Recombinant Actinobacillus pleuropneumoniae serotype 3 Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is Actinobacillus pleuropneumoniae and why is it significant in research?

Actinobacillus pleuropneumoniae (APP) is a gram-negative bacterium identified as the primary etiological agent of porcine pleuropneumonia, a highly contagious respiratory disease causing significant economic losses in the global pork industry . The bacterium has 15 documented serotypes, all capable of infecting pigs regardless of age . Its clinical significance stems from its ability to establish both acute infections with high mortality rates and chronic carrier states where infected animals can shed the pathogen without showing clinical symptoms . The pathogenic capabilities of APP are largely attributed to its production of four major RTX toxins (ApxI, ApxII, ApxIII, and ApxIV), which are secreted in various combinations depending on the serotype . Research into APP is significant both for veterinary medicine and as a model for studying bacterial respiratory pathogens, host-pathogen interactions, and virulence mechanisms.

What is the role of UbiB in ubiquinone biosynthesis?

UbiB is classified as a probable ubiquinone biosynthesis protein, though its precise function remains incompletely characterized. Unlike other enzymes in the ubiquinone biosynthesis pathway that have well-defined catalytic activities, UbiB lacks the conserved motifs characteristic of hydroxylases that were once thought to be its primary function . Current evidence suggests UbiB may function as a putative kinase involved in Coenzyme Q (CoQ) synthesis, though definitive studies confirming this activity are still lacking . The uncertainty surrounding UbiB's exact role has complicated efforts to fully elucidate the ubiquinone biosynthesis pathway. In Escherichia coli, where ubiquinone biosynthesis has been more extensively studied, mutants lacking functional UbiB show decreased production of CoQ8, suggesting its essential role in this pathway despite the ambiguity regarding its precise enzymatic function .

How does ubiquinone biosynthesis differ between aerobic and anaerobic conditions?

Ubiquinone biosynthesis demonstrates remarkable adaptability to oxygen availability, with distinct aerobic and anaerobic pathways identified particularly in Escherichia coli and related proteobacteria. Under aerobic conditions, the classic oxygen-dependent pathway operates, utilizing molecular oxygen as a substrate for hydroxylation reactions catalyzed by monooxygenases like UbiI . In contrast, anaerobic conditions activate an oxygen-independent pathway controlled by the ubiT, ubiU, and ubiV genes . This anaerobic pathway performs hydroxylation of ubiquinone precursors through a unique oxygen-independent process, with UbiU and UbiV contributing to this oxygen-independent hydroxylation . The differential regulation is primarily controlled by the oxygen-sensing Fnr transcriptional regulator, which coordinates the expression of UbiTUV genes under anaerobic conditions . This dual pathway system enables facultative anaerobes to maintain ubiquinone synthesis across varying environmental oxygen levels, providing metabolic flexibility essential for adaptation to changing respiratory conditions.

What experimental animal models are available for studying Actinobacillus pleuropneumoniae?

Recent advances have established both pig and mouse models for studying Actinobacillus pleuropneumoniae infections, each with distinct advantages for different research applications. The natural host model using pigs provides high clinical relevance but presents challenges in terms of experimental operation, cost, and genetic manipulation. The recently developed mouse model offers several advantages including ease of experimental operation, lower costs, greater availability of genetic resources, and simplified disease progression monitoring .

A key breakthrough in the mouse model was the development of an effective airborne transmission method for APP serotype 1 (APP1), which overcame the challenges of bacterial host tropism and the small respiratory tract of mice . In the validated BALB/c mouse model, APP1 infection resulted in 60% mortality within three days, with bacterial dissemination from the lungs to other organs over the first 5 days post-infection . Histological and morphological examinations confirmed lung damage patterns similar to those observed in porcine infections. This mouse model has proven effective for antibacterial therapy testing, with ampicillin demonstrating full protection against APP1 infection . The model has also been used to compare virulence between different APP strains (APP1, APP2, and APP5) based on survival rates, establishing it as a reliable system for APP research and therapeutic development .

What methods are most effective for expressing and purifying recombinant Actinobacillus pleuropneumoniae UbiB protein?

The expression and purification of recombinant Actinobacillus pleuropneumoniae UbiB presents several technical challenges due to its membrane-associated nature and putative kinase activity. Based on successful approaches with related proteins, an effective expression protocol would likely employ a bacterial expression system using E. coli BL21(DE3) cells transformed with a vector containing the APP ubiB gene optimized with rare codon optimization for improved expression. The gene should be cloned into a vector with an N- or C-terminal affinity tag (6xHis or GST tag) to facilitate purification while minimizing interference with protein function.

For optimal expression, cultures should be grown at reduced temperatures (16-18°C) after induction with a low IPTG concentration (0.1-0.5 mM) to prevent inclusion body formation. Since UbiB is likely membrane-associated, inclusion of mild detergents (0.1-1% Triton X-100 or 0.5-2% CHAPS) during cell lysis is advisable. Purification would typically involve immobilized metal affinity chromatography (IMAC) using Ni-NTA resins for His-tagged constructs, followed by size exclusion chromatography to improve purity.

For functional studies, it's crucial to verify that the recombinant protein retains its native conformation and activity. This can be assessed through kinase activity assays measuring ATP consumption if the putative kinase function is being investigated. Additionally, co-expression with chaperone proteins may improve solubility and proper folding, particularly if expression yields are initially low.

How do mutations in the ubiB gene affect ubiquinone biosynthesis and bacterial virulence?

Mutations in the ubiB gene significantly impact ubiquinone biosynthesis, with consequent effects on bacterial metabolism and potential virulence alterations. While the precise function of UbiB remains incompletely characterized, studies in E. coli have shown that ubiB mutants exhibit decreased production of Coenzyme Q8 (CoQ8) . This reduction in ubiquinone synthesis affects the electron transport chain functionality, potentially compromising energy generation through oxidative phosphorylation.

The relationship between UbiB function and virulence is complex. In facultative anaerobes like E. coli, UbiB-dependent ubiquinone synthesis contributes to bacterial multiplication in the mouse gut, though this contribution appears modest . More significantly, the oxygen-independent ubiquinone synthesis pathway (involving proteins like UbiU and UbiV) has been shown to be essential for nitrate respiration and uracil biosynthesis under anaerobic conditions . These metabolic capabilities are particularly relevant for bacterial persistence in low-oxygen environments encountered during infection.

What is the relationship between ubiquinone biosynthesis and Apx toxin production in Actinobacillus pleuropneumoniae?

The relationship between ubiquinone biosynthesis and Apx toxin production in Actinobacillus pleuropneumoniae represents an intriguing but incompletely explored area of research. While no direct studies explicitly connecting these two processes are available in the current literature, several plausible hypotheses can be formulated based on the known functions of both systems.

Apx toxins (ApxI, ApxII, ApxIII, and ApxIV) are essential virulence factors of APP, with different serotypes producing various combinations of these toxins . These RTX (Repeats in Toxin) proteins require energy-dependent secretion systems for their export from the bacterial cell. Ubiquinone, as a central component of the electron transport chain, plays a crucial role in energy generation through aerobic and anaerobic respiration.

A potential relationship exists through the energy requirements for toxin synthesis and secretion. Disruptions in ubiquinone biosynthesis could lead to reduced ATP production, potentially limiting the energy available for Apx toxin synthesis, assembly, and secretion. Furthermore, ubiquinone biosynthesis and Apx toxin production may be co-regulated in response to environmental conditions encountered during infection. The oxygen-sensing transcriptional regulator Fnr, known to control anaerobic ubiquinone biosynthesis in E. coli , could potentially influence virulence factor expression in APP under the variable oxygen conditions encountered during respiratory infection.

Experimental approaches to investigate this relationship could include creating conditional ubiB mutants in APP and measuring Apx toxin production under various conditions, or examining transcriptional profiles to identify potential co-regulation of ubiquinone biosynthesis and toxin production genes.

How can recombinant UbiB protein be utilized in developing new vaccines against Actinobacillus pleuropneumoniae?

Recombinant UbiB protein from Actinobacillus pleuropneumoniae presents a novel antigen candidate for next-generation vaccines against porcine pleuropneumonia. Unlike traditional approaches focusing primarily on Apx toxins, targeting metabolic proteins like UbiB offers several potential advantages.

For vaccine development utilizing recombinant UbiB, several approaches could be considered:

Evaluation of such vaccines would require specialized ELISA methods to measure specific immune responses, similar to those developed for Apx toxins . The effectiveness of UbiB-targeting vaccines could potentially be assessed using the recently established mouse model of APP infection , which provides a cost-effective preliminary screening platform before proceeding to pig challenge studies.

What techniques are available for detecting and measuring ubiquinone production in bacterial cultures?

Several analytical techniques are available for detecting and quantifying ubiquinone in bacterial cultures, each with specific advantages for different research applications. These methodologies can be broadly categorized into chromatographic, spectroscopic, and biological approaches.

Chromatographic Methods:

• High-Performance Liquid Chromatography (HPLC): This represents the gold standard for ubiquinone quantification. Typically employing a C18 reverse-phase column with isocratic or gradient elution using methanol/ethanol and isopropanol mixtures. Detection can be performed using UV absorption (275 nm for ubiquinone) or electrochemical detection for enhanced sensitivity. HPLC has been successfully used to analyze ubiquinone intermediates in studies of ubiquinone biosynthesis .

• Ultra-Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS): Offers superior resolution and sensitivity compared to conventional HPLC, allowing identification and quantification of not only ubiquinone but also biosynthetic intermediates. This technique is particularly valuable for monitoring changes in the ubiquinone pathway when studying UbiB function.

Spectroscopic Methods:

• UV-Visible Spectroscopy: Ubiquinones have characteristic absorption peaks at approximately 275 nm in the oxidized form. This method is less specific but can be useful for rapid analysis of purified samples.

• Fluorescence Spectroscopy: Though ubiquinone itself is not strongly fluorescent, some intermediates in the biosynthetic pathway exhibit fluorescence, allowing for their detection and relative quantification.

Extraction Protocol:
For accurate analysis, proper extraction is critical. A typical protocol involves:

• Harvesting bacterial cells at appropriate growth phase

• Extraction with organic solvent mixtures (hexane:ethanol 5:2 v/v)

• Concentration under nitrogen

• Reconstitution in appropriate solvent for analysis

Table 1: Comparison of Methods for Ubiquinone Detection

When specifically studying UbiB function in ubiquinone biosynthesis, analysis of pathway intermediates becomes particularly important. Accumulation of specific precursors in UbiB mutants compared to wild-type can provide insights into the precise step where UbiB functions in the biosynthetic pathway.

How can researchers evaluate the functional activity of recombinant UbiB protein?

Evaluating the functional activity of recombinant UbiB protein presents unique challenges due to its incompletely characterized enzymatic function. Based on current understanding of UbiB as a putative kinase involved in ubiquinone biosynthesis , several complementary approaches can be employed to assess its functional activity.

In vitro Kinase Activity Assays:
Since UbiB is hypothesized to function as a kinase, ATP consumption assays represent a logical first approach. These can be performed using:

• Luminescence-based ATP detection: Commercial kits measuring residual ATP following incubation with recombinant UbiB and potential substrates.

• Radioactive assays: Using [γ-32P]ATP to detect phosphorylation of potential substrates, followed by SDS-PAGE and autoradiography or scintillation counting.

• Phosphate release assays: Colorimetric methods to detect inorganic phosphate released during kinase activity, such as malachite green-based assays.

The major challenge is identifying the correct physiological substrate. Candidates could include intermediates in the ubiquinone biosynthetic pathway or other proteins involved in this pathway.

Complementation Studies:
Genetic complementation provides powerful evidence of functional activity and can be conducted as follows:

• Transform UbiB-deficient bacterial strains (E. coli ubiB mutants or APP ubiB knockouts) with a plasmid expressing recombinant UbiB.

• Measure restoration of ubiquinone production using HPLC or other analytical methods described in section 3.1.

• Assess growth characteristics under conditions requiring functional ubiquinone, such as aerobic respiration-dependent growth.

Protein-Protein Interaction Analysis:
UbiB may function as part of a complex with other ubiquinone biosynthesis proteins. Methods to investigate these interactions include:

• Co-immunoprecipitation: Using antibodies against recombinant UbiB to pull down interacting proteins.

• Bacterial two-hybrid assays: To screen for potential interaction partners.

• Mass spectrometry following cross-linking: To identify proteins in close proximity to UbiB in vivo.

Table 2: Evaluation Methods for Recombinant UbiB Activity

A comprehensive assessment would ideally combine multiple approaches, correlating in vitro biochemical activity with in vivo functional complementation to provide convincing evidence of recombinant UbiB activity.

What are the most effective immunological methods for detecting Actinobacillus pleuropneumoniae infection in research settings?

Effective immunological detection of Actinobacillus pleuropneumoniae infection is crucial for both research applications and diagnostic purposes. Several methods have been developed, with particular success in targeting APP-specific antigens to avoid cross-reactivity with other Actinobacillus species.

ELISA-Based Detection Methods:
Enzyme-linked immunosorbent assay (ELISA) represents the most widely used immunological detection method for APP. Significant advances have been made in developing specific ELISA approaches:

• ApxIV-Specific ELISA: This approach targets the ApxIV toxin, which is uniquely produced by all 15 serotypes of APP only during infection and not under in vitro conditions . Unlike antibodies against ApxI, ApxII, and ApxIII, which may cross-react with toxins from less pathogenic Actinobacillus species (A. rossii and A. suis), ApxIV-specific antibodies are exclusively found in pigs infected with APP . Recombinant proteins based on the N-terminal part of ApxIV have been successfully used in both immunoblot and indirect ELISA formats to detect APP-specific infections .

• Serotype-Specific Apx Toxin ELISAs: Researchers have developed methods to measure immune responses to individual Apx toxins separately, addressing the challenge of antibody cross-reactivity. This involves identifying specific antigen regions among the toxins, cloning them, and developing ELISA methods that can detect specific immune responses to each Apx toxin . These methods are particularly valuable for evaluating vaccine efficacy.

Immunoblot Analysis:
Western blot analysis using recombinant ApxIV has been shown to detect APP infections in experimentally infected pigs by the second to third week post-infection . This approach provides high specificity but is more labor-intensive than ELISA methods.

Temporal Considerations:
When designing immunological detection studies, the temporal dynamics of antibody development must be considered. Detection of ApxIV-specific antibodies typically becomes possible 2-3 weeks post-infection , which may limit early detection capabilities.

Table 3: Comparison of Immunological Detection Methods for APP

These immunological approaches can be complemented with molecular methods such as PCR for comprehensive detection strategies, particularly in research settings requiring early detection before seroconversion occurs.

What bioinformatic approaches can identify potential functions and interactions of UbiB protein?

Bioinformatic analysis provides powerful tools to predict potential functions and interactions of incompletely characterized proteins like UbiB. These computational approaches can guide experimental design and generate testable hypotheses about UbiB's role in ubiquinone biosynthesis.

Sequence-Based Functional Prediction:

• Homology-Based Annotation: Identifying homologs with known functions through BLAST searches against curated databases (UniProt, KEGG). While UbiB lacks conserved hydroxylase motifs, it may share structural features with kinase families .

• Domain and Motif Analysis: Tools like PROSITE, Pfam, and InterPro can identify functional domains and motifs within the UbiB sequence. The presence of nucleotide-binding motifs would support its putative kinase function.

• Phylogenetic Analysis: Constructing phylogenetic trees of UbiB homologs across species can reveal evolutionary relationships and functional conservation, particularly comparing UbiB between E. coli (where it's better studied) and Actinobacillus pleuropneumoniae.

Structural Prediction and Analysis:

• 3D Structure Prediction: Using tools like AlphaFold2, RoseTTAFold, or I-TASSER to generate structural models of UbiB. These models can be compared with known structures of kinases and other enzymes involved in ubiquinone biosynthesis.

• Molecular Docking: Performing in silico docking studies with potential substrates, including ATP and ubiquinone precursors, to identify potential binding sites and catalytic residues.

• Molecular Dynamics Simulations: Investigating the dynamic behavior of UbiB, particularly regions predicted to be involved in substrate binding or catalysis.

Network and Systems Biology Approaches:

• Gene Neighborhood Analysis: Examining the genomic context of ubiB in various bacterial species to identify consistently co-localized genes, which may suggest functional relationships.

• Co-expression Network Analysis: Identifying genes with similar expression patterns across various conditions, which may indicate functional relationships.

• Protein-Protein Interaction Prediction: Tools like STRING and STITCH can predict interaction partners based on various evidence types, including genomic context, co-expression, and text mining of scientific literature.

Table 4: Recommended Bioinformatic Tools for UbiB Analysis

The integration of these various bioinformatic approaches can provide a comprehensive prediction of UbiB function, generating specific hypotheses that can be experimentally validated using the biochemical and genetic methods discussed in previous sections.