Recombinant Pasteurella multocida Uncharacterized protein PM1934 (PM1934)

What is Pasteurella multocida PM1934 protein and what is its biological significance?

Pasteurella multocida PM1934 is an uncharacterized protein from strain Pm70 that has gained research interest for potential applications in vaccine development. Pasteurella multocida is a Gram-negative, nonmotile, penicillin-sensitive coccobacillus responsible for a range of diseases in mammals and birds, including fowl cholera in poultry, atrophic rhinitis in pigs, and bovine hemorrhagic septicemia in cattle and buffalo. PM1934 represents one of many proteins being investigated for immunogenic properties, though its specific biological function remains incompletely characterized compared to other Pasteurella multocida proteins like VacJ, PlpE, and OmpH which have demonstrated immunogenic potential .

How is recombinant PM1934 protein typically produced for research applications?

Recombinant PM1934 protein production typically employs expression systems similar to those used for other Pasteurella multocida proteins. The protein is commonly expressed in Escherichia coli expression systems, although yeast, baculovirus, or mammalian cell expression systems may also be utilized depending on research requirements. The general methodology involves gene cloning, expression vector construction, host cell transformation, protein expression induction, and subsequent purification. Researchers often amplify the PM1934 gene from Pasteurella multocida genomic DNA using PCR with specific primers, then clone it into an appropriate expression vector with a histidine tag for purification purposes .

What expression systems yield optimal recombinant PM1934 protein production?

Based on protocols for similar Pasteurella multocida proteins, Escherichia coli expression systems are most commonly employed for PM1934 production. Specifically, BL21(DE3) strains are frequently used with pET expression vectors (such as pET43.1a) for efficient recombinant protein production. These systems offer high expression levels, ease of genetic manipulation, and cost-effectiveness. The choice between E. coli, yeast, baculovirus, or mammalian cell expression systems depends on specific research requirements, particularly concerning protein folding, post-translational modifications, and downstream applications .

What purification strategies yield the highest purity of recombinant PM1934 protein?

Purification of recombinant PM1934 protein typically follows standardized protocols established for other Pasteurella multocida proteins. The most effective purification approach employs affinity chromatography utilizing the histidine tag incorporated during cloning. This method involves lysing the bacterial cells, clarifying the lysate through centrifugation, and passing the supernatant through a nickel or cobalt affinity column. The bound protein is then eluted with increasing concentrations of imidazole. Further purification may involve ion exchange chromatography or size exclusion chromatography to achieve higher purity. Quality assessment typically includes SDS-PAGE analysis to confirm protein size (approximately 94 amino acids, with varying molecular weight depending on fusion tags) and Western blotting to verify identity .

What are the critical factors affecting recombinant PM1934 stability during purification and storage?

Several critical factors influence PM1934 stability during purification and storage, similar to those affecting other recombinant Pasteurella multocida proteins. Temperature control is essential, with most purification steps performed at 4°C to minimize proteolytic degradation. Buffer composition significantly impacts stability, with optimal pH typically in the range of 7.0-8.0, and the inclusion of protease inhibitors prevents degradation during extraction and purification. Storage conditions typically involve keeping the purified protein at -80°C for long-term storage or at -20°C for short-term storage, often with the addition of glycerol (10-20%) as a cryoprotectant. Repeated freeze-thaw cycles should be avoided as they can lead to protein aggregation and loss of biological activity .

How should researchers design experiments to evaluate PM1934 immunogenicity in animal models?

Experimental design for evaluating PM1934 immunogenicity should follow established protocols used for other Pasteurella multocida proteins. Such studies typically involve:

• Animal selection: Ducks are appropriate models due to their susceptibility to Pasteurella multocida infections, though mice may be used for preliminary studies.

• Vaccination protocol: Animals receive purified recombinant PM1934 formulated with appropriate adjuvants (water-in-oil or oil-coated adjuvants have shown efficacy with similar proteins).

• Sampling schedule: Serum collection at regular intervals (pre-immunization, 7, 14, 21, and 28 days post-immunization) to measure antibody responses.

• Immune response assessment: ELISA to quantify antigen-specific antibody titers, including IgG levels.

• Challenge model: Intraperitoneal challenge with virulent Pasteurella multocida (typically at 20 LD50 doses) to evaluate protective efficacy.

• Protection assessment: Survival rates, histopathological examination, and bacterial load determination in tissues to comprehensively evaluate vaccine efficacy .

How does PM1934 compare structurally and functionally to well-characterized Pasteurella multocida proteins like VacJ, PlpE, and OmpH?

While PM1934 remains less characterized than other Pasteurella multocida proteins, comparative analysis provides insights into its potential functions. Unlike the better-studied VacJ (a lipoprotein of approximately 27.5 kDa associated with bacterial virulence), PlpE (a 38 kDa lipoprotein with established immunogenicity), and OmpH (a 33.8 kDa major outer membrane protein), PM1934 is a smaller protein consisting of 94 amino acids from strain Pm70. Structurally, sequence homology analysis would be required to determine relatedness to these better-characterized proteins. Functionally, while VacJ has been shown to elicit humoral immune responses and protective immunity, and both PlpE and OmpH have demonstrated significant protective efficacy (83.33% protection in duck models), the specific immunogenic properties of PM1934 require further investigation to determine if it shares similar protective potential .

What is the potential role of PM1934 in the pathogenesis of Pasteurella multocida infections?

The specific role of PM1934 in Pasteurella multocida pathogenesis remains to be fully elucidated. Pasteurella multocida causes a spectrum of diseases, including highly contagious duck cholera (duck hemorrhagic septicemia), with serotype A being the predominant pathogenic serotype. By analogy with other membrane-associated proteins, PM1934 may potentially contribute to bacterial adhesion, host immune evasion, or nutrient acquisition. The protein might function similarly to other membrane proteins like VacJ, which plays a virulence-associated role in most Gram-negative bacteria. Comprehensive functional studies involving gene knockout, complementation, and virulence assessment would be necessary to definitively establish PM1934's role in pathogenesis. Such studies would typically examine bacterial colonization, tissue damage, and host immune responses in appropriate animal models .

How might post-translational modifications affect PM1934 function and immunogenicity?

Post-translational modifications (PTMs) potentially play significant roles in PM1934 function and immunogenicity, though specific modifications have not been extensively documented in the available literature. By analogy with other bacterial membrane proteins, possible modifications might include glycosylation, lipidation, or phosphorylation. These modifications could significantly impact protein folding, stability, subcellular localization, and recognition by the host immune system. For instance, lipidation of bacterial proteins often enhances recognition by pattern recognition receptors, potentially increasing immunogenicity. When producing recombinant PM1934, researchers should consider that expression systems like E. coli may not reproduce the native PTM profile, potentially affecting functional studies. Mass spectrometry analysis of native PM1934 isolated from Pasteurella multocida would be necessary to identify specific modifications and their positions within the protein sequence .

What adjuvants are most effective when formulating vaccines with recombinant PM1934?

The selection of appropriate adjuvants for PM1934-based vaccines should be guided by successful formulations used with other Pasteurella multocida proteins. Based on research with similar proteins, water-in-oil adjuvants have demonstrated particular efficacy for subunit vaccines containing recombinant Pasteurella multocida proteins. For killed vaccines containing Pasteurella multocida components, oil-coated adjuvants have shown effectiveness. These adjuvant formulations enhance immune responses by creating a depot effect, prolonging antigen presentation, and stimulating innate immune responses. When developing PM1934-based vaccines, researchers should conduct comparative adjuvant studies measuring parameters such as antibody titers, isotype distributions, cell-mediated immune responses, and ultimately protection rates following challenge to determine optimal formulations .

How does PM1934 perform in multi-component vaccine formulations with other Pasteurella multocida proteins?

Multi-component vaccines containing several Pasteurella multocida proteins have demonstrated enhanced efficacy compared to single-protein formulations. While specific data on PM1934 in such combinations is limited, research with other Pasteurella multocida proteins provides a valuable model. For example, a combination of recombinant VacJ, PlpE, and OmpH proteins with adjuvant achieved 100% protection in duck models, significantly outperforming individual protein vaccines (which provided 33.3%, 83.33%, and 83.33% protection, respectively) and killed vaccines (50% protection). This synergistic effect suggests that combining PM1934 with other immunogenic proteins might similarly enhance protective efficacy. When developing such combinations, researchers should evaluate potential protein-protein interactions, stability in combination, and immune response profiles to ensure compatibility and optimal efficacy .

What are the comparative protective efficacies of different Pasteurella multocida protein vaccines?

The table below compares the protective efficacies of various Pasteurella multocida protein-based vaccines based on challenge studies in duck models:

This comparative data indicates that while single recombinant protein vaccines can provide substantial protection, combination vaccines offer superior efficacy. These findings suggest that PM1934, when appropriately formulated and potentially combined with other immunogenic proteins, might contribute to effective vaccine development against Pasteurella multocida infections .

What gene expression systems and vectors are optimal for high-yield PM1934 production?

For high-yield PM1934 production, several expression systems and vectors warrant consideration based on successful approaches with similar Pasteurella multocida proteins. The pET expression system, particularly pET43.1a vector in E. coli BL21(DE3) cells, has demonstrated efficacy for recombinant Pasteurella multocida protein expression. This system offers tight regulation through the T7 promoter and high expression levels upon IPTG induction. Alternative systems include pGEX vectors for GST-fusion proteins that may enhance solubility, or pMAL systems for maltose-binding protein fusions. For challenging expression cases, codon optimization for E. coli usage can significantly improve yields. Expression conditions typically require optimization of induction temperature (often reduced to 16-25°C to enhance solubility), IPTG concentration, and induction duration. Shake flask cultures are sufficient for initial studies, while bioreactor systems may be employed for scaled-up production with controlled dissolved oxygen and pH parameters .

How should researchers design challenge studies to evaluate PM1934-based vaccine candidates?

Challenge studies evaluating PM1934-based vaccine candidates should follow established protocols used for other Pasteurella multocida proteins. A comprehensive challenge study design includes:

• Immunization protocol: Animals receive purified recombinant PM1934 (typically 50-200 μg per dose) formulated with appropriate adjuvants, administered in 2-3 doses at 2-week intervals.

• Control groups: Include positive controls (commercial vaccines or established efficacious formulations), negative controls (adjuvant-only), and unvaccinated controls.

• Challenge strain selection: Use virulent Pasteurella multocida strains relevant to the target host species, preferably homologous to the strain from which PM1934 was derived.

• Challenge dose determination: Establish LD50 through preliminary studies and challenge with 10-20× LD50 to ensure robust testing of vaccine efficacy.

• Challenge route: Intraperitoneal administration is common for laboratory studies, though natural infection routes (intranasal, oral) may better reflect field conditions.

• Monitoring parameters: Track survival rates, clinical signs, body temperature, weight changes, and feed intake daily for 7-14 days post-challenge.

• Tissue sampling: Collect samples from liver, spleen, lungs, and blood for bacterial isolation, quantification, and histopathological examination .

What analytical techniques are most informative for characterizing PM1934 structure-function relationships?

A comprehensive analytical approach to characterizing PM1934 structure-function relationships should employ multiple complementary techniques:

• Protein structure analysis: X-ray crystallography or NMR spectroscopy to determine three-dimensional structure, coupled with circular dichroism to assess secondary structure elements and thermal stability.

• Protein-protein interaction studies: Pull-down assays, co-immunoprecipitation, or surface plasmon resonance to identify binding partners within bacterial cells or host tissues.

• Epitope mapping: Peptide arrays or hydrogen-deuterium exchange mass spectrometry to identify immunodominant regions recognized by antibodies from vaccinated or naturally infected animals.

• Functional assays: Adhesion assays with host cells, complement resistance testing, and phagocytosis assays to assess functional contributions to virulence.

• Genetic approaches: Site-directed mutagenesis targeting predicted functional domains followed by in vitro and in vivo phenotypic characterization.

• Immunological assays: T-cell proliferation assays, cytokine profiling, and antibody isotyping to characterize the type of immune response elicited.

• Computational analysis: Homology modeling, molecular dynamics simulations, and bioinformatic analysis to predict structural features and evolutionary relationships with characterized proteins .

What are the most promising future research avenues for PM1934 vaccine development?

Several promising research directions could advance PM1934-based vaccine development. First, comprehensive immunogenicity studies comparing PM1934 with established immunogens like PlpE and OmpH would clarify its potential value in vaccine formulations. Second, exploration of combination formulations containing PM1934 alongside other protective antigens merits investigation, given the demonstrated synergistic protection observed with other protein combinations. Third, alternative delivery platforms, such as viral vectors or DNA vaccines expressing PM1934, might enhance immune responses beyond those achieved with purified protein formulations. Fourth, structure-function studies identifying immunodominant epitopes could guide the development of epitope-focused vaccines with potentially enhanced efficacy and cross-protection. Finally, field trials in natural host species under varied environmental conditions would be essential to validate laboratory findings and establish real-world efficacy parameters before commercial development consideration .

How might cross-serotype protection be achieved with PM1934-based vaccines?

Achieving cross-serotype protection represents a significant challenge and opportunity in Pasteurella multocida vaccine development. PM1934's potential for cross-serotype protection would depend on its conservation across serotypes and the immunological relevance of conserved regions. A systematic approach to developing cross-protective PM1934-based vaccines would involve: (1) sequence analysis of PM1934 across multiple serotypes to identify conserved regions; (2) epitope mapping to determine if conserved regions contain protective epitopes; (3) design of recombinant constructs focusing on conserved, immunogenic regions; (4) heterologous challenge studies testing protection against multiple serotypes; and (5) potential combination with other conserved antigens to broaden protection. The relatively high conservation observed in other Pasteurella multocida outer membrane proteins like PlpE suggests that similar approaches might be successful with PM1934, potentially yielding vaccines with broader protection than current serotype-specific formulations .

What technological innovations might enhance PM1934 vaccine efficacy and delivery?

Several technological innovations could significantly enhance PM1934 vaccine efficacy and delivery:

• Nanoparticle-based delivery systems could improve antigen stability and controlled release while enhancing uptake by antigen-presenting cells.

• Adjuvant innovations, including toll-like receptor agonists or cytokine-adjuvant combinations, might elicit more robust and appropriate immune responses.

• Reverse vaccinology approaches utilizing genome-wide epitope prediction could identify optimal PM1934 epitopes for inclusion in subunit vaccines.

• CRISPR-Cas9 engineering of attenuated Pasteurella multocida strains expressing modified PM1934 might yield live-attenuated vaccines with enhanced safety and efficacy.

• Plant-based expression systems could enable cost-effective production of edible vaccines for oral administration to livestock and poultry.

• mRNA vaccine technology could be adapted to deliver PM1934-encoding transcripts, potentially eliciting strong cellular and humoral responses.

• Structure-based design informed by detailed PM1934 structural analysis could yield optimized immunogens with enhanced stability and immunogenicity .