Recombinant Pasteurella multocida Uncharacterized protein PM1386 (PM1386)

What are the basic structural characteristics of PM1386?

PM1386 is an uncharacterized protein from Pasteurella multocida with a full sequence length of 112 amino acids . The protein is available in recombinant form as a His-tagged protein expressed in E. coli expression systems . While detailed structural information remains limited, the relatively small size of PM1386 (112 amino acids) suggests it may function as part of a larger protein complex or serve as a regulatory protein within bacterial systems. Researchers should conduct preliminary structural analyses using techniques such as circular dichroism (CD) spectroscopy to determine secondary structure elements before proceeding to more advanced structural characterization methods.

What bioinformatic approaches can predict potential functions of PM1386?

When working with uncharacterized proteins like PM1386, researchers should employ a multi-faceted bioinformatic approach. Begin with sequence homology searches using BLAST against non-redundant protein databases, followed by domain prediction using tools like SMART or Pfam. Structural homology modeling can be performed using platforms like I-TASSER or Phyre2, which may reveal structural similarities to functionally characterized proteins. Additionally, genomic context analysis examining the neighboring genes of pm1386 in the P. multocida genome can provide functional hints, as genes in the same operon often participate in related functions. For more advanced analysis, machine learning-based function prediction tools like DeepFRI can be employed to suggest potential molecular functions based on predicted structural features.

How does PM1386 compare to other uncharacterized proteins in Pasteurella multocida?

Pasteurella multocida contains numerous uncharacterized proteins that require systematic characterization approaches. Comparative analysis should include sequence alignment of PM1386 with other uncharacterized proteins in the P. multocida genome to identify potential paralogues and orthologues. Researchers should examine the distribution of PM1386 across different strains and isolates of P. multocida using the PmGT platform, which can provide genotyping information for P. multocida strains . Phylogenetic analysis should be performed to determine evolutionary relationships between PM1386 and similar proteins. The expression patterns of PM1386 compared to other uncharacterized proteins should be analyzed under various growth conditions and infection models to identify co-regulated proteins, potentially suggesting functional relationships.

What expression systems are optimal for producing recombinant PM1386?

The recombinant PM1386 protein is currently available as a His-tagged protein expressed in E. coli . For optimal production of functional PM1386, researchers should consider several expression strategies. E. coli BL21(DE3) remains the preferred expression system for initial attempts due to its high yield and straightforward protocols. Optimize expression conditions by testing various induction temperatures (16°C, 25°C, 30°C, 37°C), IPTG concentrations (0.1-1 mM), and induction durations (4-24 hours). For proteins exhibiting poor solubility, consider fusion partners beyond the His-tag, such as MBP, GST, or SUMO, which may enhance solubility. Alternative expression systems including Pseudomonas-based systems might provide more native-like post-translational modifications if E. coli expression proves problematic. Purification should employ multi-step chromatography combining affinity (Ni-NTA), ion-exchange, and size-exclusion approaches to achieve >95% purity needed for structural and functional studies.

What experimental approaches should be used to identify potential interaction partners of PM1386?

To characterize protein-protein interactions of PM1386, researchers should implement both in vitro and in vivo approaches. Begin with pull-down assays using purified His-tagged PM1386 as bait with P. multocida cell lysates, followed by mass spectrometry to identify bound proteins. For validation, employ techniques such as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to determine binding affinities between PM1386 and candidate interacting proteins. In vivo approaches should include bacterial two-hybrid systems, which are more suitable for bacterial protein interactions than yeast two-hybrid systems. Co-immunoprecipitation from P. multocida cultures using antibodies against PM1386 can identify physiologically relevant interactions. Additionally, proximity-based labeling methods like BioID can identify proteins in close proximity to PM1386 within the bacterial cell. Cross-referencing these results with the interactome networks of P. multocida may reveal functional pathways involving PM1386.

How can researchers determine the cellular localization of PM1386 in Pasteurella multocida?

Determining the subcellular localization of PM1386 requires multiple complementary approaches. Generate fluorescent protein fusions (e.g., PM1386-GFP) for expression in P. multocida, followed by confocal microscopy to visualize localization patterns. Validate microscopy results with subcellular fractionation experiments separating cytoplasmic, periplasmic, membrane, and secreted fractions, followed by Western blot analysis using anti-PM1386 antibodies. For more precise localization, employ immunogold electron microscopy using anti-PM1386 antibodies on fixed P. multocida cells. Computational prediction tools such as PSORTb, SignalP, and TMHMM should be used to predict possible localization signals within the PM1386 sequence. Combine these experimental and computational approaches to build a comprehensive understanding of PM1386's cellular compartmentalization, which will provide significant insights into its potential functional roles.

What gene knockout strategies are most effective for studying PM1386 function in Pasteurella multocida?

For functional characterization of PM1386 through gene knockout, researchers should implement a multi-step strategy. Begin with precise gene deletion using homologous recombination methods optimized for P. multocida, targeting the pm1386 gene without affecting neighboring genes. The lambda Red recombinase system adapted for P. multocida offers efficient knockout generation. Alternatively, CRISPR-Cas9 systems optimized for Pasteurellaceae provide more precise genome editing capabilities. Verify knockouts through both PCR and sequencing, followed by comprehensive phenotypic characterization. Compare growth rates in various media compositions, stress resistance (oxidative, pH, temperature), biofilm formation capacity, and virulence in infection models. Complement knockout strains by introducing the pm1386 gene on an expression plasmid to confirm phenotypic restoration. For temporal control of gene expression, consider inducible knockdown systems using antisense RNA or CRISPR interference (CRISPRi). These approaches collectively provide a robust framework for determining PM1386 function in P. multocida biology.

How can transcriptomic analysis help uncover the function of PM1386?

Transcriptomic analysis provides valuable insights into the functional role of PM1386 through expression profiling and regulatory network analysis. Compare RNA-seq data between wild-type and pm1386 knockout strains under multiple conditions, including standard laboratory growth, various stress conditions (oxidative, nutrient limitation, host-mimicking environments), and different growth phases. Identify differentially expressed genes between wild-type and knockout strains to reveal potential pathways affected by PM1386 absence. Perform time-course experiments during infection models to identify infection-specific expression patterns. Co-expression network analysis can identify genes with similar expression patterns to pm1386, suggesting functional relationships. Additionally, conduct ChIP-seq experiments if PM1386 is predicted to have DNA-binding properties to identify potential binding sites and regulated genes. Integration of transcriptomic data with other omics data (proteomics, metabolomics) will provide a systems-level understanding of PM1386's role in P. multocida biology.

What approaches should be used to investigate the role of PM1386 in Pasteurella multocida virulence?

Investigating PM1386's role in virulence requires a comprehensive experimental strategy spanning in vitro and in vivo models. Begin with adhesion and invasion assays using relevant cell lines (respiratory epithelial cells for respiratory isolates, endothelial cells for septicemic strains) comparing wild-type and pm1386 knockout strains. Quantify bacterial survival in macrophage infection models and measure host immune responses through cytokine profiling (IL-1β, IL-6, TNF-α). Analyze biofilm formation capacity, as many virulence factors contribute to biofilm development. For in vivo analysis, utilize appropriate animal models based on host specificity of the P. multocida strain (murine models for general virulence, species-specific models for host-adapted strains), comparing colonization, persistence, and disease progression between wild-type and knockout strains. Monitor bacterial burden in tissues, histopathological changes, and host immune responses. Additionally, perform comparative virulence studies using different P. multocida genotypes (using the PmGT classification system) to determine if PM1386's role in virulence varies across different strain backgrounds .

What crystallization strategies are most effective for determining the structure of PM1386?

Determining the crystal structure of PM1386 requires systematic optimization of crystallization conditions. Begin with highly purified (>95% purity) recombinant His-tagged PM1386 , employing size-exclusion chromatography as the final purification step to ensure monodispersity. Initial screening should utilize commercial sparse matrix screens (Hampton Research, Molecular Dimensions) with both hanging drop and sitting drop vapor diffusion methods at multiple protein concentrations (5-15 mg/ml) and temperatures (4°C and 20°C). For proteins resistant to crystallization, consider surface entropy reduction (SER) approaches, introducing mutations that replace high-entropy surface residues (Lys, Glu) with alanines to promote crystal contacts. Alternatively, employ crystallization chaperones such as antibody fragments (Fab, nanobodies) that can stabilize flexible regions. For co-crystallization with potential ligands or interacting partners, conduct thermal shift assays to identify stabilizing compounds before setting up crystallization trials. If traditional crystallization proves challenging, explore alternative structural determination methods such as cryo-electron microscopy for larger complexes or NMR spectroscopy, which is well-suited for smaller proteins like PM1386 (112 amino acids).

How can computational modeling complement experimental approaches in understanding PM1386 structure?

Computational modeling provides crucial insights into PM1386 structure, especially given its uncharacterized status. Start with advanced protein structure prediction using AlphaFold2 or RoseTTAFold, which have demonstrated near-experimental accuracy for many proteins. Generate multiple models and assess their confidence scores and structural consistency. Refine these models through molecular dynamics simulations (50-100 ns) to evaluate stability and identify flexible regions. Employ binding site prediction algorithms (SiteMap, FTSite) to identify potential functional pockets that might interact with ligands or other proteins. Molecular docking studies with metabolites common in bacterial physiology may suggest potential functions. For functional annotation, conduct structural alignment against the PDB database to identify proteins with similar folds despite low sequence identity. Additionally, create homology models of PM1386 from different P. multocida strains to identify conserved structural features that might indicate functional importance. Integration of computational predictions with experimental data from limited proteolysis, hydrogen-deuterium exchange mass spectrometry, or small-angle X-ray scattering provides a more complete structural understanding than either approach alone.

What NMR spectroscopy approaches are suitable for studying the solution structure and dynamics of PM1386?

Nuclear Magnetic Resonance (NMR) spectroscopy offers powerful tools for characterizing PM1386's structure and dynamics in solution. For a protein of 112 amino acids , standard 3D NMR approaches are highly feasible. Begin with sample preparation using 15N and 13C isotope labeling during recombinant expression in minimal media. Initial 1H-15N HSQC experiments will assess protein folding and sample quality. For backbone assignment, conduct HNCA, HN(CO)CA, HNCACB, and HN(CO)CACB experiments, followed by side-chain assignment using HCCH-TOCSY and HCCH-COSY. NOE-based distance restraints, combined with dihedral angle constraints from TALOS+, enable solution structure determination. To characterize dynamics, perform 15N relaxation experiments (T1, T2, heteronuclear NOE) to identify flexible regions and potential binding interfaces. For investigating potential ligand interactions, utilize chemical shift perturbation experiments with candidate ligands or interaction partners. Residual dipolar coupling (RDC) measurements in alignment media provide additional structural restraints. For more challenging aspects, consider selective methyl labeling strategies in a deuterated background to study larger complexes of PM1386 with interaction partners. Integration of NMR-derived restraints with computational models will provide the most comprehensive structural characterization of this uncharacterized protein.

How might PM1386 contribute to host adaptation in Pasteurella multocida?

Investigating PM1386's potential role in host adaptation requires integrating genotypic, phenotypic, and functional analyses. Compare pm1386 sequence variants across P. multocida strains isolated from different host species, particularly focusing on host-specific lineages identified through the combined "capsular:LPS:MLST" genotyping system . Analyze selective pressure on pm1386 by calculating dN/dS ratios to identify signatures of positive selection that might indicate adaptation to different host environments. Conduct in vitro infection experiments using cell lines derived from different host species (bovine, porcine, avian) with wild-type and pm1386 knockout strains to determine if PM1386 contributes differentially to interaction with host cells from different species. Examine expression levels of pm1386 during infection of different host cells or in media mimicking different host environments. For in vivo analysis, compare colonization efficiency and virulence of pm1386 mutants in different host species models. Additionally, investigate interaction between PM1386 and host-specific factors like transferrin or other host proteins that display species-specific variations, as suggested by the differential distribution of virulence factors like tbpA (transferrin binding protein) across P. multocida strains from different hosts .

What experimental evidence exists for PM1386 involvement in virulence factor regulation?

While direct experimental evidence for PM1386's role in virulence regulation is currently limited, researchers can develop a systematic investigation strategy. Begin by examining transcriptomic and proteomic changes in known virulence factors between wild-type and pm1386 knockout strains. Focus particularly on virulence factors identified in P. multocida, including adhesins (ptfA, fimA, hsf-1, hsf-2, pfhA, tadD), toxins (toxA), iron acquisition proteins (exbB, exbD, tonB, hgbA, hgbB, fur, tbpA), sialidases (nanB, nanH), hyaluronidase (pmHAS), and outer membrane proteins (ompA, ompH, oma87, plpB) . Utilize quantitative RT-PCR to validate expression changes in these virulence genes. For protein-level regulation, conduct chromatin immunoprecipitation (ChIP) experiments if PM1386 is predicted to have DNA-binding properties, or protein co-immunoprecipitation if post-transcriptional regulation is suspected. Examine phenotypic virulence characteristics including biofilm formation, adhesion to host cells, resistance to serum killing, and survival within macrophages. For direct evidence of regulatory function, perform electrophoretic mobility shift assays (EMSA) with purified PM1386 and promoter regions of affected virulence genes. Integration of these approaches will elucidate if PM1386 functions within regulatory networks controlling virulence factor expression in P. multocida.

What are the optimal conditions for expressing and purifying recombinant PM1386?

For optimal recombinant PM1386 production, researchers should implement a systematic optimization approach. The currently available recombinant PM1386 is produced as a His-tagged protein in E. coli . For expression optimization, test multiple E. coli strains (BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Arctic Express) to address potential codon bias or toxic effects. Conduct small-scale expression tests varying induction temperature (16-37°C), IPTG concentration (0.1-1.0 mM), and induction duration (4-24 hours). For optimal purification, employ a multi-step approach starting with immobilized metal affinity chromatography (IMAC) using Ni-NTA resin, followed by ion-exchange chromatography and size-exclusion chromatography to achieve high purity. Buffer optimization is critical - test various pH ranges (6.5-8.5), salt concentrations (100-500 mM NaCl), and stabilizing additives (glycerol, reducing agents) to improve protein stability. Assess protein quality using dynamic light scattering to confirm monodispersity and thermal shift assays to identify stabilizing buffer conditions. For structural studies, consider tag removal using specific proteases (TEV, PreScission) followed by reverse IMAC. Implement rigorous quality control measures including SDS-PAGE, Western blotting, mass spectrometry, and N-terminal sequencing to confirm protein identity and integrity before proceeding to functional or structural studies.

What antibody development strategies are recommended for PM1386 detection and characterization?

Developing high-quality antibodies against PM1386 requires strategic immunization and screening approaches. Begin with purified recombinant His-tagged PM1386 for immunization, considering both polyclonal and monoclonal antibody development paths. For polyclonal antibodies, immunize rabbits or guinea pigs with 100-200 μg of purified PM1386 using a prime-boost regimen with appropriate adjuvants (Freund's complete for prime, incomplete for boosts). Collect pre-immune sera as controls and test antibody titers by ELISA after each boost. Affinity-purify antibodies using immobilized PM1386 to improve specificity. For monoclonal antibodies, immunize mice and conduct hybridoma generation, followed by stringent screening to identify high-affinity, specific clones. Critically, validate all antibodies for specificity using multiple methods: Western blot analysis comparing wild-type and pm1386 knockout P. multocida lysates, immunoprecipitation followed by mass spectrometry, and immunofluorescence microscopy comparing signal in wild-type versus knockout strains. Epitope mapping through peptide arrays or hydrogen-deuterium exchange mass spectrometry will identify the specific regions recognized by the antibodies, which is valuable for interpreting experimental results. These validated antibodies enable multiple applications including immunolocalization, ChIP assays, and pull-down experiments to characterize PM1386's cellular localization, potential DNA-binding activity, and protein-protein interactions.

How can mass spectrometry techniques be applied to study PM1386 post-translational modifications?

Mass spectrometry provides powerful approaches for comprehensive characterization of potential post-translational modifications (PTMs) on PM1386. Begin with bottom-up proteomics approaches, digesting purified PM1386 with multiple proteases (trypsin, chymotrypsin, Glu-C) to achieve complete sequence coverage. Analyze digested peptides using LC-MS/MS with higher-energy collisional dissociation (HCD) and electron-transfer dissociation (ETD) fragmentation to identify PTMs including phosphorylation, acetylation, methylation, and bacterial-specific modifications like glycosylation and lipidation. For phosphorylation analysis, employ titanium dioxide enrichment of phosphopeptides before MS analysis. For intact mass analysis, utilize top-down proteomics approaches with high-resolution instruments (Orbitrap, QTOF) to determine the precise molecular weight of intact PM1386 and confirm the presence and stoichiometry of modifications. Native MS can provide insights into how PTMs affect protein complex formation. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can reveal how PTMs affect protein conformation and dynamics. Compare PTM profiles of PM1386 expressed in native P. multocida versus recombinant systems to identify biologically relevant modifications that may be absent in recombinant preparations. Temporal analysis of PTMs under different growth conditions and during infection models can reveal regulatory mechanisms controlling PM1386 activity in different physiological contexts.

What evidence suggests PM1386 could be a potential vaccine candidate against Pasteurella multocida infections?

Evaluating PM1386's potential as a vaccine candidate requires systematic assessment of multiple criteria. First, analyze the conservation of pm1386 across diverse P. multocida strains representing different capsular, LPS, and MLST genotypes associated with infections in various host species . High conservation would suggest broader protection potential across different strain types. Examine surface accessibility using computational predictions and experimental approaches like surface shaving proteomics to determine if PM1386 is potentially exposed to the immune system. For immunological assessment, purified recombinant PM1386 should be evaluated for its ability to stimulate innate immune responses in appropriate cell models and adaptive immune responses in animal models. Compare survival rates and bacterial loads in vaccinated versus control animals after challenge with virulent P. multocida strains. For mechanistic understanding, characterize antibody responses against PM1386, assessing their ability to promote opsonophagocytosis, complement-mediated killing, or neutralization of potential PM1386 functions. Explore potential adjuvant formulations to enhance immunogenicity. Additionally, evaluate if PM1386 shows cross-protection against heterologous strains or even related Pasteurellaceae species. While current evidence for PM1386 as a vaccine candidate is limited, these systematic approaches will determine its potential value in vaccine development strategies against P. multocida infections.

How can synthetic biology approaches be used to investigate PM1386 function?

Synthetic biology offers innovative approaches to deciphering PM1386 function through controlled expression, domain engineering, and pathway reconstruction. Design synthetic operons placing pm1386 under inducible promoters with varying strengths to assess dose-dependent effects and complement knockout phenotypes. Construct domain-swap chimeras by fusing conserved regions of PM1386 with domains from functionally characterized proteins to test for functional conservation or to create novel functionalities that might reveal native function. Implement CRISPR interference (CRISPRi) systems in P. multocida for tunable repression of pm1386, allowing temporal control over expression during infection processes. For functional mapping, create a systematic library of PM1386 variants with alanine scanning or domain truncations to identify critical residues or regions for function. Explore optogenetic control systems adapting light-responsive elements to regulate PM1386 activity with spatial and temporal precision. For systems-level understanding, reconstruct minimal synthetic pathways incorporating PM1386 and its potential interaction partners in heterologous hosts like E. coli, allowing controlled study of pathway functionality. These synthetic biology approaches circumvent limitations of traditional genetic approaches and provide precise control over PM1386 expression, localization, and interaction with other cellular components.

What comparative genomic approaches can reveal the evolutionary history of PM1386?

Elucidating the evolutionary history of PM1386 requires comprehensive comparative genomic analysis across Pasteurellaceae and related bacterial families. Begin by identifying PM1386 homologs through sensitive sequence similarity searches (PSI-BLAST, HMMer) across diverse bacterial genomes. Construct phylogenetic trees using maximum likelihood methods to trace the evolutionary history of PM1386, comparing its phylogeny with species phylogeny to identify potential horizontal gene transfer events. Analyze synteny conservation of the genomic region containing pm1386 across different species to determine if gene neighborhood has been preserved throughout evolution. Calculate selection pressures (dN/dS ratios) across different lineages to identify potential shifts in evolutionary constraints that might indicate functional divergence. Examine variation in sequence, length, and domain architecture of PM1386 homologs across species to identify conserved regions likely critical for function. Compare the presence/absence patterns of PM1386 with known virulence factors to identify potential co-evolution that might suggest functional relationships. Correlate PM1386 sequence variations with host range or pathogenicity patterns to identify potential host-adaptation signatures. These comparative genomic approaches will reveal whether PM1386 represents an ancestral gene in Pasteurellaceae, a recent acquisition, or if it has undergone significant functional diversification across different bacterial lineages.

Data Table: PM1386 Protein Characteristics

What high-throughput approaches could accelerate PM1386 functional characterization?

Accelerating PM1386 functional characterization requires integrated multi-omics and high-throughput screening approaches. Implement CRISPR-Cas9 based functional genomics screens in P. multocida, targeting genes across the genome to identify synthetic lethal or synthetic rescue interactions with pm1386, revealing potential functional pathways. Develop high-throughput phenotypic assays measuring growth, biofilm formation, and stress resistance in pm1386 mutants across hundreds of conditions using Biolog phenotype microarrays or custom-designed condition matrices. For protein function analysis, employ protein microarray technology to screen PM1386 against libraries of metabolites, nucleic acids, and other bacterial and host proteins to identify binding partners. Implement mass spectrometry-based thermal proteome profiling (MS-TPP) to identify proteins whose thermal stability is affected by PM1386 deletion, suggesting physical or functional interactions. Develop a reporter system fusing PM1386 to split fluorescent proteins for high-throughput screening of protein-protein interactions in bacterial two-hybrid systems. For structure-function relationships, employ deep mutational scanning by creating thousands of PM1386 variants and assessing their function in parallel using next-generation sequencing. Integration of these high-throughput datasets using machine learning approaches can generate testable hypotheses about PM1386 function that can be validated through targeted experiments.

How might single-cell approaches reveal heterogeneity in PM1386 expression?

Single-cell approaches offer unprecedented insights into expression heterogeneity and functional dynamics of PM1386 within bacterial populations. Develop fluorescent reporter systems by fusing the pm1386 promoter to fluorescent proteins (GFP, mCherry) to monitor expression dynamics at the single-cell level using time-lapse microscopy. Implement single-cell RNA-sequencing (scRNA-seq) adapted for bacterial cells to characterize transcriptional heterogeneity of pm1386 and co-regulated genes across individual bacteria within populations. For protein-level analysis, develop translational fusions of PM1386 with fluorescent proteins compatible with bacterial expression to monitor protein localization and abundance dynamics in real-time. Apply flow cytometry combined with fluorescent reporters to quantify expression heterogeneity across thousands of individual cells under different conditions and sort subpopulations for further analysis. Implement CRISPRi perturbations at the single-cell level to create mosaic populations with varying levels of PM1386 expression, allowing direct comparison of phenotypic consequences within the same population. Microfluidic systems can enable precise tracking of lineages with different PM1386 expression patterns during growth and stress responses. These single-cell approaches will reveal if PM1386 expression exhibits bistability, condition-dependent regulation, or cell-cycle dependence, providing insights into its functional role in bacterial physiology and potential contribution to phenotypic heterogeneity within P. multocida populations.

What interdisciplinary approaches could provide breakthrough insights into PM1386 function?

Breakthrough insights into PM1386 function may emerge from innovative interdisciplinary approaches combining diverse scientific fields. Integrate structural biology with molecular dynamics simulations and quantum mechanics calculations to predict potential enzymatic activities based on binding pocket architecture and electronic properties. Apply systems biology approaches combining multi-omics data (transcriptomics, proteomics, metabolomics) from pm1386 mutants with computational modeling to predict metabolic and regulatory network perturbations. Develop nanotechnology-based approaches using PM1386-functionalized nanoparticles to probe interaction landscapes within bacterial and host environments. Apply advanced imaging techniques including super-resolution microscopy and cryo-electron tomography to visualize PM1386 in its native cellular context at nanometer resolution. Explore the emerging field of bacterial optogenetics to develop light-controllable versions of PM1386, enabling precise spatiotemporal control of its activity to dissect its function. Implement microfluidic organ-on-a-chip technologies to study PM1386's role during infection in more physiologically relevant host tissue models. Computational approaches using graph neural networks may identify non-obvious functional relationships by mining literature and experimental data across multiple bacterial species. These interdisciplinary approaches overcome limitations of traditional methods by examining PM1386 from multiple perspectives simultaneously, potentially revealing unexpected functions that wouldn't be discovered through conventional approaches focused on a single discipline.