Recombinant Burkholderia cepacia Nucleoside diphosphate kinase (ndk)

What is the biological function of Nucleoside diphosphate kinase in Burkholderia cepacia?

Nucleoside diphosphate kinase (Ndk) is an evolutionarily highly conserved enzyme that catalyzes γ-phosphate transfer between nucleoside triphosphates and diphosphates (NTPs/NDPs). In B. cepacia and other bacteria, Ndk exhibits remarkable promiscuity, lacking a preferential NTP source and capable of transferring phosphate to multiple NDP targets. This versatility is critical for generating high-energy NTP molecules essential for DNA and RNA synthesis, as well as polysaccharide formation, which is particularly important for opportunistic pathogens .

B. cepacia Ndk demonstrates dual modes of action, functioning at both intracellular and extracellular levels during host infection. Beyond its canonical enzymatic role, B. cepacia Ndk appears to have evolved additional functions that contribute to host colonization, including immunomodulatory capabilities that may facilitate bacterial persistence in host tissues .

How does B. cepacia Ndk differ between clinical and environmental strains?

Research has revealed significant functional differences in Ndk activity between clinical and environmental strains of B. cepacia. Environmental strains typically exhibit more active Ndk compared to clinical isolates, which correlates with distinct host responses following infection. Notably, environmental strains with higher Ndk activity are associated with enhanced host-cell survival during infection, whereas clinical strains with relatively inactive Ndk tend to induce greater cytotoxicity and cell death .

This functional differentiation suggests that Ndk activity may be a determining factor in bacterial virulence strategies. Environmental strains, which are generally less virulent during early-stage colonization, may leverage active Ndk to establish opportunistic infections by preventing extracellular ATP (eATP)-mediated host tissue destruction. This mechanism potentially allows environmental B. cepacia strains to establish more persistent infections by preserving host cell viability .

What are the optimal expression systems for producing recombinant B. cepacia Ndk?

For efficient expression of recombinant B. cepacia Ndk, E. coli-based expression systems have proven most effective in laboratory settings. When designing expression constructs, researchers should consider:

• Vector selection: pET series vectors (particularly pET28a) with T7 promoter systems offer high-yield expression

• Affinity tags: N-terminal 6xHis-tag facilitates purification without significantly affecting enzymatic activity

• Host strains: E. coli BL21(DE3) or Rosetta strains accommodate the codon usage preferences of Burkholderia species

• Induction conditions: IPTG concentrations of 0.5-1.0 mM at 25°C rather than 37°C minimize inclusion body formation

Successful expression protocols typically involve cultivation in LB medium supplemented with appropriate antibiotics, induction at mid-log phase (OD600 ≈ 0.6-0.8), and post-induction expression periods of 16-18 hours at reduced temperatures.

What techniques are available for detecting B. cepacia complex in clinical and environmental samples?

Several molecular techniques have been developed for detecting Burkholderia cepacia complex (BCC) bacteria, with varying sensitivity and specificity profiles:

• Recombinase-aided amplification (RAA) assay targeting the 16S rRNA gene: This novel approach can be completed in just 10 minutes at 39°C, with a sensitivity of 10 copies per reaction and high specificity. Clinical validation demonstrated 100% sensitivity and 98.5% specificity when tested against 269 clinical samples from bacterial pneumonia patients .

• Real-time RPA (recombinase polymerase amplification) assay targeting the secY gene: In silico validation using 1,129 representative BCC genomes showed 96.99% sensitivity with no false positives among 61,858 non-BCC bacterial genomes. The F6-R4 primer combination proved particularly effective for detecting known BCC strains .

• Whole genome sequencing (WGS): The ultimate approach for identification, typing, and characterizing virulence factors and resistance mechanisms. WGS has successfully identified more than 160 genes involved in virulence and resistance to antibiotics, disinfectants, and preservatives in B. lata and B. contaminans isolates from pharmaceutical environments .

• Traditional gene targets: The 16S rDNA gene, recA gene, fur gene, and hisA gene remain common targets for DNA typing and identification of Burkholderia species due to their genetic polymorphism .

How does B. cepacia Ndk modulate host immune responses during infection?

B. cepacia Ndk plays a significant role in modulating host immune responses through multiple mechanisms:

• eATP modulation: B. cepacia Ndk can hydrolyze extracellular ATP (eATP), which serves as a critical danger signal in host immune activation. By depleting eATP, Ndk from environmental strains may prevent eATP-mediated host tissue destruction, potentially facilitating establishment of opportunistic infections .

• Cell death regulation: The differential activity of Ndk between clinical and environmental strains appears to influence host cell survival outcomes. Environmental strains with more active Ndk are associated with reduced cytotoxicity, suggesting that Ndk activity may be a determining factor in whether infection leads to cell death or bacterial persistence .

• Comparison with other bacterial Ndk: This immunomodulatory function parallels observations in other bacterial species such as Mycobacterium bovis BCG, where secreted Ndk prevents eATP-mediated macrophage cell death, allowing phagocytes to survive and thereby supporting long-term bacterial carriage in host cells .

The specific molecular pathways through which B. cepacia Ndk interacts with host immune components warrant further investigation, as current evidence suggests both direct enzymatic effects on danger signals and potential interference with host cell signaling cascades.

What experimental models are most appropriate for studying B. cepacia Ndk function in host interactions?

Several experimental models have been validated for investigating B. cepacia Ndk function during host interactions:

• Primary human gingival epithelial cell (GEC) model: This has been successfully employed for characterizing Ndk in dynamic host-microbe interactions, offering insights into mechanisms at the epithelial barrier .

• Macrophage infection models: Given the ability of Burkholderia species to target macrophages, macrophage cell lines (such as THP-1, J774, or RAW264.7) or primary human macrophages provide valuable systems for studying Ndk's effects on phagocyte function and survival.

• Cystic fibrosis cell models: Since BCC is particularly problematic in cystic fibrosis patients, epithelial cell lines with CFTR mutations or primary bronchial epithelial cells from CF patients represent disease-relevant models for studying Ndk's role in CF-specific pathogenesis.

• Animal models: Murine models of acute and chronic respiratory infection can be utilized to investigate the in vivo relevance of Ndk, particularly through comparative studies using wild-type bacteria versus Ndk-knockout mutants.

For optimal experimental design, researchers should employ multiple complementary models and consider using the established B. cepacia experimental strain panel, which includes representative strains of the first five genomovars of the B. cepacia complex .

How can site-directed mutagenesis of B. cepacia Ndk reveal structure-function relationships?

Site-directed mutagenesis represents a powerful approach for dissecting the structure-function relationships of B. cepacia Ndk. Key considerations include:

• Catalytic site mutations: Target highly conserved residues in the active site (such as H118, which coordinates Mg2+ for phosphate transfer) to distinguish enzymatic from non-enzymatic functions.

• Oligomerization interface modifications: Ndk typically functions as a hexamer; mutations at subunit interfaces can help determine whether oligomerization is essential for all Ndk functions or if monomeric variants retain certain activities.

• Secretion signal alterations: Mutations in potential secretion signals can help elucidate the mechanisms by which Ndk is delivered extracellularly during infection.

• Comparative analysis: Design mutations based on structural comparisons with well-characterized Ndk proteins from other bacterial species (e.g., Pseudomonas aeruginosa, Mycobacterium tuberculosis) to identify unique features of B. cepacia Ndk.

When testing mutant proteins, researchers should assess multiple parameters:

• Enzymatic activity (phosphotransferase assay)

• Oligomerization state (size exclusion chromatography)

• Secretion efficiency in bacterial culture

• Interactions with host proteins (pull-down assays)

• Effects on host cell viability and immune responses

What explains the contradictory findings regarding Ndk's role in different bacterial species?

The literature contains apparent contradictions regarding Ndk's role in host-pathogen interactions across different bacterial species. These contradictions likely stem from several factors:

• Species-specific effects: Ndk from different bacterial species may have evolved distinct functions beyond their conserved enzymatic activity. For instance, Ndk from Pseudomonas aeruginosa appears to contribute to host cell death in mucoid strains, while Mycobacterium bovis BCG Ndk prevents eATP-mediated macrophage death .

• Strain variability: Even within the B. cepacia complex, significant functional differences exist between clinical and environmental isolates. Environmental strains with more active Ndk appear to promote host cell survival, whereas clinical strains with relatively inactive Ndk are associated with enhanced cytotoxicity .

• Localization differences: The subcellular localization and secretion mechanisms of Ndk may vary across bacterial species, resulting in different concentrations of the enzyme in various host compartments.

• Host cell type specificity: The effects of bacterial Ndk may depend on the target host cell type, which varies across experimental models. For example, effects on macrophages might differ from effects on epithelial cells.

• Experimental conditions: Differences in infection models, bacterial growth conditions, and analytical methods may contribute to seemingly contradictory results.

To reconcile these contradictions, researchers should conduct comparative studies using standardized experimental conditions, multiple bacterial strains, and diverse host cell types. Additionally, genetic knockout and complementation studies are essential to definitively attribute observed phenotypes to Ndk activity.

How effective is the secY gene as a molecular marker for detecting and identifying B. cepacia complex bacteria?

The secY gene has emerged as a highly effective molecular marker for detecting and identifying Burkholderia cepacia complex (BCC) bacteria. In silico evaluation using a comprehensive genomic database of 1,376 BCC and 156,793 non-BCC bacterial genomes demonstrated exceptional performance:

• Sensitivity: The secY gene was present in 99.93% of BCC bacterial genomes, with only one B. cenocepacia strain (VC2387) lacking the gene, likely due to incomplete sequencing data .

• Specificity: The full-length secY sequence showed 98.5% specificity, while focusing on the core sequence improved specificity to 99.8% by reducing false positive results from 2,355 to just 269 non-BCC strains .

• Phylogenetic resolution: Phylogenetic analysis of secY sequences effectively clustered all 24 BCC species within a single branch, clearly distinguishing them from non-BCC species of the Burkholderia genus and other closely related bacteria .

• Primer performance: The F6-R4 primer combination validated against 1,129 representative BCC genomes showed 96.99% sensitivity with no false positives among 61,858 non-BCC bacterial genomes, making it an excellent choice for BCC detection assays .

This data indicates that the secY gene provides superior discrimination compared to traditional markers such as 16S rDNA, recA, fur, and hisA genes, which often suffer from excessive genetic polymorphism that can lead to misleading results when novel BCC species are encountered .

What are the current challenges in accurately identifying and characterizing BCC species?

Despite advances in molecular methods, accurate identification and characterization of Burkholderia cepacia complex (BCC) species face several significant challenges:

• Taxonomic complexity: The BCC currently comprises 21 validated species that are phenotypically similar and difficult to differentiate using standard methods. This complexity continues to expand as new species are discovered .

• Genetic plasticity: BCC bacteria exhibit substantial genomic variation and plasticity, complicating the development of universal detection methods. Some gene targets show high polymorphism, making it challenging to design assays that cover all potential BCC strains .

• Method limitations: Traditional phenotypic methods often fail to distinguish between BCC species, while molecular methods based on single genes may yield misleading results due to genetic variations or horizontal gene transfer .

• Novel species detection: With continuous discovery of new BCC species, assays based on non-specific genes may produce misleading results when encountering previously uncharacterized strains .

• Environmental vs. clinical strains: Significant phenotypic and genotypic differences between environmental and clinical isolates of the same species further complicate identification efforts .

To address these challenges, researchers increasingly rely on multi-gene or whole-genome approaches:

• Whole genome sequencing (WGS) has emerged as the ultimate approach for identifying, typing, and characterizing virulence factors and resistance profiles .

• Novel biomarker mining strategies that identify conserved genes like secY offer improved sensitivity and specificity compared to traditional markers .

• Combining multiple molecular targets may provide more reliable identification than single-gene approaches.

How can recombinant B. cepacia Ndk be utilized in drug discovery research?

Recombinant B. cepacia Ndk offers several promising applications in drug discovery research, particularly for developing novel antimicrobial strategies:

• As a target for inhibitor development:

  • High-throughput screening assays using purified recombinant Ndk can identify small molecule inhibitors

  • Structure-based drug design leveraging crystal structures of Ndk can guide rational inhibitor optimization

  • Natural product libraries can be screened for compounds that selectively inhibit bacterial Ndk while sparing human counterparts

• As a tool for understanding host-pathogen interactions:

  • Recombinant Ndk can be used to identify host cell receptors and binding partners

  • In vitro assays with recombinant Ndk can map signaling pathways disrupted during infection

  • Comparative studies with Ndk from different BCC species can reveal species-specific virulence mechanisms

• For vaccine development:

  • Exploration of modified recombinant Ndk (enzymatically inactive mutants) as potential vaccine candidates

  • Screening of Ndk epitopes for immunogenicity and protective potential

  • Combination approaches incorporating Ndk with other BCC antigens for multivalent vaccines

• For studying antibiotic resistance mechanisms:

  • Investigating whether Ndk contributes to antimicrobial resistance through energy metabolism modulation

  • Examining potential synergies between Ndk inhibitors and conventional antibiotics

  • Exploring connections between Ndk activity and biofilm formation, which contributes to antibiotic tolerance

Future drug discovery efforts should consider the differential effects of Ndk between clinical and environmental strains, as these differences may influence the efficacy of targeting Ndk in different infection scenarios.

What novel research directions are emerging for understanding B. cepacia Ndk's role in cystic fibrosis infections?

Several novel research directions are emerging to better understand B. cepacia Ndk's role in cystic fibrosis (CF) infections:

• CF-specific microenvironment interactions:

  • Investigating how the unique biochemical environment of CF airways (increased mucus, altered pH, hypoxia) affects Ndk expression and activity

  • Examining whether Ndk contributes to BCC's ability to thrive in the competitive polymicrobial communities typical of CF airways

  • Studying how Ndk-mediated ATP modulation affects ion transport in CF epithelial cells with CFTR mutations

• Host adaptation mechanisms:

  • Tracking evolutionary changes in Ndk sequence and expression during long-term BCC colonization of CF lungs

  • Comparing Ndk variants between initial colonizing strains and isolates from chronic infections

  • Investigating whether Ndk contributes to the transition from environmental to clinical phenotypes during adaptation to the CF lung

• Interspecies interactions:

  • Exploring how B. cepacia Ndk influences co-infecting pathogens common in CF, such as Pseudomonas aeruginosa

  • Examining whether Ndk-mediated ATP modulation affects competitive dynamics in polymicrobial communities

  • Investigating potential horizontal gene transfer of ndk variants between different bacterial species in CF airways

• Advanced CF model systems:

  • Utilizing CF patient-derived lung organoids to study Ndk's effects in physiologically relevant 3D tissue environments

  • Employing advanced imaging techniques to track the spatiotemporal distribution of Ndk during infection of CF airway models

  • Leveraging single-cell RNA sequencing to characterize host cell responses to Ndk exposure in heterogeneous CF airway cell populations

These emerging research directions will benefit from interdisciplinary approaches combining molecular microbiology, immunology, and CF pathophysiology to develop a comprehensive understanding of Ndk's contribution to BCC pathogenesis in the context of cystic fibrosis.