Recombinant Haemophilus influenzae Lipid A biosynthesis lauroyl acyltransferase (htrB)

What is the molecular function of htrB in Haemophilus influenzae?

The htrB gene in Haemophilus influenzae encodes a ketodeoxyoctonate phosphate-dependent acyltransferase that is responsible for performing one of the late acylation reactions in lipid A synthesis . This enzyme plays a critical role in the complete acylation of the lipooligosaccharide (LOS) structure. Specifically, it contributes to the formation of the hexa-acylated lipid A in wild-type H. influenzae strains . The acylation process is essential for proper LOS structure and function, which in turn affects numerous bacterial phenotypes including host colonization, immune evasion, and antimicrobial peptide resistance. Functional studies have demonstrated that when htrB is mutated, the resulting bacteria produce a modified LOS with a mixture of penta- and tetra-acylated lipid A that is also hyperphosphorylated compared to the wild-type structure .

How does htrB mutation affect bacterial phenotypes in experimental models?

Mutation in the htrB gene produces multiple significant phenotypic changes in H. influenzae. In human bronchiolar xenograft models, htrB mutants demonstrate a substantial reduction in colonization capacity compared to parental strains . This decreased colonization ability suggests that proper acylation of lipid A is a key factor in the organism's successful colonization of normal airway tissues.

In in vitro experiments with human airway epithelial cells (16HBE14o- cell line), htrB mutants elicit lesser degrees of cytoskeletal rearrangement and reduced stimulation of host cell signaling pathways, which correlates with decreased intracellular survival of the bacteria . Additionally, htrB mutants exhibit significantly increased sensitivity to antimicrobial peptides, with research demonstrating greater than 45-fold increased sensitivity to human β-defensin 2 (HBD-2) compared to wild-type strains . Importantly, when complementation with functional htrB is provided in trans, restoring acylation competence, this heightened sensitivity to antimicrobial peptides is reversed . In vivo studies using infant rat models have further confirmed that htrB mutants of nontypeable H. influenzae (NTHi) show significant attenuation, highlighting the gene's importance in pathogenesis .

What experimental approaches are used to identify htrB expression during infection?

Researchers have employed differential display techniques to identify H. influenzae mRNA that reflects genes preferentially expressed during infection compared to in vitro growth conditions . In this methodology, bacterial RNA is extracted from infected human airway xenografts and compared to RNA from bacteria grown in standard laboratory media. The differential display approach led to the identification of eleven mRNA fragments that showed consistently increased expression when the bacteria grew in xenografts .

For htrB specifically, after identification through differential display, the gene's expression can be further analyzed through reverse transcription reactions using 10-mer primers, incorporating radioactive nucleotides to generate labeled cDNA fragments that can be visualized and quantified . Following confirmation of differential expression, targeted PCR using primers directed against regions flanking the htrB gene (such as the primers htr-F and htr-R corresponding to specific genome coordinates in H. influenzae) can be used to amplify the full gene for subsequent cloning and functional studies . This systematic approach allows researchers to confirm that htrB expression is upregulated during colonization of human airway tissue, supporting its role in host-pathogen interactions.

What methodologies are recommended for generating and characterizing htrB mutants?

Creating htrB mutants in H. influenzae involves several precise molecular techniques. Based on published methodologies, researchers should first identify the htrB gene sequence using the H. influenzae genome (such as strain Rd KW20) as reference . PCR primers targeting flanking regions of the gene should be designed (e.g., htr-F: AGCTTTACGCCACGAAACAAA and htr-R: CGCAAAATTCACGAATAGCA) . The amplified fragment (approximately 1.3-kb) should be gel purified, blunt-ended, and cloned into a suitable vector such as pUC19 before transformation into E. coli (strain HB101 has been used successfully) .

For mutagenesis, transposon mutagenesis using λTn5 following the method described by de Bruijn and Lupski is effective . The resulting plasmid containing the disrupted htrB gene should be linearized and used to transform competent H. influenzae cells, with transformants selected on appropriate antibiotic-containing media (kanamycin at 15 μg/ml has been used) . Allelic exchange should be confirmed through Southern blot analysis to ensure proper integration of the disrupted gene into the chromosome .

For complementation studies, the wild-type htrB gene can be excised from a carrier plasmid (such as pCRII) using restriction enzymes (EcoRI has been used) and cloned into a shuttle vector capable of replication in both E. coli and H. influenzae, such as pGB19 . The construct should then be introduced into the htrB mutant strain through electroporation to restore acylation function . This complementation approach is critical for confirming that observed phenotypic changes are specifically due to the htrB mutation rather than polar effects or secondary mutations.

How does htrB-dependent lipid A acylation affect host immune responses?

The acylation status of lipid A significantly modulates host immune responses to H. influenzae. Studies have shown that monocytes and epithelial cells challenged with LOS isolated from an htrB mutant produce significantly less proinflammatory cytokines, specifically tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), compared to those challenged with LOS from the parental strain . This indicates that the complete acylation of lipid A is crucial for the stimulation of these inflammatory pathways.

The mechanism likely involves altered recognition of under-acylated lipid A by pattern recognition receptors such as TLR4, resulting in modified downstream signaling. The hyperphosphorylated LOS with penta- and tetra-acylated lipid A produced by htrB mutants presents different molecular patterns to immune receptors compared to the hexa-acylated lipid A of wild-type strains. This structural difference translates to altered host cell signaling, which may explain the reduced cytoskeletal rearrangements observed in epithelial cells infected with htrB mutants .

The modification of lipid A acylation also affects the bacteria's susceptibility to innate immune defenses, particularly antimicrobial peptides. NTHi lipooligosaccharide htrB mutants show dramatically increased sensitivity (more than 45-fold) to human β-defensin 2 (HBD-2) . This heightened susceptibility can be reversed when acylation competence is restored through complementation with htrB in trans , confirming the direct relationship between acylation status and antimicrobial peptide resistance.

What is the relationship between htrB function and H. influenzae colonization mechanisms?

The research evidence strongly indicates that htrB-mediated lipid A acylation plays a critical role in H. influenzae colonization of human airways. Inoculation of human bronchiolar xenografts with htrB mutants revealed a significant reduction in colonization capacity compared to parental strains . This reduced colonization ability suggests that proper acylation of lipid A represents a key bacterial adaptation for successful interaction with host airway tissues.

The molecular basis for this colonization defect appears multifaceted. First, htrB mutants demonstrate decreased ability to elicit cytoskeletal rearrangements in host epithelial cells , which likely impairs the bacteria's capacity to adhere to and potentially invade these cells. Second, the mutants show less stimulation of host cell signaling pathways , which may disrupt the bacteria's ability to modulate the local cellular environment to favor colonization. Third, htrB mutants exhibit decreased intracellular survival , suggesting that even if they successfully invade host cells, their persistence is compromised.

Additionally, the increased susceptibility of htrB mutants to antimicrobial peptides such as human β-defensin 2 likely contributes to their reduced colonization capacity, as these peptides represent a critical component of the innate immune defense at mucosal surfaces. The acylation of lipid A therefore appears to serve as a protective modification that shields H. influenzae from these host defense mechanisms, allowing for more successful colonization of the human airway.

What experimental systems are most appropriate for studying htrB function in host-pathogen interactions?

Multiple experimental systems have proven valuable for investigating htrB function in host-pathogen interactions, each with specific advantages for addressing different research questions:

• Human Bronchiolar Xenograft Model: This system has been successfully employed to investigate host-bacterial interactions involved in airway colonization by H. influenzae . The model allows for the growth of bacteria in an environment that closely mimics the human airway, making it particularly valuable for studying genes differentially expressed during colonization. Using this model, researchers identified htrB as being preferentially expressed during xenograft colonization compared to in vitro growth .

• Cell Culture Systems: Immortalized human airway epithelial cell lines, such as 16HBE14o- cells, provide a controlled system for examining specific cellular responses to wild-type and mutant H. influenzae strains . These systems allow quantitative assessment of cytoskeletal rearrangements, host cell signaling activation, bacterial adherence, and invasion. For invasion studies, gentamicin-survival assays can be employed, where cells are infected with bacteria, treated with gentamicin to kill extracellular bacteria, and then lysed to enumerate surviving intracellular bacteria .

• Animal Models: The infant rat model has been used to demonstrate the attenuation of htrB mutants in vivo , providing valuable information about the gene's role in pathogenesis. While this model does not perfectly replicate human infections, it allows for assessment of bacterial fitness in a complete host organism with functional immune responses.

• Antimicrobial Peptide Susceptibility Assays: These in vitro systems permit direct testing of how htrB mutations affect susceptibility to host defense molecules such as human β-defensins . The large magnitude of effect observed with htrB mutants (>45-fold increased sensitivity) makes these assays particularly informative for understanding the role of lipid A acylation in antimicrobial peptide resistance.

For comprehensive analysis of htrB function, a combination of these experimental systems is recommended, allowing researchers to connect molecular mechanisms to phenotypic outcomes across different levels of biological complexity.

What analytical techniques can be used to characterize LOS structural changes in htrB mutants?

Characterizing the structural changes in LOS from htrB mutants requires a combination of advanced analytical techniques:

When implementing these techniques, researchers should carefully isolate LOS using methods that minimize contamination with other bacterial components, such as phenol-water extraction followed by enzymatic treatments to remove proteins and nucleic acids. For meaningful comparison, wild-type, mutant, and complemented strains should be grown under identical conditions before LOS extraction.

How can researchers quantitatively assess the impact of htrB mutation on bacterial pathogenesis?

Quantitative assessment of htrB mutation effects on bacterial pathogenesis requires multi-dimensional approaches:

• Colonization Efficiency: In human bronchiolar xenograft models, colonization capacity can be quantified by determining the number of viable bacteria recovered from the xenograft at various time points post-inoculation . This provides a direct measure of the mutant's ability to establish and maintain colonization compared to wild-type strains.

• Cellular Interaction Metrics: Several quantitative parameters can be measured in cell culture systems:

  • Adherence: Calculated as the percentage of the initial bacterial inoculum that remains associated with host cells after thorough washing .

  • Invasion: Determined using gentamicin-survival assays, where invasion is defined as the percentage of the inoculum recovered as viable CFU after gentamicin treatment, which kills extracellular but not intracellular bacteria .

  • Cytoskeletal Rearrangement: Quantified through fluorescent labeling of cytoskeletal components followed by microscopy and image analysis to measure the degree of reorganization in response to bacterial infection.

• Immunological Response Parameters:

  • Cytokine Production: ELISA or multiplex cytokine assays can measure the levels of inflammatory mediators (like TNF-α and IL-6) produced by host cells in response to wild-type versus mutant bacteria or their isolated LOS .

  • Cell Signaling Activation: Western blotting or phospho-flow cytometry can quantify the activation of specific signaling pathways in host cells.

• Antimicrobial Peptide Susceptibility: The minimum inhibitory concentration (MIC) or kill curves can be used to quantify the increased sensitivity of htrB mutants to antimicrobial peptides like human β-defensin 2 . The >45-fold increased sensitivity observed in previous studies provides a robust quantitative measure of how lipid A acylation affects resistance to these host defense molecules.

• In Vivo Virulence Metrics: In animal models such as the infant rat model, researchers can quantify bacterial loads in various tissues, survival rates, and biomarkers of inflammation to assess the attenuation of htrB mutants .

Statistical analysis of these quantitative measures, typically using paired t-tests or ANOVA depending on the experimental design, allows for rigorous evaluation of the significance of observed differences between wild-type and mutant strains .

How should researchers address variability in acylation patterns when studying htrB mutants?

Addressing variability in acylation patterns when studying htrB mutants requires systematic approaches:

• Standardized Growth Conditions: Since acylation patterns can be influenced by growth conditions, researchers should standardize parameters such as media composition, temperature, growth phase, and oxygen levels when comparing different strains. For H. influenzae, brain heart infusion broth supplemented with 10 μg of hemin/ml and 10 μg of NAD/ml (sBHI) has been successfully used .

• Multiple Mutant Clones: Researchers should analyze multiple independently derived htrB mutant clones to ensure that observed acylation patterns are consistently associated with the mutation rather than clone-specific variations. This approach strengthens the correlation between genotype and phenotype.

• Complementation Controls: Complementation of htrB mutations in trans should restore wild-type acylation patterns. This critical control, as demonstrated in studies where complementation reversed the increased sensitivity to human β-defensin 2 , confirms that observed changes are specifically due to htrB disruption rather than secondary mutations or polar effects.

• Quantitative Analytical Methods: Mass spectrometry can provide quantitative data on the proportions of different acylated species (hexa-, penta-, and tetra-acylated lipid A) in both wild-type and mutant samples. This allows for statistical comparison of acylation profiles across strains and experimental conditions.

• Heterogeneity Analysis: Since htrB mutants produce a mixture of penta- and tetra-acylated lipid A , researchers should characterize this heterogeneity and determine whether the distribution of different acylated forms varies under different conditions or in different genetic backgrounds.

What are the key considerations when interpreting contradictory findings on htrB function across different studies?

When faced with seemingly contradictory findings regarding htrB function, researchers should consider several important factors:

• Strain Differences: Different H. influenzae strains, particularly typeable versus nontypeable strains, may show variations in htrB function or in the phenotypic consequences of htrB mutation. The studies included in the search results focused primarily on nontypeable H. influenzae (NTHi) , but extrapolation to other strains should be done cautiously.

• Genetic Background Effects: The impact of htrB mutation may depend on the expression or function of other genes involved in LOS biosynthesis or related pathways. For example, the presence or absence of phosphorylcholine (ChoP) on the LOS affects the sensitivity of H. influenzae to human antimicrobial peptides , which could interact with htrB-dependent phenotypes.

• Experimental Model Variations: Different experimental systems (cell lines, animal models, xenograft models) may yield different results regarding htrB function. The human bronchiolar xenograft model provides insights specific to human airway colonization, while cell culture systems and antimicrobial peptide susceptibility assays address more specific aspects of host-pathogen interaction.

• Methodological Differences: Variations in how htrB mutants are constructed (insertion versus deletion mutations), how complementation is achieved, and how phenotypes are measured can all contribute to apparently contradictory findings. Researchers should carefully compare methodological details when interpreting cross-study differences.

• Environmental Conditions: Growth conditions, infection protocols, and host factors can all influence bacterial gene expression and phenotypes. For instance, the upregulation of htrB expression observed specifically in xenografts but not during in vitro growth highlights the importance of environmental context.

To reconcile contradictory findings, researchers should perform side-by-side comparisons using standardized methods and multiple strains. When reporting results, contextual factors should be clearly described, and claims should be appropriately scoped to the specific experimental conditions and bacterial strains used.

What emerging techniques could advance our understanding of htrB function in H. influenzae?

Several cutting-edge methodologies hold promise for deepening our understanding of htrB function:

• CRISPR-Cas9 Gene Editing: This technique could allow for more precise modification of the htrB gene, including the creation of point mutations that affect specific functional domains rather than complete gene disruption. This would enable more nuanced analysis of structure-function relationships in the htrB enzyme.

• Single-Cell RNA Sequencing: Applying this technology to infected host cells could reveal heterogeneity in host responses to wild-type versus htrB mutant bacteria, potentially uncovering subpopulations of cells with distinct response patterns that might be masked in bulk analyses.

• Advanced Live Imaging Techniques: Real-time visualization of host-pathogen interactions using fluorescently labeled bacteria and host cellular components could provide dynamic information about how htrB-dependent lipid A modifications affect the early stages of bacterial attachment, invasion, and host cell response.

• Systems Biology Approaches: Integration of transcriptomic, proteomic, and metabolomic data from both the bacteria and host cells during infection could create comprehensive models of how htrB function influences the complex interplay between H. influenzae and the human airway.

• Humanized Mouse Models: Development of mouse models with humanized respiratory epithelium could bridge the gap between in vitro human cell studies and in vivo animal models, potentially providing more translatable insights into the role of htrB in human infections.

These advanced techniques, combined with the established methodologies described in the existing literature , could significantly enhance our understanding of how htrB-mediated lipid A acylation contributes to H. influenzae pathogenesis and host interaction, potentially opening new avenues for therapeutic intervention.