Recombinant Haemophilus influenzae Lipopolysaccharide export system protein lptC (lptC)

What is LptC and what role does it play in H. influenzae?

LptC is a conserved bitopic inner membrane protein involved in the transport of lipopolysaccharide (LPS) from its site of synthesis in the cytoplasmic membrane to the outer membrane. It forms a complex with the ATP-binding cassette transporter, LptBFG, which facilitates the extraction of LPS from the inner membrane and releases it into a translocation pathway that includes the periplasmic chaperone LptA . The LptC protein is essential for the viability of H. influenzae, as it plays a critical role in maintaining the outer membrane integrity through proper LPS assembly.

Functionally, LptC acts as part of the Lpt machinery that forms a bridge connecting the inner and outer membranes, allowing for direct efflux of LPS to the cell surface . This protein-mediated transport system is vital for Gram-negative bacterial survival and pathogenicity, as LPS constitutes approximately 4% of the organism's dry weight and is a major surface antigen .

How is LptC integrated into the LPS transport system of H. influenzae?

LptC is a component of the seven-protein Lpt (LPS transport) machinery (LptABCDEFG) that accomplishes LPS transport across the periplasm to its final assembly at the cell surface. This complex is divided into three subassemblies:

• LptBCFG: Located at the inner membrane (IM)

• LptA: Positioned in the periplasm

• LptDE: Situated at the outer membrane (OM)

Within this system, LptC interacts directly with LptA. The transport model operates as follows:

• LPS extraction from the IM begins with the LptBFG complex

• The LPS molecule is transferred to the periplasmic domain of membrane-bound LptC

• LptC then transfers LPS to LptA

• LptC may initially use energy provided by ATP hydrolysis to extract LPS from the IM

• The unidirectional transit of LPS from LptC to LptA occurs via an increasing affinity gradient

Importantly, while LptC is part of the ATP-dependent transport complex, it does not affect the kinetic parameters of the ATPase activity of the LptBCFG complex .

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

Based on published protocols and commercial products, the following methodology can be employed:

Expression Systems:

• E. coli BL21(DE3) with kanamycin selection is commonly used

• Alternative expression systems include yeast, baculovirus, or mammalian cells for specific applications

Expression and Purification Protocol:

• Cloning: Insert the lptC gene into an appropriate expression vector with a His-tag or other affinity tag

• Expression:

  • Grow bacterial culture in LB medium with 50 μg/ml kanamycin

  • Induce protein expression with 1 mM IPTG

• Purification from Inclusion Bodies:

  • Lyse cells by sonication

  • Collect inclusion bodies by centrifugation

  • Wash inclusion bodies with 5 M urea

  • Dissolve in 8 M urea

• Protein Refolding:

  • Use dilution method in refolding buffer containing:

    • 50 mM Tris-HCl, pH 7.8

    • 500 mM NaCl

    • 5 mM DTT

    • 0.005% Tween 20

    • 2 M urea

Storage Conditions:

• Store in Tris-based buffer with 50% glycerol at -20°C

• For extended storage, maintain at -80°C

• Avoid repeated freezing and thawing; store working aliquots at 4°C for up to one week

How can researchers effectively study LptC-LPS interactions in vitro?

Several complementary approaches can be used to study LptC-LPS interactions:

Binding Assays:

• Kinetic Experiments:

  • Surface plasmon resonance (SPR) to determine association and dissociation rates

  • Stopped-flow fluorescence spectroscopy to monitor rapid binding events

• Saturation and Competition Experiments:

  • Equilibrium binding studies using purified components

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters

  • Various concentrations of LPS can be used to determine the equilibrium dissociation constant (Kd)

• Binding to Synthetic Ligands:

  • Testing interaction with synthetic LPS mimetics

  • Competition assays with compounds like iaxo-102 that may share the LPS binding site

Research has revealed that:

• The in vitro LptC-LPS binding is irreversible with a Kd in the μM range

• LptC can also bind to synthetic molecules like iaxo-102, though with lower affinity than LPS

• These synthetic compounds can be considered as lead compounds for developing new antibiotics targeting LPS biogenesis

What approaches can be used to investigate LptC function in bacterial cells?

Genetic Approaches:

• Mutational Analysis:

  • Generate lptC mutants using site-directed mutagenesis

  • Create C-terminally truncated LptC variants to study domain-specific functions

  • Assess the effects of mutations on bacterial growth, membrane integrity, and LPS transport

• Transformed Recombinant Enrichment Profiling (TREP):

  • Natural transformation to generate complex pools of recombinants

  • Phenotypic selection to enrich for specific recombinants

  • Deep sequencing to identify genetic variations responsible for phenotypic changes

Imaging Techniques:

• Fluorescence Microscopy:

  • Create LptC-fluorescent protein fusions (e.g., LptC-mNeonGreen)

  • Visualize LptC localization and dynamics in live cells

  • Monitor protein enrichment in specific cellular locations (such as "foci" in the cell envelope and the septum during cell division)

• Electron Microscopy:

  • Assess ultrastructural changes in LptC-deficient bacteria

  • Visualize membrane integrity and organization

Functional Assays:

• Peptidoglycan Analysis:

  • High-pressure liquid chromatography to quantify peptidoglycan levels

  • Compare wild-type and LptC-variant strains

• Flow Cytometry:

  • Analyze the transport of LptC to outer membrane vesicles (OMVs)

  • Evaluate the impact of C-terminal domain modifications on protein localization

How does LptC contribute to H. influenzae pathogenesis?

LptC contributes to H. influenzae pathogenesis through several mechanisms:

LPS Transport and Membrane Integrity:

• LptC is essential for proper LPS transport and assembly at the outer membrane

• Disruption of LPS transport affects membrane barrier function, increasing susceptibility to antibiotics and host defense mechanisms

• LptC-mediated proper LPS assembly is critical for resistance to cationic antimicrobial peptides and complement activation

Outer Membrane Vesicle Production:

• LptC influences the production and composition of outer membrane vesicles (OMVs)

• OMVs from H. influenzae play roles in:

  • Host-pathogen interactions

  • Polymicrobial cooperation

  • Antibiotic resistance

  • Interactions with B cells

  • Modulation of host immune responses

Influence on Cell Wall Integrity:

• Though LptC's primary role is in LPS transport, research suggests interconnections between LPS transport and peptidoglycan biogenesis

• In H. influenzae, LptC function may influence cell wall integrity and composition, affecting bacterial survival during infection

What is known about H. influenzae LPS structure and its relationship with LptC function?

H. influenzae LPS (often referred to as lipooligosaccharide or LOS due to its shorter oligosaccharide chains) has several distinctive features:

Structure:

• Contains lipid A linked to a core oligosaccharide but lacks the O-antigen polysaccharide found in many Gram-negative bacteria

• The core oligosaccharide of non-typeable H. influenzae (NTHi) contains unique structural features:

  • Common element: l-α-d-Hep-p-(1→2)-[PEtn→6]-l-α-d-Hep-p-(1→3)-[β-d-Glc-p-(1→4)]-l-α-d-Hep-p-(1→5)-[PPEtn→4]-α-Kdo-p-(2→6)-lipid A

  • In major glycoforms, the terminal Hep-p residue is substituted at the O-2 position by a β-d-Gal-p residue

  • The central Hep-p residue is substituted at O-3 by an α-d-Glc-p residue

  • Phosphocholine substituents at the O-6 positions of α-d-Glc-p and β-d-Gal-p

  • Acetylation sites at O-4 of Gal and O-3 of HepIII

Relationship with LptC:

• LptC binds to the lipid A portion of LPS through its β-sheet binding site

• The binding between LptC and LPS is irreversible with a Kd in the μM range

• LptC has a lower affinity for LPS than LptA, suggesting a unidirectional transport mechanism

• LptC extracts LPS from the inner membrane and transfers it to LptA via an affinity gradient

Strain Variation:

• Significant structural heterogeneity exists among H. influenzae LPS from different strains

• This variation may affect interaction with LptC and subsequent transport efficiency

• The heterogeneity aids in evasion of host immune defense mechanisms

Could targeting LptC be a viable approach for developing novel antibiotics against H. influenzae?

Targeting LptC presents a promising avenue for antibiotic development for several reasons:

Rationale for Targeting LptC:

• Essentiality: LptC is essential for bacterial viability, making it an attractive target

• Conservation: The protein is conserved across many Gram-negative pathogens

• Unique Function: LptC has no homologs in mammalian cells, reducing the risk of off-target effects

• Surface Accessibility: As a membrane protein, parts of LptC may be accessible to inhibitors

Experimental Evidence:

• Research has shown that synthetic molecules like iaxo-102 can bind to LptC, sharing the same binding site as LPS

• These compounds could serve as lead structures for developing new antibiotics targeting the biogenesis of LPS

• In vitro binding assays have confirmed that such compounds bind irreversibly to LptC, though with lower affinity than LPS-LptC interactions

Potential Approaches:

• Structure-Based Drug Design:

  • Utilize the crystal structure of LptC to design specific inhibitors

  • Focus on the LPS binding site formed by the β-sheet structure

• Peptide-Based Inhibitors:

  • Design peptides that mimic the LptA-LptC interface to disrupt protein-protein interactions

  • Develop antibodies against surface-exposed regions of LptC

• Small Molecule Inhibitors:

  • Screen for compounds that irreversibly bind to LptC and prevent LPS transport

  • Optimize lead compounds such as iaxo-102 for improved binding and specificity

• Combination Approaches:

  • Target multiple components of the Lpt machinery simultaneously

  • Combine LptC inhibitors with conventional antibiotics to enhance efficacy

Challenges:

• Developing compounds that can penetrate the bacterial outer membrane

• Achieving specificity for H. influenzae LptC versus LptC from commensal bacteria

• Preventing the development of resistance mechanisms

What are the key differences between LptC from H. influenzae and other bacterial species?

While the general function of LptC is conserved across Gram-negative bacteria, several important differences exist between H. influenzae LptC and its homologs in other species:

Structural Comparisons:

• H. influenzae LptC shares the basic twisted boat structure with E. coli LptC, but with subtle differences in the β-sheet arrangement

• The periplasmic domains show similarity, but species-specific variations exist in surface-exposed loops

Sequence Conservation:

Functional Implications:

• Species-specific differences may reflect adaptations to different LPS structures

• These variations could influence:

  • Binding affinity for LPS

  • Interaction with other Lpt machinery components

  • Susceptibility to inhibitors

  • Membrane integration and topology

Research Applications:

• Comparative studies of LptC from multiple species can reveal conserved functional motifs

• Species-specific differences may be exploited for developing narrow-spectrum antibiotics

• Understanding these differences is crucial for translating findings from model organisms to H. influenzae

How do post-translational modifications affect LptC function in H. influenzae?

While direct evidence about post-translational modifications of H. influenzae LptC is limited in the provided search results, research in related systems suggests several potential modifications that could affect function:

Potential Modifications:

• Lipidation:

  • As a lipoprotein, LptC likely undergoes N-terminal lipidation for membrane anchoring

  • The N-terminal sequence "MIEIKMNIRWNVILGVIALCALAWFYSLNQE" contains the membrane-spanning region

• Phosphorylation:

  • Serine, threonine, and tyrosine residues could undergo phosphorylation

  • These modifications might regulate:

    • Protein-protein interactions within the Lpt complex

    • LPS binding affinity

    • Conformational changes associated with the transport cycle

• Acetylation:

  • N-terminal or lysine acetylation could affect protein stability and function

  • H. influenzae is known to use acetylation on other components related to LPS biosynthesis

Regulation Mechanisms:

• Post-translational modifications may serve as regulatory mechanisms to control LPS transport in response to environmental conditions

• These modifications could be part of stress responses or adaptation to different growth phases

Methodological Approaches to Study Modifications:

• Mass Spectrometry:

  • LC-MS/MS analysis of purified LptC to identify modified residues

  • Comparison of modification patterns under different growth conditions

• Site-Directed Mutagenesis:

  • Mutation of potential modification sites to non-modifiable residues

  • Assessment of functional consequences in vivo and in vitro

• Protein-Protein Interaction Studies:

  • Investigation of how modifications affect interactions with other Lpt components

  • Pull-down assays with modified and unmodified LptC

What are the current hypotheses regarding the atomic-level mechanism of LPS transfer by LptC?

Based on the search results and current understanding of the Lpt system, several hypotheses exist regarding the atomic-level mechanism of LPS transfer by LptC:

The Bridge Model:

• LptC, along with LptA, forms a protein bridge that physically connects the inner and outer membranes

• This bridge allows for direct efflux of LPS from its site of synthesis to the cell surface

• Physical interactions between all seven Lpt proteins support this model

Molecular Mechanisms:

• Hydrophobic Slide Mechanism:

  • The hydrophobic grooves formed by the β-sheets of LptC create a slide for the lipid A portion of LPS

  • The acyl chains of lipid A interact with hydrophobic residues in the groove

  • This interaction facilitates extraction from the membrane and transport across the periplasm

• Affinity Gradient Hypothesis:

  • LptA binds LPS with higher affinity than LptC

  • This affinity gradient ensures unidirectional transport of LPS from LptC to LptA

  • The energy for initial extraction comes from ATP hydrolysis by the LptBFG complex, but subsequent transfer may be driven by the affinity gradient

• Conformational Change Model:

  • LptC undergoes conformational changes upon LPS binding

  • These changes may expose interaction surfaces for LptA binding

  • The conformational cycle couples ATP hydrolysis to LPS transport

Evidence and Experimental Approaches:

• X-ray crystallography has revealed the structure of LptC and its potential binding site for LPS

• In vitro binding assays have demonstrated irreversible binding between LptC and LPS with Kd in the μM range

• Competition experiments with synthetic ligands like iaxo-102 have shown that these compounds share the same binding site as LPS

Future Research Directions:

• High-resolution structures of LptC in complex with LPS fragments

• Single-molecule studies to observe the dynamic process of LPS transfer

• Molecular dynamics simulations to model the atomic-level details of LPS extraction and transfer

What are the major technical challenges in studying LptC function and structure?

Researchers face several significant challenges when investigating LptC:

Expression and Purification Challenges:

• Membrane Protein Solubility:

  • As a membrane protein, LptC is inherently hydrophobic and difficult to work with

  • Aggregation and improper folding during recombinant expression are common issues

  • Inclusion body formation necessitates refolding procedures that may not yield native conformations

• Maintaining Native Conformation:

  • Detergents required for solubilization may disrupt native protein-protein interactions

  • The transmembrane domain often poses challenges for structural studies

  • Ensuring proper folding of the periplasmic domain can be difficult

Structural Analysis Limitations:

• Crystallization Difficulties:

  • Membrane proteins are notoriously difficult to crystallize

  • The flexible regions of LptC may impede crystal formation

  • The need for detergents or lipid environments complicates crystallization conditions

• Dynamic Nature:

  • LptC likely undergoes conformational changes during the LPS transport cycle

  • Capturing these different states for structural analysis is challenging

  • The transient nature of protein-protein interactions within the Lpt machinery complicates studies

Functional Assays:

• In vitro Reconstitution:

  • Recapitulating the complete LPS transport process in vitro remains challenging

  • Multiple components of the Lpt machinery must be correctly assembled

  • Membrane environments must be properly mimicked

• Real-time Monitoring:

  • Following LPS movement in real-time is technically demanding

  • Distinguishing between LptC's direct interactions and its role in the larger complex is difficult

Emerging Solutions:

• Nanodiscs and Liposome Systems:

  • Embedding LptC in nanodiscs or liposomes better mimics its native environment

  • These systems allow for functional studies in a membrane-like context

• Cryo-Electron Microscopy:

  • Cryo-EM bypasses the need for crystallization

  • Recent advances allow for high-resolution structures of membrane protein complexes

• Fluorescence-Based Assays:

  • LptC-fluorescent protein fusions enable visualization of localization and dynamics

  • FRET-based approaches can monitor protein-protein interactions and conformational changes

How can researchers differentiate between direct and indirect effects when studying LptC mutants?

Differentiating between direct and indirect effects in LptC mutant studies requires multiple complementary approaches:

Comprehensive Phenotypic Analysis:

• Growth and Viability Assessment:

  • Compare growth rates and viability of wild-type and mutant strains

  • Use multiple growth conditions to identify condition-specific effects

  • Quantify colony-forming units to assess viable cell numbers

• Membrane Integrity Assays:

  • Measure sensitivity to detergents, antibiotics, and other membrane-perturbing agents

  • Use fluorescent dyes to assess membrane permeability

• Ultrastructural Analysis:

  • Employ electron microscopy to visualize membrane organization and defects

  • Quantify changes in cell morphology and envelope architecture

Molecular and Biochemical Approaches:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation to assess interactions between LptC mutants and other Lpt components

  • Bacterial two-hybrid or FRET assays to quantify interaction strengths

  • Pull-down assays with purified components to confirm direct interactions

• LPS Analysis:

  • Characterize LPS profiles using mass spectrometry and gel electrophoresis

  • Quantify LPS in different cellular compartments to track transport defects

  • Assess modifications to LPS structure that might arise as compensatory mechanisms

• Site-Directed Mutagenesis:

  • Create specific single-amino acid mutations rather than domain deletions

  • Design mutations that target specific functions (e.g., LPS binding vs. protein-protein interactions)

  • Generate a series of related mutations to establish structure-function relationships

Complementation and Suppressor Analyses:

• Genetic Complementation:

  • Reintroduce wild-type lptC on a plasmid to confirm phenotype reversal

  • Use domain swapping between species to identify functional conservation

• Suppressor Screens:

  • Identify second-site mutations that suppress lptC mutant phenotypes

  • These suppressors can reveal functional relationships and compensatory pathways

• Controlled Expression:

  • Use inducible promoters to control expression levels of wild-type or mutant LptC

  • Establish dose-response relationships between expression level and phenotype

Control Experiments:

• Multiple Independent Mutants:

  • Generate and characterize multiple independent mutants affecting the same function

  • Consistent phenotypes across different mutational strategies strengthen direct effect claims

• Off-Target Effect Controls:

  • Include mutations in unrelated systems to control for general stress responses

  • Perform transcriptomic or proteomic analyses to identify broader cellular responses

• Time-Course Analyses:

  • Monitor phenotypic changes over time following induction of mutations

  • Primary effects typically manifest before secondary consequences

What new technologies are emerging for studying LptC and the LPS transport system?

Several cutting-edge technologies are transforming research on LptC and the LPS transport system:

Advanced Structural Biology Techniques:

• Single-Particle Cryo-Electron Microscopy:

  • Allows visualization of membrane protein complexes without crystallization

  • Can capture different conformational states of the Lpt machinery

  • Recent advances enable near-atomic resolution of large complexes

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps protein dynamics and solvent accessibility

  • Identifies regions involved in protein-protein interactions

  • Detects conformational changes upon substrate binding

• Integrative Structural Biology:

  • Combines multiple techniques (X-ray crystallography, cryo-EM, NMR, crosslinking-MS)

  • Generates comprehensive structural models of the entire Lpt machinery

  • Bridges resolution gaps between different methods

Advanced Imaging Technologies:

• Super-Resolution Microscopy:

  • Techniques like PALM, STORM, and STED bypass the diffraction limit

  • Enable visualization of protein clusters and dynamics at nanometer scale

  • Can track LptC localization and movement in live cells

• Single-Molecule Tracking:

  • Follows individual LptC molecules in living bacteria

  • Reveals diffusion rates, confinement zones, and interaction kinetics

  • Provides insights into the dynamic behavior of the LPS transport system

• Correlative Light and Electron Microscopy (CLEM):

  • Combines fluorescence localization with ultrastructural context

  • Links protein distribution to membrane organization

  • Provides multi-scale visualization of LPS transport

Functional and High-Throughput Approaches:

• Transformed Recombinant Enrichment Profiling (TREP):

  • Uses natural transformation to generate complex pools of recombinants

  • Phenotypic selection enriches for specific recombinants

  • Deep sequencing identifies genetic variations responsible for phenotypes

• CRISPR-Cas9 Gene Editing:

  • Enables precise genome modifications in H. influenzae

  • Facilitates creation of tagged proteins and specific mutations

  • Allows for simultaneous modification of multiple Lpt components

• Microfluidics and Bacterial Microcolonies:

  • Provides controlled environments for single-cell studies

  • Enables real-time monitoring of LPS transport in living cells

  • Facilitates high-throughput screening of conditions and mutations

Computational and Systems Biology Approaches:

• Molecular Dynamics Simulations:

  • Models atomic-level details of LPS-protein interactions

  • Simulates conformational changes during transport

  • Predicts effects of mutations on structure and function

• Machine Learning for Structural Prediction:

  • AlphaFold and similar tools predict protein structures with high accuracy

  • Helps model structures of LptC variants and complexes

  • Guides experimental design for validation

• Systems Biology of LPS Transport:

  • Integrates transcriptomics, proteomics, and metabolomics data

  • Maps the regulatory networks controlling LPS synthesis and transport

  • Predicts system-wide responses to perturbations in LptC function

These emerging technologies provide researchers with unprecedented capabilities to study LptC and the LPS transport system at multiple scales, from atomic structures to whole-cell functions.