Recombinant Pasteurella multocida Uncharacterized protein PM1123 (PM1123)

What is PM1123 and what organism does it originate from?

PM1123 is an uncharacterized protein from Pasteurella multocida strain Pm70, a Gram-negative, nonmotile coccobacillus . This bacterium is commonly found in the upper respiratory tract of domestic animals and can cause various diseases including fowl cholera in poultry, atrophic rhinitis in pigs, and bovine hemorrhagic septicemia in cattle . The protein is identified by the UniProt ID Q9CLT6 and is classified as "uncharacterized," meaning its precise biological function remains undetermined .

How can recombinant PM1123 be expressed and purified?

For recombinant expression, several systems have been demonstrated effective:

The protein has been successfully expressed in E. coli with an N-terminal His-tag, which facilitates purification using immobilized metal affinity chromatography (IMAC) . For optimal results, researchers should consider:

• Using BL21(DE3) or similar expression strains

• Optimizing induction conditions (temperature, IPTG concentration)

• Employing a two-step purification protocol (IMAC followed by size exclusion chromatography)

What computational approaches can predict the function of PM1123?

As an uncharacterized protein, several bioinformatic approaches should be employed to generate functional hypotheses:

• Homology-based approaches: Perform BLAST, HHpred, and HMMER searches against characterized protein databases, including distantly related sequences .

• Domain and motif analysis: Scan for conserved domains using Pfam, PROSITE, and InterPro to identify functional motifs.

• Structural prediction and comparison: Use AlphaFold2, I-TASSER, or SWISS-MODEL to generate structural models, followed by structural alignment with characterized proteins using DALI or TM-align.

• Genomic context analysis: Examine neighboring genes in the P. multocida genome, as functionally related genes are often co-located or co-expressed.

• Protein-protein interaction prediction: Use tools like STRING or PSICQUIC to predict potential interaction partners that might suggest function.

The COMBREX project approach demonstrates that existing experimental information can provide functional insights for more than half of all uncharacterized proteins through sequence and domain-composition similarity analyses .

How might PM1123 contribute to P. multocida pathogenicity?

While the specific role of PM1123 in pathogenicity remains unknown, several methodological approaches can elucidate its potential involvement:

• Gene knockout studies: Generate PM1123 deletion mutants and assess virulence in appropriate animal models.

• Transcriptomic analysis: Compare PM1123 expression levels under various conditions (e.g., infection vs. laboratory culture) using RNA-Seq.

• Localization studies: Determine cellular localization using fluorescent protein fusions or immunogold electron microscopy.

• Host interaction assays: Investigate if recombinant PM1123 interacts with host proteins or affects host cell processes such as cytokine production, phagocytosis, or apoptosis.

• Immunological studies: Assess whether PM1123 elicits protective immunity in animal models, which would support its role in pathogenesis and potential as a vaccine candidate .

Given that P. multocida causes various diseases across multiple host species, PM1123 may have host-specific functions that should be investigated in relevant experimental systems .

What experimental approaches can determine the cellular function of PM1123?

To elucidate the cellular function of PM1123, a multi-faceted experimental approach is recommended:

• Subcellular localization: Fractionate bacterial cells to determine if PM1123 is cytoplasmic, membrane-associated, or secreted.

• Protein-protein interaction studies:

  • Pull-down assays with His-tagged PM1123

  • Bacterial two-hybrid systems

  • Cross-linking followed by mass spectrometry

  • Co-immunoprecipitation with anti-PM1123 antibodies

• Phenotypic characterization of mutants:

  • Analyze growth curves in various media

  • Assess biofilm formation capability

  • Measure resistance to environmental stresses

  • Evaluate membrane integrity and permeability

• Metabolomic profiling: Compare metabolite profiles between wild-type and PM1123 knockout strains to identify affected pathways.

• Structural determination: X-ray crystallography or NMR spectroscopy may reveal structural homology to proteins of known function.

How should I design experiments to validate predicted functions of PM1123?

Validation of predicted functions requires a systematic approach:

• Start with in silico predictions: Generate specific hypotheses about PM1123 function using computational methods described in section 2.1.

• Design targeted biochemical assays:

  • If predicted to be an enzyme, design activity assays with potential substrates

  • If predicted to bind specific molecules, perform binding assays (e.g., SPR, ITC)

  • If predicted to have structural roles, assess effects on membrane integrity or cell morphology

• Include proper controls:

  • Positive controls: proteins with known activities similar to those predicted

  • Negative controls: mutated versions of PM1123 with altered predicted active sites

  • Expression controls: ensure consistent protein levels across experiments

• Follow with in vivo validation:

  • Generate complemented mutants to confirm phenotype rescue

  • Use conditional expression systems to study essential functions

  • Employ CRISPR interference for partial knockdown if complete deletion is lethal

• Confirm specificity:

  • Test related proteins from other bacterial species

  • Create point mutations in predicted functional domains

What are the critical considerations for structural studies of PM1123?

Structural characterization requires careful planning:

• Protein preparation optimization:

  • Test multiple constructs with different boundaries and tags

  • Screen buffer conditions (pH, salt, additives) for stability

  • Assess protein homogeneity by dynamic light scattering

• Crystallization strategy:

  • Begin with commercial screening kits

  • Optimize promising conditions systematically

  • Consider surface entropy reduction mutations for challenging proteins

• NMR considerations:

  • Produce isotopically labeled protein (15N, 13C)

  • Perform preliminary 1D experiments to assess feasibility

  • Consider TROSY techniques if molecular weight exceeds 20 kDa

• Computational approaches:

  • Generate models using AlphaFold2 to guide experimental design

  • Validate experimental structures against computational predictions

  • Identify potential functional sites through conservation mapping

• Data analysis workflow:

  • Establish protocols for data processing and refinement

  • Plan for validation using tools like MolProbity

  • Design follow-up experiments to test structure-based hypotheses

How can I integrate multiple datasets to resolve contradictory results about PM1123 function?

Contradictory results are common when studying uncharacterized proteins. A systematic integration approach includes:

• Construct a data matrix:

  • List all experiments performed and their results

  • Identify conflicts and consistencies

  • Weigh evidence based on experimental rigor and reproducibility

• Consider contextual factors:

  • Examine experimental conditions (pH, temperature, media composition)

  • Assess protein constructs used (full-length vs. truncated, tag position)

  • Consider strain-specific differences in Pasteurella multocida

• Apply Bayesian reasoning:

  • Start with prior probabilities based on computational predictions

  • Update with experimental evidence, accounting for technique reliability

  • Calculate posterior probabilities for competing functional hypotheses

• Design discriminatory experiments:

  • Identify experiments that can specifically distinguish between competing hypotheses

  • Prioritize orthogonal techniques (e.g., if structural studies and binding assays conflict, try genetic approaches)

• Collaborate with specialists:

  • Consult with experts in particular techniques for alternative interpretations

  • Consider reproducibility in different laboratories

What comparative genomic approaches can provide insights into PM1123 function?

Comparative genomics offers powerful tools for functional prediction:

• Phylogenetic profiling:

  • Map presence/absence of PM1123 homologs across bacterial species

  • Correlate with ecological niches or pathogenicity patterns

  • Identify co-evolving gene families

• Synteny analysis:

  • Examine conservation of genomic context across related species

  • Identify operonic structures that suggest functional relationships

  • Look for horizontally transferred genomic islands

• Evolutionary rate analysis:

  • Calculate selective pressure (dN/dS ratios) to identify conserved functional regions

  • Perform codon-based Z-tests for selection

  • Identify lineage-specific accelerated evolution

• Structural comparison across homologs:

  • Align predicted or determined structures from multiple species

  • Identify conserved pockets or interfaces

  • Map conservation onto structural models

• Pan-genome analysis:

  • Determine if PM1123 belongs to core or accessory genome of Pasteurella

  • Correlate gene presence with phenotypic traits across strains

How might PM1123 serve as a target for vaccine or therapeutic development?

As an uncharacterized protein, PM1123's potential as a therapeutic target requires systematic evaluation:

• Immunogenicity assessment:

  • Test recombinant PM1123 for antibody production in animal models

  • Evaluate T-cell responses to PM1123 epitopes

  • Compare protection levels against different P. multocida strains

• Conservation analysis:

  • Determine sequence conservation across pathogenic strains

  • Identify strain-specific variations that might affect vaccine efficacy

  • Map epitopes onto structural models to predict accessibility

• Target validation studies:

  • Confirm whether antibodies against PM1123 neutralize bacterial function

  • Determine if passive immunization provides protection

  • Assess if PM1123 is essential for virulence or survival in vivo

• Formulation optimization:

  • Test different adjuvants for enhanced immunogenicity

  • Evaluate delivery systems for optimal immune response

  • Consider combination with other P. multocida antigens

• Cross-protection potential:

  • Assess protection against heterologous strains

  • Evaluate efficacy across different host species

  • Determine duration of protective immunity

Given that P. multocida causes significant economic losses in livestock industries and occasional human infections, developing effective vaccines has substantial value .