterial species can reveal targetable differences:

• Sequence and structural comparison:

  • Perform phylogenetic analysis of lepB across diverse bacterial pathogens

  • Identify regions of sequence divergence, particularly near the catalytic site

  • Model structures to visualize species-specific surface features

• Substrate specificity analysis:

  • Compare cleavage preferences using diverse signal peptide substrates

  • Identify amino acid positions that determine species-specific recognition patterns

  • Develop synthetic peptides that interact preferentially with H. influenzae lepB

• Inhibitor screening approach:

  • Design focused compound libraries targeting H. influenzae-specific features

  • Test candidates against a panel of purified lepB proteins from multiple species

  • Validate in cellular systems using the lepB under-expression approach

• Experimental validation:

  • Use site-directed mutagenesis to convert residues between species and test the impact on inhibitor sensitivity

  • Employ structural biology techniques to visualize inhibitor binding modes

  • Validate specificity in mixed bacterial culture models

This approach could yield inhibitors with narrower spectrum activity, potentially reducing impacts on beneficial microbiota compared to broad-spectrum antimicrobials.