Recombinant Putative antitoxin VapB18 (vapB18)

What is the VapB18 antitoxin and what is its role in bacterial cells?

VapB18 is a putative antitoxin protein that belongs to the VapBC (Virulence associated protein B and C) family of type II toxin-antitoxin (TA) systems. In bacterial cells, VapB18 functions to neutralize the toxic effects of its cognate toxin, likely VapC18. Under normal growth conditions, the antitoxin binds to and inhibits the activity of the toxin, preventing it from damaging the cell. Based on studies of similar VapB antitoxins, VapB18 likely also acts as a transcriptional regulator, binding to the promoter region of the vapBC18 operon to repress its expression . This dual functionality—toxin neutralization and transcriptional regulation—is characteristic of type II antitoxins, allowing bacteria to finely tune their stress response mechanisms.

How do VapB antitoxins like VapB18 neutralize their cognate toxins?

VapB antitoxins, including VapB18, neutralize their cognate toxins through direct protein-protein interaction. This interaction prevents the toxin from exerting its ribonuclease activity on cellular RNA targets. From studies on other VapB antitoxins, we understand that antitoxins typically bind to toxins via their C-terminal domains, forming a stable complex. For instance, research on the Rv2018 antitoxin demonstrated that it effectively neutralizes the toxic effect of the Rv2019 toxin when co-expressed in E. coli, restoring normal colony formation and growth . This neutralization mechanism is likely consistent across the VapBC family, suggesting that VapB18 would similarly counteract VapC18 activity by forming a tight complex that blocks the toxin's active site.

What is the genomic organization of the vapB18-vapC18 operon?

The vapB18-vapC18 operon, like other vapBC operons, is likely organized with the antitoxin gene (vapB18) preceding the toxin gene (vapC18). This genetic arrangement is a common feature of type II TA systems. Based on studies of similar VapBC modules, we can infer that the vapB18-vapC18 genes are co-transcribed from a single promoter, with the expression tightly regulated by the VapB18 antitoxin itself, which binds to the operator site to repress transcription. In Mycobacterium tuberculosis, which contains approximately 50 putative VapBC TA systems, such operons are distributed throughout the genome . The conserved organization facilitates coordinated expression and regulation of the toxin and antitoxin components, ensuring proper functioning of the TA system in response to environmental cues.

What are the optimal conditions for recombinant expression of VapB18?

The optimal conditions for recombinant expression of VapB18 would likely parallel those used for similar antitoxins. Based on methodologies applied to other VapB proteins, expression in E. coli BL21(DE3) using a pET system (such as pET21c) with IPTG induction at concentrations around 0.05-0.5 mM has proven effective . The expression should be carried out at temperatures ranging from 16-30°C to promote proper folding and solubility. For VapB18, an induction period of 4-6 hours at 30°C or overnight at 16°C would likely yield good protein expression. When co-expressing with its cognate toxin, it's crucial to ensure that the antitoxin is expressed prior to or simultaneously with the toxin to prevent growth inhibition of the host cells, as demonstrated with the Rv2018-Rv2019 pair .

What purification strategies are most effective for obtaining pure VapB18 protein?

Effective purification of VapB18 would likely involve a multi-step approach, similar to strategies employed for other VapB antitoxins. Initially, affinity chromatography using a His-tag system would facilitate initial capture of the protein. This could be followed by ion-exchange chromatography to separate the antitoxin from contaminants with different charge properties. Size exclusion chromatography would serve as a final polishing step to achieve high purity. Based on purification protocols for related antitoxins, the following conditions may be effective:

It's important to note that VapB antitoxins often have a tendency to aggregate or form insoluble complexes, so adding stabilizing agents such as 5-10% glycerol to all buffers may improve protein stability and yield .

How can researchers overcome stability issues when working with recombinant VapB18?

Overcoming stability issues with recombinant VapB18 requires several strategic approaches. First, consider co-expression with its cognate toxin VapC18, as the complex is often more stable than the antitoxin alone. Studies on other VapBC systems have demonstrated that the toxin-antitoxin complex can be more readily purified than individual components . Second, optimize buffer conditions: include stabilizing agents such as 5-10% glycerol, 1-5 mM DTT or TCEP to prevent oxidation, and consider adding metal ions (Mg²⁺ or Mn²⁺) that might stabilize the protein structure. Third, maintain lower temperatures (4°C) throughout purification processes to minimize degradation. Finally, consider fusion tags that enhance solubility, such as MBP (maltose-binding protein) or SUMO, which have been effective for other challenging proteins. If proteolytic degradation is an issue, adding protease inhibitors to all buffers and minimizing the time from cell lysis to final purification can significantly improve yields of intact VapB18.

What assays can be used to confirm the antitoxin activity of VapB18?

The antitoxin activity of VapB18 can be confirmed using several complementary approaches. A primary assay involves growth rescue experiments in bacterial expression systems. By co-expressing VapB18 with its cognate toxin VapC18 in E. coli, researchers can observe whether VapB18 neutralizes the growth inhibition caused by VapC18, similar to how Rv2018 counteracted the toxic effect of Rv2019 . This can be quantified through measurements of optical density, colony formation, and cell viability.

In vitro confirmation can be obtained through ribonuclease inhibition assays. If VapC18 exhibits ribonuclease activity like other VapC toxins, researchers can assess whether purified VapB18 inhibits this activity using purified rRNA or synthetic RNA substrates as targets. The inhibition can be visualized using gel electrophoresis to detect RNA degradation patterns .

Biophysical interaction studies using techniques such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), or microscale thermophoresis (MST) can determine the binding affinity between VapB18 and VapC18, providing quantitative data on their interaction strength and stoichiometry.

How can researchers investigate the DNA-binding properties of VapB18?

Investigating the DNA-binding properties of VapB18 requires several specialized techniques. Electrophoretic mobility shift assays (EMSA) provide a straightforward approach to detect VapB18 binding to its presumed operator DNA. This involves incubating purified VapB18 with labeled DNA fragments containing the putative binding site and observing the mobility shift on a native polyacrylamide gel.

DNase I footprinting can be employed to precisely identify the DNA sequence protected by VapB18 binding. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) offers an in vivo approach to map VapB18 binding sites across the genome, providing insights into potential regulatory roles beyond its own operon.

For quantitative analysis, fluorescence anisotropy or surface plasmon resonance can determine binding affinities and kinetics of the VapB18-DNA interaction. These techniques would reveal the strength of binding under various conditions, potentially elucidating how environmental factors influence VapB18's regulatory function. Based on studies of other VapB antitoxins, researchers should focus on the promoter region of the vapBC18 operon as the primary binding site for initial investigations .

What techniques can determine the stoichiometry of VapB18-VapC18 complexes?

The stoichiometry of VapB18-VapC18 complexes can be determined using several complementary biophysical techniques. Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) provides accurate molecular weight determination of the complex in solution, allowing researchers to calculate the ratio of components. Analytical ultracentrifugation (AUC), particularly sedimentation velocity and equilibrium experiments, offers high-resolution analysis of complex formation and can distinguish between different oligomeric states.

Native mass spectrometry provides precise mass measurements of intact complexes, revealing their composition and stoichiometry. X-ray crystallography, while more challenging, would provide atomic-level details of the complex structure, including the binding interface and stoichiometry. Based on studies of related VapBC complexes, the expected stoichiometry might be 2:2 or 4:4 (antitoxin:toxin), forming heterotetramers or heterooctamers .

How does VapB18 contribute to bacterial stress response and persistence?

VapB18, like other antitoxins in TA systems, likely plays a crucial role in bacterial stress response and persistence through dynamic regulation of its cognate toxin. Under stress conditions, cellular proteases such as Lon and ClpP degrade antitoxins like VapB18 more rapidly than their toxin counterparts due to the antitoxins' intrinsically disordered regions. This degradation releases the toxin (VapC18) from inhibition, allowing it to exert its ribonuclease activity on cellular RNAs. The resulting reduction in translation and metabolism helps bacteria enter a dormant or persistent state that enhances survival under adverse conditions.

In pathogenic bacteria, TA systems including VapBC pairs have been associated with adaptation to hostile environments encountered during infection. For instance, in Mycobacterium tuberculosis, which contains an unusually large number of TA systems (approximately 79), these modules are believed to contribute to the pathogen's remarkable ability to persist within host tissues . The conservation of these systems in pathogenic mycobacteria but not in non-pathogenic relatives suggests a potential role in virulence, persistence, and biofilm formation .

The controlled activation and deactivation of VapB18-VapC18, in coordination with other TA systems, likely allows for fine-tuned responses to specific stressors, facilitating bacterial adaptation to changing environments without committing to programmed cell death.

What is the relationship between VapB18 and bacterial virulence in pathogenic species?

The relationship between VapB18 and bacterial virulence in pathogenic species is likely complex and multifaceted. While no direct evidence specifically for VapB18 is available in the search results, insights can be drawn from studies of similar VapB antitoxins and TA systems generally. In Mycobacterium tuberculosis, the remarkable conservation of numerous TA systems across pathogenic mycobacterial species (M. tuberculosis, M. microti, M. bovis, M. africanum, and M. canetti) but not in non-pathogenic relatives strongly suggests their involvement in pathogenesis .

TA systems, including VapBC pairs, may contribute to virulence through several mechanisms:

• Facilitating persistence during host-imposed stress conditions, including nutrient limitation, oxidative stress, and antibiotic exposure

• Contributing to biofilm formation, which enhances bacterial resistance to host defenses and antimicrobials

• Modulating bacterial metabolism to adapt to the host environment

• Potentially regulating the expression of virulence factors through their ribonuclease activity and transcriptional regulation

The presence of the unusually large number of TA systems (79) in M. tuberculosis compared to related non-pathogenic mycobacterial species underscores their potential importance in pathogenesis . While the specific contribution of VapB18-VapC18 to virulence requires further investigation, its role as part of this broader network of stress-response elements suggests its involvement in the complex adaptation strategies that successful pathogens employ during infection.

How do mutations in the vapB18 gene affect toxin-antitoxin dynamics and bacterial fitness?

Mutations in the vapB18 gene could significantly alter toxin-antitoxin dynamics and bacterial fitness through several mechanisms. Based on studies of other VapB antitoxins, we can predict that mutations affecting the toxin-binding domain would reduce VapB18's ability to neutralize VapC18, potentially leading to unregulated toxin activity and growth inhibition or cell death. This is supported by observations that mutations in the Rv2019 toxin (E41K and D98K) affected its toxicity , suggesting that complementary mutations in its antitoxin would similarly disrupt their interaction.

Mutations in the DNA-binding domain of VapB18 would likely impair its ability to regulate transcription of the vapBC18 operon, potentially leading to overexpression of both components or dysregulated expression patterns. This could disrupt the fine balance needed for proper stress response.

Mutations affecting protein stability might lead to more rapid degradation of VapB18, even under non-stress conditions. This premature degradation would mimic stress conditions, potentially triggering inappropriate activation of persistence mechanisms and reducing bacterial fitness during normal growth.

Interestingly, studies in M. smegmatis showed that cells transformed with the Rv2019 toxin gene developed spontaneous mutations within the open reading frame , suggesting a selection pressure against fully functional toxins when not properly regulated by their antitoxins. This indicates that bacteria actively maintain the integrity of TA systems, highlighting their importance for fitness.

How does VapB18 structurally and functionally compare to other VapB antitoxins?

VapB18 likely shares structural and functional characteristics with other VapB antitoxins, while maintaining specificity for its cognate toxin. Structurally, VapB antitoxins typically possess a bipartite organization: an N-terminal DNA-binding domain that interacts with operator sequences, and a C-terminal domain that binds to and neutralizes the toxin. The structures of several VapBC complexes from M. tuberculosis have been determined, including VapBC3, VapBC5, VapBC15, and VapBC30 , providing templates for comparative analysis of VapB18.

Regulatory mechanisms appear consistent across VapBC systems, with the antitoxin serving as a transcriptional repressor of its own operon. This auto-regulation allows for rapid response to changing environmental conditions. The metal ion dependencies observed in VapC toxins, which require divalent cations like Mg²⁺ or Mn²⁺ for activity , suggest that VapB18 would need to interfere with these interactions to effectively neutralize VapC18.

What cross-reactivity exists between VapB18 and other VapC toxins?

Cross-reactivity between VapB18 and other VapC toxins is likely limited but possible in some cases. While toxin-antitoxin pairs typically show high specificity for their cognate partners, recent research has revealed that some non-cognate interactions can occur. The search results indicate that "a toxin and non-cognate antitoxin interact, and therefore, TA families are independently divided into 12 super-families of Type II toxins and 20 super-families of Type II antitoxins" .

This cross-reactivity may explain some experimental observations where expected toxicity was not observed. For instance, the search results mention that "neither native nor highly expressed GST-fused Rv3180c was toxic to E. coli," suggesting possible neutralization by non-cognate antitoxins present in the host .

For VapB18 specifically, any cross-reactivity would likely be limited to VapC toxins that share significant structural similarity at the antitoxin-binding interface. Experimental approaches to investigate this cross-reactivity could include:

• Co-expression studies pairing VapB18 with various VapC toxins to assess neutralization

• In vitro binding assays measuring affinity between VapB18 and different VapC toxins

• Structural studies identifying the binding interfaces that determine specificity

Understanding this cross-reactivity has implications for bacterial physiology, as it suggests potential backup mechanisms where one antitoxin might compensate for the loss of another, providing redundancy in stress response systems.

How is VapB18 expression regulated in different bacterial species and under various stress conditions?

VapB18 expression regulation likely follows patterns observed in other VapB antitoxins, with species-specific adaptations and stress-responsive elements. The primary regulation mechanism involves autoregulation, where the VapB18 antitoxin (possibly in complex with VapC18) binds to the promoter region of its own operon to repress transcription. This creates a negative feedback loop that maintains appropriate toxin-antitoxin levels under normal conditions.

Under stress conditions, regulation shifts dramatically. Various cellular proteases (particularly Lon and ClpP) are activated or redirected to degrade antitoxins like VapB18, which are typically more labile than their toxin counterparts. This differential stability is a key regulatory feature of TA systems, allowing rapid response to environmental changes.

Different bacterial species likely show variations in VapB18 regulation based on their ecological niches and lifestyle. In pathogenic species like M. tuberculosis, which contains approximately 50 putative VapBC TA systems , these modules appear to be important for adaptation to the host environment. The expression patterns might differ significantly between pathogenic and non-pathogenic species, as suggested by the observation that the Rv3180c toxin was toxic to M. smegmatis but not to E. coli .

Specific stress conditions that might trigger VapB18-VapC18 activation include:

What are the main challenges in distinguishing VapB18's effects from those of other antitoxins in complex bacterial systems?

Distinguishing VapB18's effects from those of other antitoxins presents several significant challenges. First, functional redundancy among multiple TA systems can mask the phenotypic effects of VapB18 manipulation. For example, M. tuberculosis contains approximately 79 TA systems , many of which may have overlapping functions, making it difficult to isolate the specific contribution of VapB18-VapC18.

Second, cross-reactivity between non-cognate toxins and antitoxins can confound experimental results. As noted in the search results, "a toxin and non-cognate antitoxin interact" , which means that manipulating VapB18 levels might affect not only VapC18 but potentially other toxins as well.

Third, the integration of TA systems into broader regulatory networks creates complex downstream effects that may be difficult to attribute specifically to VapB18. For instance, ribonuclease activity of toxins can affect global gene expression patterns, creating cascading effects throughout the cell.

To overcome these challenges, researchers should consider:

• Creating clean deletion mutants of vapB18 in combination with deletions of other TA systems to assess compound effects

• Using inducible expression systems with tightly controlled promoters to manipulate VapB18 levels with temporal precision

• Developing VapB18-specific antibodies or epitope tags for tracking protein levels under various conditions

• Employing RNA-seq and proteomics approaches to capture global effects of VapB18 manipulation

• Utilizing single-cell analyses to account for population heterogeneity in TA system activation

How can researchers effectively study VapB18-VapC18 interactions in vivo without disrupting normal cellular functions?

Effectively studying VapB18-VapC18 interactions in vivo while maintaining normal cellular functions requires sophisticated approaches that provide minimal disruption. One promising strategy is to use fluorescence resonance energy transfer (FRET) with fluorescent protein fusions to VapB18 and VapC18. This allows real-time monitoring of their interactions without significantly altering their function, particularly if the fluorescent tags are attached via flexible linkers to minimize structural interference.

Another approach involves split fluorescent protein complementation, where fragments of a fluorescent protein are fused to VapB18 and VapC18. When the two proteins interact, the fragments come together to form a functional fluorescent protein, providing a visual signal of interaction.

For minimal disruption of native expression patterns, CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) systems allow modulation of endogenous gene expression without permanent genetic modification. This approach enables researchers to tune VapB18 or VapC18 levels within physiological ranges.

Proximity-based labeling techniques (like BioID or APEX) can identify proteins in close proximity to VapB18 in living cells, providing insights into its interaction network. These methods involve fusing an enzyme to VapB18 that biotinylates nearby proteins, which can then be purified and identified.

To minimize disruption when studying the effects of VapB18-VapC18 on cell physiology, researchers can use inducible degron systems that allow rapid and reversible depletion of the proteins. This approach provides temporal control over protein levels while avoiding the compensatory mechanisms that often develop in knockout strains.

What controls should be included when designing experiments to study VapB18's role in bacterial stress response?

Designing rigorous experiments to study VapB18's role in bacterial stress response requires comprehensive controls to account for various confounding factors. Essential controls include:

• Genetic Controls:

  • Single gene deletion mutants (ΔvapB18, ΔvapC18)

  • Double deletion mutant (ΔvapB18ΔvapC18)

  • Complementation strains (ΔvapB18 + vapB18)

  • Point mutants affecting specific functions (DNA-binding, toxin-binding)

• Expression Controls:

  • Empty vector controls for all expression constructs

  • Non-toxic point mutants of VapC18 (based on mutations like E41K and D98K found in Rv2019 )

  • Non-binding point mutants of VapB18

  • Equivalent expression levels verified by western blot

• Stress Response Controls:

  • Multiple stress conditions (nutrient limitation, oxidative stress, antibiotic exposure)

  • Time course experiments to distinguish immediate from adaptive responses

  • Recovery conditions to assess reversibility

• Cross-System Controls:

  • Comparison with other VapBC systems

  • Chimeric constructs with domains from other TA systems

  • Epistasis analysis with other stress response pathways

• Species Controls:

  • Parallel experiments in pathogenic and non-pathogenic species

  • Heterologous expression in distinct bacterial hosts (similar to the differential effects of Rv3180c in E. coli versus M. smegmatis )

These controls help distinguish VapB18-specific effects from general stress responses, account for potential cross-reactivity between TA systems, and ensure observed phenotypes are directly attributable to VapB18 function rather than experimental artifacts or secondary effects.

What are promising therapeutic applications targeting VapB18-VapC18 interactions?

Promising therapeutic applications targeting VapB18-VapC18 interactions focus primarily on disrupting bacterial persistence and resensitizing pathogens to conventional antibiotics. Since TA systems like VapBC contribute to persistence and antibiotic tolerance, compounds that interfere with VapB18's ability to neutralize VapC18 could potentially activate the toxin and induce self-intoxication in pathogens. Conversely, molecules that mimic VapB18 and constitutively inhibit VapC18 could prevent the bacterium from entering a persistent state, maintaining its sensitivity to antibiotics.

Small-molecule inhibitors designed to disrupt the VapB18-VapC18 interaction interface represent a promising approach. Structural studies of related VapBC complexes from M. tuberculosis (VapBC3, VapBC5, VapBC15, and VapBC30) provide templates for rational drug design targeting conserved interaction motifs. Peptide-based inhibitors derived from the binding interface of VapB18 could also serve as lead compounds.

Another innovative approach involves manipulating the stability of VapB18. Compounds that prevent antitoxin degradation during stress would keep the toxin permanently neutralized, potentially blocking persistence. Conversely, molecules that selectively accelerate VapB18 degradation could activate VapC18 toxicity at will.

Given the observation that toxin expression can trigger growth arrest rather than cell death , combination therapies pairing TA system modulators with conventional antibiotics might be particularly effective, with the former preventing persistence and the latter eliminating the active population.

How might CRISPR-Cas technologies be applied to study VapB18 function and regulation?

CRISPR-Cas technologies offer powerful approaches to study VapB18 function and regulation with unprecedented precision. CRISPR interference (CRISPRi) utilizing a catalytically inactive Cas9 (dCas9) fused to a transcriptional repressor domain can be employed to selectively downregulate vapB18 expression without affecting the adjacent vapC18 gene. This approach overcomes the limitations of traditional knockout methods, which might disrupt the entire operon.

Conversely, CRISPR activation (CRISPRa) systems can upregulate vapB18 expression by targeting dCas9 fused to transcriptional activators to the promoter region. This allows researchers to assess the effects of VapB18 overexpression on cellular physiology and stress response.

For precise genome editing, CRISPR-Cas9 can be used to introduce specific mutations in the vapB18 gene to study structure-function relationships. For example, mutations affecting the DNA-binding domain or the toxin-binding interface can be introduced with minimal off-target effects.

Live-cell imaging of VapB18 dynamics can be achieved by using CRISPR to insert fluorescent protein tags at the endogenous vapB18 locus, ensuring physiologically relevant expression levels. Similarly, CRISPR can be used to insert affinity tags for chromatin immunoprecipitation (ChIP) studies to identify VapB18 binding sites across the genome.

Base editing and prime editing CRISPR systems allow for even more precise modifications without introducing double-strand breaks, making them ideal for studying the effects of single amino acid changes in VapB18 on its function and interactions with VapC18.

What technological advances would facilitate better understanding of VapB18's role in bacterial physiology and pathogenesis?

Several technological advances would significantly enhance our understanding of VapB18's role in bacterial physiology and pathogenesis. High-resolution single-cell analysis techniques would enable researchers to observe heterogeneity in VapB18-VapC18 activation within bacterial populations, potentially revealing subpopulations poised for persistence. This includes advances in single-cell proteomics, transcriptomics, and microfluidic platforms that can track individual cell fates over time.

Improved structural biology techniques, particularly cryo-electron microscopy (cryo-EM) methods that can resolve smaller complexes, would facilitate determination of the VapB18-VapC18 complex structure without the need for crystallization. This would provide insights into the molecular basis of their interaction and potential targets for therapeutic intervention.

In vivo biosensors that can detect VapB18-VapC18 activity in real-time during infection would bridge the gap between in vitro studies and actual pathogenesis. These might include FRET-based sensors that respond to changes in VapB18-VapC18 interaction or reporters sensitive to the ribonuclease activity of VapC18.

Advanced animal models with humanized immune systems would provide more realistic environments for studying the role of VapB18-VapC18 in pathogenesis. Complementing these, tissue-on-chip technologies could simulate specific infection microenvironments where TA systems are activated.

Computational advances, particularly in systems biology and machine learning approaches, would help integrate the vast amounts of data generated and predict the complex network effects of manipulating VapB18-VapC18, accounting for interactions with other TA systems and regulatory networks. This would be particularly valuable given the large number of TA systems in pathogens like M. tuberculosis .