Recombinant Pasteurella multocida UPF0114 protein PM1258 (PM1258)

How is recombinant PM1258 typically expressed and purified for laboratory studies?

Recombinant PM1258 is typically expressed in E. coli expression systems using vectors such as pET series plasmids. The protein can be tagged (commonly with histidine tags) to facilitate purification. The process generally involves:

• Cloning the PM1258 gene into an appropriate expression vector

• Transforming E. coli (commonly BL21 DE3 strains) with the recombinant plasmid

• Inducing protein expression using IPTG or auto-induction systems

• Cell lysis followed by affinity chromatography (typically using nickel columns for His-tagged proteins)

• Further purification may include ion exchange or size exclusion chromatography

• Storage in optimized buffer conditions, typically Tris-based buffer with 50% glycerol

For optimal stability, the purified protein should be stored at -20°C for short-term use or -80°C for extended storage, with working aliquots maintained at 4°C for up to one week to avoid freeze-thaw cycles that may compromise protein integrity .

What is the current understanding of PM1258's function in Pasteurella multocida?

While the precise function of PM1258 remains under investigation, structural analysis indicates it belongs to the UPF0114 protein family. The amino acid sequence suggests multiple membrane-spanning domains, potentially indicating a role in membrane integrity or transport. Based on patterns observed in similar bacterial proteins, PM1258 may be involved in:

• Membrane stability or organization

• Small molecule transport across membranes

• Signaling pathways related to environmental adaptation

• Potential virulence mechanisms

Further functional studies involving gene knockouts or protein-protein interaction analyses are required to definitively characterize this protein's role in P. multocida physiology .

What are the recommended methodologies for studying PM1258's role in Pasteurella multocida pathogenesis?

To investigate PM1258's role in pathogenesis, researchers should consider a multi-faceted approach:

• Gene knockout studies: Using techniques like TargeTron mutagenesis as described for other P. multocida proteins. The plasmid pAL953 can be modified for targetron-based disruption of the PM1258 gene .

• Complementation analysis: Following knockout creation, complementation can be performed using expression plasmids such as pAL99, pAL99S, or pAL99T. These plasmids place gene expression under control of the constitutive P. multocida tpiA promoter .

• Virulence assessment: Compare wild-type, knockout, and complemented strains in appropriate animal models to assess virulence changes. Duck models have been successfully used for P. multocida virulence studies .

• Protein-protein interaction studies: Techniques such as co-immunoprecipitation or bacterial two-hybrid systems can identify potential interaction partners.

• Membrane localization confirmation: Fractionation studies combined with Western blotting can confirm the predicted membrane localization of PM1258 .

How can researchers assess potential immunogenic properties of recombinant PM1258?

To evaluate PM1258's immunogenicity, researchers should implement the following methodological approach:

• Antibody production: Immunize appropriate animal models with purified recombinant PM1258 formulated with suitable adjuvants. Studies with other P. multocida proteins have successfully used water-in-oil adjuvants .

• ELISA assessment: Develop and optimize an ELISA system to measure antibody titers against PM1258 in sera from immunized animals.

• Western blot analysis: Confirm antibody specificity through Western blotting against both recombinant protein and native protein from P. multocida lysates.

• Challenge studies: Following immunization, challenge animals with virulent P. multocida to assess protective capacity. The standard approach involves intraperitoneal challenge with defined LD50 doses (often 20 LD50, as used with other P. multocida proteins) .

• Histopathological examination: Analyze tissue samples post-challenge to assess bacterial colonization and tissue damage in control versus immunized groups .

This systematic approach allows for comprehensive evaluation of PM1258's potential as an immunogen or vaccine component.

How might PM1258 interact with the lipopolysaccharide (LPS) biosynthesis pathway in P. multocida?

Given the importance of LPS in P. multocida virulence and immunogenicity, potential interactions between PM1258 and LPS biosynthesis represent an important research direction. Methodological approaches should include:

• Co-localization studies: Determine if PM1258 co-localizes with known LPS biosynthesis machinery using fluorescently tagged proteins or immunofluorescence microscopy.

• Protein-protein interaction analysis: Employ pull-down assays, bacterial two-hybrid systems, or crosslinking studies to identify interactions between PM1258 and LPS biosynthetic enzymes.

• LPS analysis in PM1258 mutants: Analyze LPS profiles from wild-type bacteria versus PM1258 knockout mutants using techniques such as silver staining, Western blotting with anti-LPS antibodies, or mass spectrometry analysis of purified LPS .

• Complementation studies: Introduce modified versions of PM1258 with targeted mutations to identify critical regions for any observed LPS-related functions.

The LPS of P. multocida is known to be essential for virulence, and disruptions in LPS biosynthesis genes have dramatic effects on pathogenicity. Understanding potential interactions between PM1258 and LPS could reveal important aspects of bacterial membrane biology .

What structural biology approaches are most appropriate for elucidating PM1258's three-dimensional structure?

Given PM1258's predicted membrane association, specialized structural biology approaches are required:

• Protein purification optimization: Membrane proteins require specific detergents for solubilization and stability. A screening approach testing multiple detergents (DDM, LDAO, etc.) is recommended.

• Crystallography considerations: For X-ray crystallography, techniques specifically developed for membrane proteins should be employed, including:

  • Lipidic cubic phase crystallization

  • Use of antibody fragments to increase polar surface area

  • Crystallization in the presence of stabilizing ligands

• Cryo-electron microscopy: A potentially valuable alternative to crystallography, especially if PM1258 forms higher-order structures.

• NMR approaches: For specific domains, solution NMR might be applicable. For the full protein, solid-state NMR techniques may be required.

• Computational prediction: In parallel with experimental approaches, homology modeling and ab initio structure prediction (especially using AlphaFold2) can provide initial structural insights.

Structural information would significantly advance understanding of PM1258's function and potential as a therapeutic target .

How can researchers investigate potential functional redundancy between PM1258 and related proteins in P. multocida?

Functional redundancy is an important consideration in bacterial systems. To address this question:

• Bioinformatic analysis: Identify proteins with similar sequence, domain architecture, or predicted structure within the P. multocida genome.

• Double/multiple knockout studies: Create single, double, and multiple knockouts of PM1258 and related proteins to identify synergistic or redundant effects.

• Transcriptomic profiling: Compare gene expression patterns between wild-type and PM1258 knockout strains to identify compensatory expression changes.

• Heterologous complementation: Test whether related proteins can complement PM1258 knockouts when expressed from plasmids.

• Phenotypic microarrays: Use systems like Biolog to comprehensively assess metabolic capabilities across multiple conditions in wild-type versus mutant strains.

This approach would help establish the unique versus redundant functions of PM1258 within the bacterial cell .

What are the critical considerations for designing expression constructs for functional studies of PM1258?

Effective expression construct design is crucial for PM1258 functional studies:

• Expression vector selection: For E. coli expression, pET series vectors (such as pET43.1a) have been successfully used for P. multocida proteins. For expression in P. multocida itself, vectors like pAL99 with the constitutive tpiA promoter are recommended .

• Tag selection and placement: Consider both N- and C-terminal tagging options, as membrane protein topology may make one terminus inaccessible. Commonly used tags include:

  • His6 tags for purification

  • Fluorescent protein fusions for localization studies

  • Epitope tags (FLAG, Myc, HA) for detection

• Codon optimization: Adjust codon usage when expressing in heterologous systems to enhance expression levels.

• Inclusion of native signal sequences: For proper membrane targeting, native signal sequences may need to be preserved.

• Solubility enhancement: Consider fusion partners like MBP, SUMO, or Thioredoxin if solubility is problematic.

The table below summarizes successful expression systems used for P. multocida proteins:

Selection of appropriate expression systems is critical for downstream functional studies .

How should researchers approach PM1258 epitope mapping for immunological studies?

For comprehensive epitope mapping of PM1258, researchers should employ multiple complementary techniques:

• Peptide array analysis: Synthesize overlapping peptides spanning the entire PM1258 sequence and test reactivity with immune sera.

• Truncation mutant analysis: Create a series of N- and C-terminal truncations to narrow down immunoreactive regions.

• Site-directed mutagenesis: Once candidate epitopes are identified, create point mutations to confirm specific amino acid contributions.

• Phage display technology: Screen phage-displayed peptide libraries with anti-PM1258 antibodies to identify mimotopes.

• Structural biology integration: Map identified epitopes onto structural models to understand accessibility and structural context.

This approach has proven effective for epitope mapping of other P. multocida antigens such as OmpH, which has been extensively studied for vaccine development .

How can PM1258 research be integrated with broader studies on P. multocida pathogenesis?

Integrating PM1258 research into broader pathogenesis studies requires:

• Comparative genomics: Analyze PM1258 conservation across P. multocida strains of different serotypes and hosts to determine if variations correlate with host specificity or virulence patterns.

• Multi-omics integration: Combine transcriptomic, proteomic, and metabolomic data to place PM1258 function within broader cellular networks.

• In vivo expression technology (IVET): Determine if PM1258 expression changes during infection using techniques that monitor gene expression in vivo.

• Animal model studies: Incorporate PM1258 research into established animal models for duck cholera, fowl cholera, or other P. multocida diseases .

• Vaccine development pipeline: Evaluate PM1258 alongside established protective antigens like VacJ, PlpE, and OmpH using standardized immunization and challenge protocols .

Research has shown that combining multiple P. multocida antigens can provide enhanced protection compared to single-antigen formulations. For example, a combination of rVacJ, rPlpE, and rOmpH provided 100% protection in duck models compared to 33.3-83.33% with individual antigens .

What are the optimal approaches for studying potential interactions between PM1258 and host immune components?

To study PM1258-host immune interactions:

• Macrophage interaction studies: Assess binding, uptake, and survival of wild-type versus PM1258 knockout bacteria in macrophage cell lines.

• Cytokine profiling: Measure cytokine responses in immune cells exposed to purified PM1258 using ELISA or multiplex bead arrays.

• Toll-like receptor (TLR) activation assays: Test PM1258's ability to stimulate different TLRs using reporter cell lines.

• T cell epitope prediction and validation: Use computational tools to predict potential T cell epitopes, followed by experimental validation using synthetic peptides and T cell assays.

• Antibody-dependent mechanisms: Investigate whether anti-PM1258 antibodies can mediate opsonophagocytosis or complement-mediated killing.

Understanding these interactions is critical for rational vaccine design, particularly given that P. multocida vaccines must stimulate appropriate immune responses for protection. Current research with other P. multocida antigens suggests that protective immunity requires both humoral and cellular components .