Recombinant Major outer membrane protein (ompH)

What is OmpH and which organisms express it?

OmpH is one of the main outer-membrane proteins found in a wide array of Gram-negative bacteria including Pasteurella multocida, Aeromonas salmonicida, Shigella dysenteriae, and Escherichia coli. It serves critical structural and functional roles in bacterial cell membranes . In pathogenic bacteria such as P. multocida, OmpH contributes significantly to virulence mechanisms while maintaining immunogenic properties .

What are the primary physiological functions of OmpH in bacterial systems?

OmpH serves multiple essential physiological functions including maintenance of structural integrity and morphology of bacterial cells, porin activity (facilitating the passage of small molecules across the membrane), and roles in conjugation and bacteriophage binding . These functions make OmpH critical for bacterial survival and pathogenicity, particularly in host environments where structural stability and nutrient acquisition are essential for persistence.

What are proven methodologies for cloning the ompH gene from bacterial sources?

Successful cloning of the ompH gene typically follows these research-validated steps:

• Design primers specific to the ompH gene sequence (as demonstrated with P. multocida)

• PCR amplification of the target gene from bacterial genomic DNA

• Insertion into expression vectors such as pET-3d plasmid systems

• Transformation into competent E. coli cells (commonly BL21(DE3) strains)

• Selection of positive transformants using appropriate antibiotic resistance markers

• Verification of successful cloning through restriction enzyme analysis and sequencing

What expression systems yield optimal recombinant OmpH production?

E. coli expression systems have been extensively validated for recombinant OmpH production. The pET expression system with BL21(DE3) host strains has proven particularly effective as it allows for high-level protein expression under control of T7 promoters . This approach facilitates the production of sufficient quantities of recombinant protein for subsequent purification and analysis, making it the preferred system for most OmpH research applications.

How can researchers troubleshoot common challenges in recombinant OmpH expression?

Common challenges in recombinant OmpH expression include protein insolubility and formation of inclusion bodies. Researchers can address these issues through:

• Optimization of induction conditions (temperature, IPTG concentration, and duration)

• Implementation of specialized solubilization-renaturation procedures for isolating protein from inclusion bodies

• Use of fusion partners to enhance solubility

• Testing different host strains with varying genetic backgrounds

• Modifying buffer compositions during purification to maintain protein stability and native conformation

How does OmpH contribute to bacterial virulence mechanisms?

OmpH contributes to bacterial virulence through multiple mechanisms as evidenced by comparative studies between wild-type and OmpH-deficient strains:

• Facilitates bacterial colonization and persistence in host tissues

• Contributes to protection against host immune responses

• Influences bacterial dissemination throughout host tissues

• Affects severity of pathological changes in infected tissues

Research with OmpH gene deletion mutants in P. multocida has demonstrated that bacterial loads are significantly lower in tissues infected with OmpH-deficient strains compared to wild-type strains, confirming OmpH's role in pathogenesis .

What protein interaction networks are associated with OmpH during host infection?

OmpH engages in complex protein interaction networks during host infection. Proteomic analyses have revealed that OmpH deletion affects 57 of 773 expressed proteins in P. multocida . These interactions involve both coding proteins (ropE, HSPBP1, FERH, ATP10A, ABCA13) and immune response-related proteins (RRP7A, IL-10, IFN-γ, IL-17A, EGFR, dnaJ), forming an intricate network that influences host-pathogen interactions .

How can researchers quantitatively assess the impact of OmpH on bacterial proliferation in host tissues?

Researchers can quantitatively assess OmpH's impact on bacterial proliferation through systematic tissue sampling and bacterial enumeration studies. The table below demonstrates a comparative analysis of bacterial loads in various organs from yaks infected with wild-type (P0910) versus OmpH-deficient (ΔOmpH) P. multocida:

This data demonstrates that bacterial loads are generally higher in wild-type infections, particularly in the spleen, which appears to be a primary target organ for P. multocida .

What is the optimal methodology for constructing OmpH gene deletion mutants?

The research-validated methodology for constructing OmpH gene deletion mutants involves:

• PCR amplification of homologous recombination arms upstream (approximately 362 bp) and downstream (approximately 462 bp) of the OmpH gene using specific primers

• Connection of these arms via overlapping PCR

• Insertion into a suitable suicide vector (such as pEX18AP) using appropriate restriction sites (EcoRI and BamHI)

• Transformation into E. coli DH5α for plasmid propagation

• Transfer of the recombinant plasmid into target bacteria via electroporation

• Selection of mutants using appropriate antibiotic resistance markers

• Verification of gene deletion through PCR analysis

How does OmpH deletion affect the pathological progression of bacterial infections in animal models?

OmpH deletion significantly alters the pathological progression of bacterial infections:

• Delayed onset of clinical symptoms (16 hours post-infection for ΔOmpH versus 11 hours for wild-type)

• Milder clinical manifestations including reduced fever, less pronounced anorexia and respiratory distress

• Significantly reduced pathological changes in tissues and organs

• Reduced hemorrhage and tissue damage in organs such as thymus, heart, liver, spleen, and kidney

• Lower bacterial loads across multiple organ systems

These findings indicate that OmpH contributes substantially to the virulence and tissue damage associated with P. multocida infection.

What molecular pathways are affected by OmpH deletion based on proteomics analysis?

Proteomics analysis has revealed that OmpH deletion affects multiple molecular pathways:

KEGG pathway enrichment analysis identified 20 major pathways regulated by 57 differentially expressed proteins between wild-type and ΔOmpH strains, including:

• ABC transportation (ko02010)

• Two-component system (ko02020)

• RNA degradation (ko03018)

• RNA polymerase (ko03020)

• Glycolysis/gluconeogenesis (ko00010)

• Ubiquinone and terpenoid-quinone biosynthesis (ko00130)

• Citrate cycle (TCA cycle) (ko00020)

• Fructose and mannose metabolism (ko00051)

These findings suggest that OmpH influences multiple fundamental cellular processes beyond its structural role in the outer membrane.

What purification strategies yield highest purity recombinant OmpH protein?

Highly effective purification strategies for recombinant OmpH include:

• Isolation of protein from inclusion bodies using a solubilization-renaturation procedure

• Ion exchange chromatography using Q-Sepharose to achieve >95% pure monomeric protein

• His-tag affinity chromatography when using histidine-tagged constructs

• Size exclusion chromatography for final polishing steps

• Quality assessment via SDS-PAGE and Western blot analysis

These methods consistently yield high-purity OmpH suitable for downstream applications including immunological studies and structural analyses.

How can researchers validate differentially expressed genes in OmpH-deletion studies?

Validation of differentially expressed genes in OmpH-deletion studies can be accomplished through:

• Real-time quantitative PCR (qPCR) to confirm expression changes observed in proteomics studies

• Western blot analysis to verify protein-level changes

• Immunohistochemistry to assess spatial distribution of differentially expressed proteins

• Functional assays to determine the biological significance of expression changes

Researchers have successfully validated differential expression of genes including FERH, HSPBP1, ABCA13, ATP10A, RRP7A, IL-10, IFN-γ, and dnaJ in OmpH deletion studies using qPCR, confirming findings from label-free proteomic analyses .

What strategies ensure FAIR (Findable, Accessible, Interoperable, Reusable) compliance for OmpH research data?

To ensure FAIR compliance for OmpH research data, researchers should implement:

• Standardized experimental data tables with comprehensive metadata

• Protocols based on Design of Experiments (DoE) methodology

• Deposition of data in appropriate public repositories with persistent identifiers

• Use of standardized vocabulary and ontologies for describing experimental conditions

• Documentation of all data processing steps and analysis methods

These practices facilitate data sharing, reproducibility, and reuse while satisfying key FAIR criteria without imposing insurmountable burdens on researchers .

How can OmpH-based vaccine candidates be developed and evaluated?

Development and evaluation of OmpH-based vaccine candidates should follow these methodological steps:

• Expression and purification of recombinant OmpH protein using optimized protocols

• Creation of OmpH gene deletion mutants as potential live attenuated vaccines

• Formulation with appropriate adjuvants to enhance immunogenicity

• Evaluation of humoral and cell-mediated immune responses

• Challenge studies to assess protective efficacy

• Safety assessment through histopathological examination and clinical monitoring

• Cross-protection studies against heterologous strains

Research has demonstrated that OmpH deletion mutants maintain immunogenicity while exhibiting reduced virulence, suggesting potential as live attenuated vaccine candidates .

What experimental designs best evaluate the structure-function relationship of OmpH?

Optimal experimental designs for evaluating OmpH structure-function relationships include:

• Site-directed mutagenesis of conserved residues to identify functional domains

• Truncation studies to delineate regions responsible for specific functions

• Chimeric protein construction combining domains from different species

• Structural analysis through X-ray crystallography or cryo-electron microscopy

• Molecular dynamics simulations to understand conformational changes

• Binding studies to identify interaction partners and binding kinetics

• In vivo assessment of mutant proteins to correlate structural changes with functional outcomes

Such approaches help establish causal relationships between specific structural elements and the diverse functions of OmpH proteins.

How can systems biology approaches enhance understanding of OmpH's role in host-pathogen interactions?

Systems biology approaches can significantly enhance understanding of OmpH's role through:

• Integration of multi-omics data (genomics, transcriptomics, proteomics, metabolomics)

• Network analysis to identify key interaction nodes and regulatory mechanisms

• Temporal studies examining dynamic changes during infection progression

• In silico modeling of host-pathogen interactions

• Comparative analysis across multiple bacterial species and host systems

• Single-cell analysis to identify heterogeneous responses

• Machine learning applications to predict OmpH functions in different contexts

Research has already identified complex protein interaction networks associated with OmpH, involving both coding and immune response-related proteins that form an intricate system influencing host-pathogen interactions .