Recombinant Haemophilus parasuis serovar 5 UPF0283 membrane protein HAPS_0079 (HAPS_0079)

What is Haemophilus parasuis and why is the UPF0283 membrane protein HAPS_0079 significant in research?

Haemophilus parasuis (HPS) is the causative agent of Glässer's disease in pigs, characterized by fibrinous polyserositis, arthritis, and meningitis. It often presents as a mixed infection with other upper respiratory tract pathogens, causing significant economic losses to the pig industry . The UPF0283 membrane protein HAPS_0079 is a membrane-associated protein found in serovar 5, one of the 15 known serovars of H. parasuis. Membrane proteins are particularly important in bacterial pathogenesis as they often mediate critical functions including adhesion, invasion, and evasion of host immune responses. Outer membrane proteins like HAPS_0079 may play roles similar to other characterized H. parasuis membrane proteins, such as OMP P2, which has been shown to affect adhesion to host cells and resistance to serum killing .

What isolation methods are most effective for studying H. parasuis membrane proteins?

For effective isolation of membrane proteins from H. parasuis, a modified centrifugation-based method similar to that used for other gram-negative bacteria is recommended. The protocol typically involves:

• Bacterial culture harvesting by centrifugation (1000 g, 5 min, 4°C)

• Homogenization in buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 250 mM sucrose, 1 mM EDTA, and protease inhibitor cocktail

• Initial centrifugation at 10,000 g for 10 min at 4°C to remove cellular debris

• Ultracentrifugation of supernatant at approximately 430,000 g for 30 min at 4°C

• Resuspension of the membrane-containing pellet in appropriate buffer

Further enrichment can be achieved through additional washing steps, such as urea wash or alkaline wash, which can significantly improve the purity of membrane protein preparations. Each washing method has distinct advantages for different experimental goals:

For subsequent analysis, LC-MS/MS is typically employed with proper validation using multiple technical replicates to ensure reliability of identification .

How can researchers differentiate H. parasuis serotype 5 from other serotypes for HAPS_0079 studies?

Serotyping of H. parasuis isolates is critical for studying serovar-specific proteins like HAPS_0079. Currently, 15 serovars have been identified, with serovar 5 being among the more virulent types . The differentiation methods include:

• Immunodiffusion assays: Using serovar-specific antisera for gel precipitation reactions

• PCR-based methods: Targeting serovar-specific genes

• Indirect hemagglutination test: A standard serotyping method for H. parasuis

• MALDI-TOF mass spectrometry: Allows rapid identification of H. parasuis serovars based on protein profile differences

For confirming the presence of the HAPS_0079 gene specifically, PCR amplification using primers targeting the UPF0283 gene followed by sequencing is recommended. Additionally, whole-genome sequencing can provide definitive identification of the strain and confirm the presence and sequence of the HAPS_0079 gene within the isolate.

What expression systems are optimal for producing recombinant HAPS_0079 protein?

When expressing recombinant membrane proteins from H. parasuis, several expression systems can be considered:

For HAPS_0079 specifically, an E. coli expression system with a His-tag for purification has been documented . The protocol should include:

• Gene synthesis or amplification optimized for the expression host

• Cloning into an appropriate vector with an inducible promoter

• Expression in the selected host under optimized conditions

• Membrane fraction isolation by ultracentrifugation

• Solubilization using appropriate detergents (DDM, LDAO, or CHAPS)

• Purification via affinity chromatography using the His-tag

What structural characterization techniques are most informative for studying HAPS_0079?

For comprehensive structural characterization of HAPS_0079, a multi-technique approach is recommended:

For membrane proteins like HAPS_0079, integrating computational approaches such as molecular dynamics simulations with experimental data can provide more comprehensive structural insights, particularly regarding membrane interactions and conformational flexibility.

How can HAPS_0079 function be assessed in the context of H. parasuis virulence?

To assess the function of HAPS_0079 in H. parasuis virulence, the following methodological approaches are recommended:

• Gene knockout studies: Create HAPS_0079 deletion mutants using homologous recombination or CRISPR-Cas9 systems. Comparison of the ΔHP0079 mutant with wild-type strains in various virulence assays would reveal the protein's contribution to pathogenicity, similar to studies performed with other H. parasuis genes .

• In vitro virulence assays:

  • Adhesion and invasion assays using porcine cell lines (e.g., PK-15, PUVEC, 3D4/21)

  • Serum resistance assays to evaluate complement evasion

  • Biofilm formation assays

  • Resistance to phagocytosis by porcine alveolar macrophages

• Protein-protein interaction studies:

  • Pull-down assays to identify host targets

  • Protein microarrays to screen for interactions with host factors

  • Yeast two-hybrid or bacterial two-hybrid systems for interaction mapping

• In vivo infection models:

  • Pig infection models with wild-type versus ΔHAPS_0079 mutants

  • Tissue colonization and pathology assessment

  • Immune response evaluation

Based on studies with other H. parasuis virulence factors, it would be valuable to examine whether HAPS_0079 contributes to adhesion to epithelial cells, evasion of phagocytosis, or resistance to serum killing - all critical virulence mechanisms in this pathogen .

What bioinformatics approaches can predict functional domains of HAPS_0079?

A comprehensive bioinformatic analysis of HAPS_0079 should include:

• Sequence-based predictions:

  • Transmembrane topology prediction using TMHMM, TOPCONS, or Phobius

  • Signal peptide prediction with SignalP

  • Conserved domain analysis using InterProScan, PFAM, and CDD

  • Sequence alignment with homologous proteins across bacterial species

• Structural predictions:

  • Ab initio modeling using tools like AlphaFold2 or RoseTTAFold

  • Template-based modeling if structural homologs exist

  • Molecular dynamics simulations to assess stability in membrane environments

• Functional predictions:

  • Gene neighborhood analysis to identify functionally related genes

  • Co-expression network analysis using existing transcriptomic data

  • Phylogenetic profiling to identify patterns of evolutionary conservation

• Binding site prediction:

  • Protein-ligand binding site prediction using tools like COACH or FTSite

  • Protein-protein interaction surface prediction using SPPIDER or PredUs

Combining these computational approaches with experimental validation can provide robust hypotheses about HAPS_0079 function and potential roles in bacterial virulence or survival.

How do proteomics approaches help identify interaction partners of HAPS_0079?

To identify interaction partners of HAPS_0079, several complementary proteomics approaches should be employed:

• Co-immunoprecipitation coupled with mass spectrometry (Co-IP-MS):

  • Express tagged HAPS_0079 in H. parasuis

  • Crosslink protein complexes in vivo (optional step)

  • Immunoprecipitate using tag-specific antibodies

  • Identify co-precipitated proteins via LC-MS/MS

  • Validate interactions with reverse Co-IP and Western blotting

• Proximity-dependent biotin identification (BioID) or APEX2:

  • Fuse HAPS_0079 with a biotin ligase (BioID) or an engineered peroxidase (APEX2)

  • Express the fusion protein in H. parasuis

  • Activate enzyme to biotinylate nearby proteins

  • Purify biotinylated proteins and identify by MS

• Chemical crosslinking mass spectrometry (XL-MS):

  • Apply crosslinking reagents to intact cells or purified membrane fractions

  • Digest crosslinked protein complexes

  • Identify crosslinked peptides by specialized MS methods

  • Map interaction interfaces at amino acid resolution

• Quantitative interactomics:

  • Compare protein interactions under different conditions (e.g., infection vs. non-infection)

  • Use SILAC, TMT, or label-free quantification

  • Apply statistical analysis to identify significant interaction changes

The identified interaction partners should be categorized into functional groups (e.g., host receptors, immune factors, other bacterial proteins) and validated through independent methods such as bacterial two-hybrid assays or fluorescence resonance energy transfer (FRET).

What experimental designs can elucidate the role of HAPS_0079 in vaccine development?

For investigating HAPS_0079 as a potential vaccine antigen, a systematic experimental approach is recommended:

• Immunogenicity assessment:

  • Immunize mice or pigs with purified recombinant HAPS_0079

  • Measure antibody titers by ELISA

  • Assess T-cell responses through proliferation assays and cytokine profiling

  • Compare responses across different adjuvant formulations

• Cross-protection analysis:

  • Evaluate antibody cross-reactivity against multiple H. parasuis serovars

  • Perform sequence and structural analysis of HAPS_0079 across serovars

  • Identify conserved epitopes that could provide broad protection

• Functional antibody assays:

  • Bacterial opsonization assays

  • Complement-mediated killing assays

  • Adhesion inhibition assays with relevant cell lines

• Challenge studies:

  • Design vaccination-challenge trials in pigs

  • Compare HAPS_0079 alone versus in combination with other antigens

  • Include appropriate controls (commercial vaccines, inactivated whole-cell preparations)

  • Assess protection based on clinical signs, bacterial load, and pathological findings

• Delivery system optimization:

  • Test different formulations (soluble protein, liposomes, virus-like particles)

  • Evaluate mucosal versus parenteral administration routes

  • Consider prime-boost strategies

This methodical approach would provide comprehensive data on whether HAPS_0079 has potential as a component in subunit vaccines against Glässer's disease, similar to other outer membrane proteins that have shown promise as vaccine candidates .

What are the optimal conditions for culturing H. parasuis for membrane protein studies?

For successful membrane protein studies, optimized culture conditions are essential. H. parasuis has specific growth requirements:

Batch-to-batch variation should be minimized by standardizing inoculum size, monitoring growth curves, and harvesting at consistent optical densities. For membrane protein studies specifically, adding a membrane stabilizing agent like sucrose (250 mM) to the harvesting buffer helps maintain protein integrity during isolation procedures .

How can researchers overcome challenges in HAPS_0079 solubilization and purification?

Membrane protein solubilization and purification present significant challenges. For HAPS_0079, the following optimized protocol is recommended:

• Solubilization screening:

  • Test multiple detergents (DDM, LDAO, CHAPS, Triton X-100) at various concentrations

  • Evaluate solubilization efficiency by Western blot

  • Assess protein stability over time in each detergent

• Optimal solubilization conditions:

  • Buffer: 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol

  • Detergent: Start with 1% DDM (w/v) for initial solubilization

  • Incubation: 1 hour at 4°C with gentle rotation

  • Clearance: Ultracentrifugation at 100,000 g for 1 hour

• Purification strategy:

  • IMAC (Immobilized Metal Affinity Chromatography) using Ni-NTA for His-tagged protein

  • Buffer containing reduced detergent concentration (0.05-0.1% DDM)

  • Stepwise elution with imidazole gradient

  • Size exclusion chromatography as a polishing step

• Common challenges and solutions:

• Quality control:

  • SDS-PAGE to assess purity

  • Western blotting for identity confirmation

  • Dynamic light scattering to evaluate monodispersity

  • Circular dichroism to confirm proper folding

This systematic approach maximizes the chances of obtaining functional, properly folded HAPS_0079 protein for subsequent studies.

What controls should be included in experiments evaluating HAPS_0079 function?

Rigorous experimental design requires appropriate controls. For HAPS_0079 functional studies, the following controls should be included:

• Genetic controls:

  • Wild-type H. parasuis serovar 5 strain (positive control)

  • HAPS_0079 knockout mutant (negative control)

  • Complemented mutant (restored HAPS_0079 expression)

  • Heterologous expression control (e.g., E. coli expressing HAPS_0079)

• Protein-level controls:

  • Denatured HAPS_0079 (to confirm structure-dependent functions)

  • Related membrane protein from H. parasuis (specificity control)

  • Membrane protein from unrelated bacteria (non-specific control)

• Host interaction controls:

  • Uninfected host cells

  • Host cells treated with inhibitors of specific pathways

  • Host cells with knockdown/knockout of suspected interaction partners

• Technical controls:

  • Multiple biological replicates (minimum n=3)

  • Technical replicates for each biological sample

  • Vehicle/buffer-only controls

  • Isotype controls for antibody-based assays

• Data analysis controls:

  • Appropriate statistical tests with multiple testing correction

  • Blinded analysis where applicable

  • Validation with independent methodologies

Including these comprehensive controls ensures that observed effects can be specifically attributed to HAPS_0079 function rather than experimental artifacts or non-specific effects.

How can researchers design experiments to assess HAPS_0079 contribution to H. parasuis virulence?

To systematically evaluate HAPS_0079's role in virulence, a multi-faceted experimental design is necessary:

• Construction of isogenic mutants:

  • Generate clean deletion of HAPS_0079 (ΔHAPS_0079)

  • Create complemented strain (ΔHAPS_0079+pHAPS_0079)

  • Develop point mutants targeting predicted functional domains

• In vitro virulence phenotype assessment:

• Ex vivo tissue models:

  • Primary porcine respiratory epithelial cell cultures

  • Porcine precision-cut lung slices

  • Measure bacterial adherence, tissue damage, and inflammatory responses

• In vivo infection models:

  • Intranasal challenge in piglets (ethical considerations necessary)

  • Monitor clinical signs, bacterial loads in tissues

  • Assess histopathological changes and inflammatory markers

• Transcriptomic and proteomic analyses:

  • Compare gene/protein expression profiles between wild-type and ΔHAPS_0079

  • Identify compensatory mechanisms or affected pathways

  • Examine host response differences

This comprehensive approach would provide robust evidence regarding the contribution of HAPS_0079 to H. parasuis virulence, similar to studies conducted with other virulence factors such as polysaccharide biosynthetic proteins and CDT toxins .

What statistical approaches are appropriate for analyzing HAPS_0079 experimental data?

• For comparative studies (wild-type vs. mutant):

  • Student's t-test for single comparisons between two groups

  • ANOVA with appropriate post-hoc tests (Tukey, Bonferroni) for multiple group comparisons

  • Non-parametric alternatives (Mann-Whitney, Kruskal-Wallis) if normality assumptions are violated

• For time-course experiments:

  • Repeated measures ANOVA

  • Mixed-effects models to account for both fixed and random effects

  • Area under the curve (AUC) analysis followed by appropriate comparisons

• For dose-response studies:

  • Non-linear regression to fit appropriate models

  • EC50/IC50 determination with confidence intervals

  • Comparison of curve parameters across experimental conditions

• For high-dimensional data (proteomics, transcriptomics):

  • Multiple testing correction (Benjamini-Hochberg, Bonferroni)

  • False discovery rate (FDR) control

  • Dimension reduction techniques (PCA, t-SNE)

  • Clustering algorithms for pattern identification

• Sample size and power considerations:

  • A priori power analysis to determine required sample sizes

  • Effect size estimation based on preliminary data

  • Minimum biological replicates: n=3, preferably n≥5 for robust analysis

Statistical analysis should be performed using established software such as GraphPad Prism, R, or specialized packages depending on the experimental design . All analyses should include appropriate measures of central tendency and dispersion, with clear reporting of p-values and confidence intervals.

How can researchers integrate HAPS_0079 findings with broader H. parasuis pathogenesis research?

Integrating HAPS_0079 research into the broader context of H. parasuis pathogenesis requires a systematic approach: