Recombinant Probable ribonuclease VapC35 (vapC35)

What is VapC35 and what is its role in Mycobacterium tuberculosis?

VapC35 is a toxin component of the VapBC35 toxin-antitoxin (TA) system present in Mycobacterium tuberculosis. It belongs to the VapBC family, which is notably abundant in members of the M. tuberculosis complex. VapC35 functions as a ribonuclease that targets specific RNA substrates, thereby regulating cellular processes during stress conditions .

The toxin plays a significant role in stress adaptation, particularly during oxidative stress. Studies demonstrate that the VapBC35 system is necessary for M. tuberculosis to adapt under oxidative stress conditions, suggesting its importance in the pathogen's survival mechanism during host immune responses . Interestingly, while it is crucial for stress adaptation, VapBC35 appears to be dispensable for M. tuberculosis growth in guinea pig infection models, indicating functional redundancy among the multiple TA systems within the pathogen .

VapC35 is expressed as part of a stress response mechanism and contributes to the bacterial persistence that characterizes M. tuberculosis infections. The toxin's activity is normally neutralized by its cognate antitoxin VapB35, maintaining a balanced cellular state under normal conditions .

What is the enzymatic mechanism of VapC35?

VapC35 exhibits Mg²⁺-dependent ribonuclease activity, targeting RNA substrates such as MS2 RNA. Its catalytic function depends on a conserved PIN domain, with the glutamic acid residue E5 being critical for enzymatic activity. The ribonuclease mechanism involves the following key aspects:

• Metal ion dependency: VapC35 requires magnesium ions (Mg²⁺) as cofactors for catalytic activity. This dependency is evidenced by complete inhibition of RNA cleavage when EDTA (a chelating agent) is present.

• Substrate specificity: Like other VapC family toxins, VapC35 appears to be sequence-selective in its ribonuclease activity, targeting specific RNA sequences rather than degrading RNA indiscriminately.

• Catalytic residues: The glutamic acid at position 5 (E5) in the PIN domain is essential for catalysis. Mutation of this residue to alanine (E5A) completely abolishes the ribonuclease activity and toxicity of VapC35 .

The following table summarizes experimental evidence of VapC35's ribonuclease activity:

These findings confirm VapC35's role as a sequence-selective ribonuclease, similar to other VapC family members in M. tuberculosis.

How does the VapBC35 toxin-antitoxin system function?

The VapBC35 toxin-antitoxin system operates through a tightly regulated molecular mechanism that balances the potentially harmful effects of the VapC35 toxin. The system functions through the following processes:

• Autoregulation: The VapBC35 complex regulates its own expression by binding to the promoter-operator DNA region. Research indicates that increasing the VapB35 antitoxin to VapC35 toxin ratio results in stronger binding affinity to the promoter-operator DNA, leading to tighter autoregulation .

• Toxin inhibition: Under normal conditions, the VapB35 antitoxin directly binds to VapC35, neutralizing its ribonuclease activity. This protein-protein interaction prevents VapC35 from cleaving cellular RNA, thereby maintaining normal cell growth and division .

• Stress response: Under stress conditions, cellular proteases degrade the more labile VapB35 antitoxin, releasing active VapC35. The free toxin then cleaves specific RNA targets, temporarily halting translation and cell growth, which may contribute to stress adaptation and persistence .

• Cross-interactions: Interestingly, VapC35 can also interact with non-cognate antitoxins such as VapB3. These cross-interactions between different TA systems suggest the existence of complex regulatory networks within M. tuberculosis, potentially providing redundancy and fine-tuning of stress responses .

The toxin-antitoxin balance is critical for bacterial physiology, as demonstrated by co-expression studies showing that VapB35 antitoxin can restore normal growth in bacteria where VapC35 is overexpressed .

What experimental models are used to study VapC35?

Researchers employ various experimental models to investigate VapC35 function and regulation. The primary models include:

• Recombinant protein expression systems: VapC35 is typically expressed with an N-terminal 6X-His tag in E. coli for biochemical assays and purification using standard protocols . This approach allows for in vitro characterization of enzymatic activity and protein-protein interactions.

• Mycobacterium smegmatis as a surrogate host: M. smegmatis is frequently used as a non-pathogenic surrogate for studying VapC35 function. Using inducible expression systems (such as tetracycline-inducible promoters), researchers can control VapC35 expression to examine its effects on bacterial growth, morphology, and viability .

• In vitro RNA cleavage assays: MS2 RNA is commonly used as a substrate to assess the ribonuclease activity of purified VapC35 under various conditions, including the presence or absence of magnesium ions and EDTA .

• M. tuberculosis deletion mutants: Researchers generate VapBC35 deletion mutants in M. tuberculosis to study the system's role in stress adaptation, virulence, and in vivo growth in animal infection models such as guinea pigs .

• Co-expression systems: Dual inducible systems allowing separate control of toxin and antitoxin expression help investigate interactions between VapC35 and various antitoxins (both cognate and non-cognate) .

These models collectively provide insights into the biochemical properties, cellular effects, and physiological roles of VapC35 in mycobacterial biology and pathogenesis.

How can VapC35 ribonuclease activity be measured in vitro?

Measuring VapC35 ribonuclease activity in vitro requires careful experimental design and appropriate controls. The following methodological approach is recommended:

• Substrate preparation: MS2 RNA is commonly used as a substrate due to its well-characterized structure. RNA substrate should be purified and quantified spectrophotometrically. Consider using 5′-end labeled RNA with fluorescent or radioactive tags for more sensitive detection of cleavage products .

• Reaction setup: A standard ribonuclease assay typically contains:

  • Purified recombinant VapC35 (2 μM final concentration)

  • MS2 RNA substrate (typically 0.5-1 μg)

  • Reaction buffer containing Mg²⁺ (usually 5-10 mM)

  • Optional additives to test specificity (e.g., other divalent cations)

• Control reactions:

  • Negative control: Reaction without VapC35

  • EDTA inhibition control: Reaction with VapC35 plus EDTA (12 mM) to chelate Mg²⁺

  • Specificity control: VapC35 E5A mutant protein to confirm the role of the catalytic residue

  • Antitoxin inhibition control: Reaction with VapC35 pre-incubated with purified VapB35 antitoxin

• Reaction conditions: Incubate reactions at 37°C for 30-60 minutes. Take time-course samples at various intervals (e.g., 0, 5, 15, 30, 60 min) to capture the kinetics of RNA degradation.

• Analysis of cleavage products: Analyze the reaction products using denaturing polyacrylamide gel electrophoresis followed by staining with ethidium bromide or visualization of radioactive/fluorescent labels. Intact RNA and cleaved fragments can be quantified using densitometry or phosphorimaging .

This methodology allows for quantitative assessment of VapC35's ribonuclease activity and can be adapted to investigate substrate specificity, catalytic efficiency, and inhibition by various compounds or antitoxins.

What are the optimal conditions for expressing and purifying recombinant VapC35?

Expressing and purifying recombinant VapC35 requires specific conditions to maximize yield while maintaining protein stability and activity. Based on established protocols, the following approaches are recommended:

• Expression system:

  • Host strain: E. coli BL21(DE3) or similar expression strains are preferred

  • Vector: pET-based vectors with N-terminal 6X-His tag for affinity purification

  • Co-expression: Consider co-expressing with VapB35 antitoxin initially to reduce toxicity, followed by separation during purification

• Induction conditions:

  • Culture density: Induce at OD₆₀₀ of 0.6-0.8

  • Inducer: IPTG at 0.5-1.0 mM concentration

  • Temperature: Lower post-induction temperature to 18-20°C to enhance solubility

  • Duration: Extended expression (16-18 hours) at lower temperature often yields better results

• Cell lysis and extraction:

  • Buffer composition: Tris-HCl (50 mM, pH 8.0), NaCl (300 mM), imidazole (10 mM), glycerol (10%), and protease inhibitors

  • Lysis method: Sonication or high-pressure homogenization

  • Clarification: Centrifugation at high speed (≥20,000 × g) to remove cell debris

• Purification strategy:

  • Primary purification: Ni-NTA affinity chromatography

  • Washing: Gradual increase in imidazole concentration (20-50 mM) to reduce non-specific binding

  • Elution: Higher imidazole concentration (250-300 mM)

  • Secondary purification: Size exclusion chromatography to remove aggregates and achieve high purity

  • Buffer optimization: Include 5-10 mM MgCl₂ in final buffer to stabilize the enzyme

• Quality control:

  • Purity assessment: SDS-PAGE and western blotting

  • Activity testing: MS2 RNA cleavage assay to confirm enzymatic function

  • Protein concentration: Bradford or BCA assay

  • Storage: Small aliquots at -80°C in buffer containing 10% glycerol to prevent freeze-thaw damage

These optimized conditions should yield purified VapC35 suitable for in vitro enzymatic assays, structural studies, and interaction analyses with antitoxins or potential inhibitors.

How can researchers study VapC35-mediated growth inhibition in mycobacteria?

Investigating VapC35-mediated growth inhibition in mycobacteria requires controlled expression systems and appropriate phenotypic assays. The following methodology has been successfully employed:

• Vector construction and transformation:

  • Cloning: Insert the vapC35 gene into an anhydrotetracycline (Atc) inducible episomal (pTetR) or integrative vector (pTetR-int)

  • Control constructs: Create inactive mutant (vapC35 E5A) and empty vector controls

  • Transformation: Electroporate constructs into M. smegmatis

  • Selection: Culture transformants on MB7H11 plates with appropriate antibiotics (hygromycin for pTetR or kanamycin for pTetR-int constructs)

• Induction and growth monitoring:

  • Initial culture: Grow cultures to early-log phase (OD₆₀₀ ~0.2-0.3)

  • Induction: Add anhydrotetracycline (50 ng/ml) to induce vapC35 expression

  • Growth measurement: Monitor OD₆₀₀ at regular intervals (e.g., 0, 3, 6, 9, 24 hours post-induction)

  • Viability assessment: Determine bacterial counts (CFU/ml) at key timepoints

  • Spotting assay: Perform serial dilution spotting on solid media to visualize growth inhibition

• Co-expression studies:

  • Vector construction: Clone antitoxin genes (vapB35 for cognate, vapB3 for non-cognate) into acetamide-inducible vectors (e.g., pLam12)

  • Dual induction: Add both Atc (for toxin) and acetamide (for antitoxin) to early-log phase cultures

  • Analysis: Compare growth patterns and viability with toxin-only expression

• Cellular morphology analysis:

  • Microscopy: Prepare samples for light or fluorescence microscopy at defined intervals

  • Cell size measurement: Measure cell length using calibrated microscopy software

  • Statistical analysis: Analyze at least 100 cells per condition for statistical significance

Results from such studies typically show data similar to the following table:

This comprehensive approach allows researchers to quantify growth inhibition, assess bacteriostatic effects, and observe morphological changes induced by VapC35 activity.

What techniques are used to investigate VapC35 interactions with cognate and non-cognate antitoxins?

Investigating interactions between VapC35 and various antitoxins requires a combination of in vivo and in vitro approaches. The following techniques are commonly employed:

• Co-expression and rescue assays:

  • Dual vector system: Express VapC35 from one inducible promoter and antitoxins from another

  • Growth rescue: Quantify the ability of different antitoxins to rescue VapC35-mediated growth inhibition

  • Titration experiments: Vary the expression levels of antitoxins to determine minimum ratios needed for neutralization

• Protein-protein interaction assays:

  • Pull-down assays: Use His-tagged VapC35 to pull down interacting antitoxins

  • Size-exclusion chromatography: Analyze complex formation by shifts in elution profiles

  • Isothermal titration calorimetry (ITC): Determine binding affinities and thermodynamic parameters

  • Surface plasmon resonance (SPR): Measure association and dissociation kinetics between VapC35 and antitoxins

• Structural characterization:

  • X-ray crystallography: Determine atomic structures of VapC35-antitoxin complexes

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Identify binding interfaces

  • Circular dichroism (CD): Assess changes in secondary structure upon complex formation

• Functional inhibition assays:

  • Ribonuclease activity: Test the ability of different antitoxins to inhibit VapC35 enzymatic activity

  • Dose-response relationships: Determine IC₅₀ values for different antitoxins

  • Competition assays: Assess whether non-cognate antitoxins can displace the cognate antitoxin

• Advanced screening methods:

  • High-throughput display methods: Identify residues involved in cognate and non-cognate interactions

  • Screening mutagenesis libraries: Map critical interaction residues

  • Flow cytometry and next-generation sequencing: Analyze large numbers of variants to identify binding determinants

These techniques collectively provide a comprehensive understanding of the specificity, affinity, and functional consequences of interactions between VapC35 and various antitoxins. The research indicates that VapC35 exhibits cross-interactions with non-cognate antitoxins like VapB3, highlighting the complex regulatory networks that exist between different TA systems in M. tuberculosis .

How should researchers account for Mg²⁺ dependency when studying VapC35 activity?

The Mg²⁺ dependency of VapC35 ribonuclease activity is a critical factor that must be carefully controlled in experimental designs. Here are key considerations and methodological approaches:

• Buffer composition optimization:

  • Include MgCl₂ at optimal concentrations (typically 5-10 mM) in reaction buffers

  • Avoid phosphate buffers which can precipitate magnesium ions

  • Consider potential interference from other buffer components (certain buffering agents can chelate divalent cations)

  • Maintain consistent pH (typically 7.5-8.0) as pH affects metal ion availability

• Experimental controls:

  • Negative control with EDTA (12 mM) to demonstrate Mg²⁺ dependency

  • Concentration dependency experiments with varying Mg²⁺ levels (1-20 mM)

  • Alternative divalent cation experiments (Mn²⁺, Ca²⁺, Zn²⁺) to test specificity

  • Include controls with catalytically inactive VapC35 E5A mutant under identical conditions

• Storage and handling considerations:

  • Include MgCl₂ in protein storage buffers to maintain stability

  • Avoid repeated freeze-thaw cycles which can affect metal binding

  • Use freshly prepared MgCl₂ solutions to ensure accurate concentrations

  • Consider pre-incubation of VapC35 with Mg²⁺ before adding substrate

• Analytical considerations:

  • Monitor free vs. bound Mg²⁺ concentrations when relevant

  • Account for Mg²⁺ concentration effects on substrate structure (particularly for structured RNAs)

  • Consider competitive binding when studying inhibitors (some may function by chelating Mg²⁺)

  • Use appropriate statistical methods to analyze dose-dependent effects

• In vivo implications:

  • Consider intracellular Mg²⁺ concentrations when interpreting in vivo data

  • Account for potential variations in metal ion availability under different stress conditions

  • Design experiments that examine VapC35 activity across physiologically relevant Mg²⁺ concentrations

Properly accounting for the Mg²⁺ dependency ensures reliable and reproducible results when studying VapC35 activity, particularly when comparing results across different experimental conditions or when screening for inhibitors.

What controls are essential in VapC35 overexpression studies?

Overexpression studies with VapC35 require careful control design to ensure valid interpretation of results. The following controls are essential:

• Vector controls:

  • Empty vector control: Cells transformed with the same expression vector lacking the vapC35 gene

  • This controls for effects of the induction system, antibiotic selection, and metabolic burden of plasmid maintenance

  • Data shows vector control M. smegmatis reaches OD₆₀₀ of 1.2 ± 0.1 at 24h compared to 0.6 ± 0.1 for vapC35 expression

• Catalytic mutant controls:

  • VapC35 E5A mutant: Expression of catalytically inactive VapC35 variant

  • Controls for non-specific effects of protein overexpression separating them from enzymatic activity

  • Research shows the E5A mutant abrogates growth inhibition (OD₆₀₀ of 1.1 ± 0.1 at 24h)

• Induction controls:

  • Uninduced cultures: Parallel cultures without inducer addition

  • Dose-response: Multiple inducer concentrations to establish relationship between expression level and phenotype

  • Time-course controls: Samples collected at multiple time-points (0, 9, 24h post-induction) to track progression of effects

• Neutralization controls:

  • Co-expression with cognate antitoxin: VapB35 co-expression reverses growth inhibition

  • Non-cognate antitoxin: VapB3 partially mitigates toxicity, establishing specificity of interaction

  • Antitoxin-only expression: Controls for potential effects of antitoxin alone

• Phenotypic validation controls:

  • Multiple phenotypic measurements: Track both optical density (OD₆₀₀) and viable counts (CFU/ml)

  • Morphological analysis: Microscopy to confirm cell elongation phenotype (average 6.2 ± 0.6 μm for VapC35 overexpression vs. 4.0 ± 0.5 μm for vector control)

  • Recovery assessment: Ability of cells to resume growth after inducer removal

These controls collectively establish causality between VapC35 activity and observed phenotypes, rule out non-specific effects, and provide quantitative benchmarks for comparing experimental conditions. Properly designed controls are particularly important when investigating potential cross-interactions with other TA systems or when evaluating the effects of mutations on VapC35 function .

What are the key considerations when studying VapBC35 in stress response models?

When investigating the role of VapBC35 in stress response, researchers must carefully design experiments that accurately model physiological conditions while allowing for precise measurement of system dynamics. Key considerations include:

• Stress condition relevance and standardization:

  • Select stress conditions relevant to M. tuberculosis pathogenesis (oxidative stress, nutrient limitation, hypoxia, acid stress)

  • Standardize stress application protocols (e.g., H₂O₂ concentration, exposure time)

  • Include graduated stress levels to detect threshold effects

  • Control for secondary effects of stress conditions on experimental systems

• Genetic manipulation approaches:

  • Generate clean deletion mutants (ΔvapBC35) using specialized techniques for mycobacteria

  • Create complemented strains to verify phenotype specificity

  • Consider conditional expression systems to control timing of VapBC35 expression

  • Account for potential polar effects on adjacent genes

• Comparative analysis with other TA systems:

  • Include mutants of other VapBC systems as comparators

  • Generate multiple TA system knockouts to address functional redundancy

  • Measure differential expression of other TA systems in ΔvapBC35 mutants

  • Consider potential cross-talk between systems under stress conditions

• Temporal considerations:

  • Monitor stress responses across multiple time points

  • Consider both immediate and adaptive responses

  • Account for potential differences between acute and chronic stress

  • Design recovery experiments to assess post-stress adaptation

• In vivo relevance:

  • Bridge in vitro findings with appropriate animal models

  • Consider host-relevant stressors in infection models

  • Compare in vitro stress responses with in vivo expression patterns

  • Research shows VapBC35 is needed for adaptation to oxidative stress but dispensable for guinea pig infection, suggesting context-dependent functions

• Molecular mechanism investigation:

  • Monitor VapB35:VapC35 ratio under stress conditions

  • Identify specific RNA targets affected during stress

  • Measure antitoxin stability under different stress conditions

  • Examine promoter activity and transcriptional regulation during stress response

These considerations help ensure that experiments accurately characterize the physiological role of VapBC35 in stress adaptation while accounting for the complex network of redundant and interacting TA systems in M. tuberculosis. The apparent disconnect between oxidative stress requirement and in vivo dispensability highlights the importance of comprehensive experimental design in this area .

How can researchers quantify VapC35-induced cell morphology changes?

Quantifying morphological changes induced by VapC35 expression requires systematic imaging and analysis approaches. The following methodology provides a robust framework:

• Microscopy acquisition protocols:

  • Sample preparation: Culture cells with and without VapC35 induction, collect at defined timepoints (e.g., 9h, 24h post-induction)

  • Fixation and staining: Use gentle fixation (2% paraformaldehyde) and appropriate stains (e.g., DAPI for nucleoids, membrane dyes)

  • Imaging parameters: Capture phase contrast and fluorescence images at consistent magnification (typically 100x oil immersion)

  • Field selection: Randomly select multiple fields (≥10) per sample to avoid bias

  • Technical replicates: Perform independent biological replicates (n≥3)

• Image analysis methodology:

  • Software selection: Use calibrated imaging software (e.g., ImageJ/FIJI with appropriate plugins)

  • Automated measurement: Develop macros for high-throughput analysis where possible

  • Manual verification: Verify subset of automated measurements manually

  • Measurement parameters: Cell length, width, aspect ratio, and area

  • Population analysis: Measure ≥100 cells per condition for statistical power

• Statistical analysis approaches:

  • Distribution analysis: Generate histograms of cell length distributions

  • Descriptive statistics: Calculate mean, median, standard deviation, and coefficient of variation

  • Statistical tests: Apply appropriate tests (t-test for two conditions, ANOVA for multiple conditions)

  • Correlation analysis: Correlate morphological changes with growth parameters

• Advanced analysis techniques:

  • Time-lapse imaging: Monitor morphological changes in real-time

  • Single-cell tracking: Follow individual cells through division cycles

  • Machine learning classification: Train algorithms to categorize morphological phenotypes

  • 3D imaging: Consider Z-stack acquisition for volume measurements

• Data reporting standards:

  • Include representative images with scale bars

  • Present quantitative data in tables (as shown below) and distribution plots

  • Report exact p-values and confidence intervals

  • Include effect size measurements (Cohen's d or similar)

Based on published data, VapC35 overexpression produces significant morphological changes:

This quantitative approach enables researchers to precisely characterize the cell elongation phenotype associated with VapC35 expression, which likely reflects interference with cell division processes.

What statistical approaches are recommended for analyzing VapC35 growth inhibition data?

• Growth curve analysis:

  • Area under the curve (AUC) calculation for comprehensive comparison

  • Growth rate determination during exponential phase

  • Lag phase duration measurement

  • Maximum OD (carrying capacity) comparison

  • Repeated measures ANOVA for time-course data

• Colony forming unit (CFU) analysis:

  • Log₁₀ transformation of CFU data for normality

  • Two-way ANOVA with time and strain as factors

  • Post-hoc tests with appropriate correction for multiple comparisons

  • Effect size calculation to quantify magnitude of growth inhibition

  • Power analysis to ensure adequate sample size

• Bacteriostatic vs. bactericidal determination:

  • Time-kill curves with extended timepoints

  • Minimum duration for 3-log reduction calculation

  • Recovery assays after toxin expression

  • Statistical comparison of killing kinetics

  • Survival ratio analysis at key timepoints

• Experimental design considerations:

  • Minimum of three biological replicates

  • Technical replicates within each biological replicate

  • Inclusion of appropriate positive and negative controls

  • Randomization and blinding where possible

  • Sample size determination based on preliminary data

• Advanced statistical approaches:

  • Mixed-effects models for complex experimental designs

  • Non-parametric alternatives when normality assumptions are violated

  • Bayesian analysis for integrating prior knowledge

  • Meta-analysis when combining data across experiments

Example statistical reporting from VapC35 studies:

These statistical approaches provide rigorous quantification of VapC35-mediated growth inhibition, allowing for precise comparisons between experimental conditions and mutant variants. Proper statistical analysis is particularly important when evaluating subtle phenotypes or when comparing the effects of different antitoxins on VapC35 activity .

How should researchers interpret apparent contradictions in VapC35 function across different studies?

When encountering seemingly contradictory findings about VapC35 function, researchers should systematically analyze potential sources of variation and apply a structured interpretive framework. Consider the following approaches:

• Experimental context differences:

  • Host organism variations: Results from E. coli vs. M. smegmatis vs. M. tuberculosis may differ due to physiological differences

  • Growth conditions: Media composition, oxygen availability, and growth phase can significantly impact TA system function

  • Expression systems: Different promoters, copy numbers, and induction methods alter toxin levels

  • Genetic background: Strain-specific differences in TA system networks may affect outcomes

• Methodological variations:

  • Construct design: Presence of tags, fusion proteins, or mutations may affect function

  • Assay sensitivity: Different methods have varying detection thresholds

  • Timeframes: Short-term vs. long-term experiments may reveal different aspects of function

  • Data analysis: Variations in normalization, statistical approaches, or threshold definitions

• Functional redundancy considerations:

  • Single vs. multiple TA system analysis: M. tuberculosis contains numerous TA systems with potential overlapping functions

  • Compensatory mechanisms: Deletion of one system may trigger upregulation of others

  • Stress-specific roles: Each TA system may have context-specific functions

  • The apparent contradiction between VapBC35's necessity for oxidative stress survival yet dispensability for in vivo growth illustrates this concept

• Physiological state dependencies:

  • Active growth vs. dormancy: TA systems may have different roles depending on bacterial metabolic state

  • Acute vs. chronic stress: Immediate and adaptive responses may involve different mechanisms

  • Host environment variations: Different animal models or cell types may exert different selective pressures

• Reconciliation approach:

  • Construct a hierarchical model incorporating context-dependent functions

  • Design experiments that bridge contradictory findings

  • Consider non-canonical functions beyond growth inhibition

  • Evaluate interactions with other cellular systems

A comprehensive example is the apparent contradiction between VapBC35's requirement for oxidative stress survival and its dispensability in guinea pig infection. This can be reconciled by considering:

• Redundancy among multiple TA systems during infection

• Potential differences in oxidative stress levels between in vitro models and in vivo conditions

• Temporal aspects of infection where different TA systems may be important at different stages

• Compensatory upregulation of other stress response systems in the ΔvapBC35 mutant during infection

This structured approach helps researchers develop more nuanced models of VapC35 function that accommodate seemingly contradictory findings within a coherent theoretical framework.

What are the current limitations in understanding VapC35 function in vivo?

Despite significant progress in characterizing VapC35, several limitations constrain our understanding of its function in vivo. These challenges include:

• Physiological target identification:

  • The natural RNA targets of VapC35 in M. tuberculosis remain largely unknown

  • Sequence specificity has been studied with artificial substrates (MS2 RNA) but not comprehensively with cellular RNAs

  • Global transcriptome effects of VapC35 activation have not been fully characterized

  • Connecting specific RNA targeting to physiological outcomes remains challenging

• Regulatory network complexity:

  • M. tuberculosis possesses numerous TA systems with potential functional overlap

  • The relative contribution of VapBC35 within this network is difficult to isolate

  • Cross-talk between different TA systems complicates interpretation of single-system studies

  • Environmental signals that specifically modulate VapBC35 activity in vivo remain poorly defined

• Methodological constraints:

  • Limited tools for monitoring toxin-antitoxin dynamics in real-time within live bacteria

  • Challenges in distinguishing direct from indirect effects of VapC35 activation

  • Difficulties in precisely controlling VapC35 activity levels in vivo

  • Technical challenges in studying slow-growing pathogenic mycobacteria

• In vivo context limitations:

  • Disconnect between in vitro phenotypes and in vivo requirements (e.g., essentiality for oxidative stress response but dispensability in guinea pig infection)

  • Limited understanding of VapC35 activity during different infection phases

  • Variation in results between different animal models

  • Challenges in extrapolating from model systems to human tuberculosis infection

• Structural and mechanistic gaps:

  • Limited structural information about VapC35-substrate interactions

  • Incomplete understanding of mechanisms governing antitoxin degradation in vivo

  • Minimal information about post-translational modifications affecting VapC35 activity

  • Limited insight into the spatial distribution and localization of VapC35 within cells

These limitations highlight the need for integrated approaches that combine biochemical, genetic, and in vivo studies to develop a more complete understanding of VapC35 function in the context of M. tuberculosis pathogenesis. Future studies should address these knowledge gaps to better define the physiological role of this important toxin-antitoxin system .

How might researchers address functional redundancy among toxin-antitoxin systems?

Addressing functional redundancy among toxin-antitoxin systems presents a significant challenge in understanding their individual and collective roles. Researchers can employ the following strategies:

• Systematic combinatorial deletion approaches:

  • Generate multiple TA system deletion mutants in defined combinations

  • Create comprehensive deletion libraries using CRISPR-Cas9 or recombineering technologies

  • Apply hierarchical deletion strategies targeting functionally related TA systems

  • Develop inducible deletion systems to circumvent potential lethality of multiple deletions

• Transcriptomic and proteomic profiling:

  • Perform RNA-seq of single TA system mutants to identify compensatory changes

  • Use ribosome profiling to detect translational effects across the TA network

  • Apply proteomics to measure changes in toxin and antitoxin levels following deletion of specific systems

  • Employ ChIP-seq to map regulatory network alterations in response to TA system perturbations

• Advanced phenotypic characterization:

  • Develop high-sensitivity assays to detect subtle phenotypic changes

  • Utilize competitive fitness assays to reveal small fitness differences

  • Apply single-cell analysis to identify population heterogeneity in TA system activation

  • Design multi-stress challenge experiments to reveal condition-specific redundancy patterns

• Network modeling approaches:

  • Construct mathematical models of TA system interactions and regulation

  • Develop predictive algorithms for TA system functional overlap

  • Apply machine learning to identify patterns in large-scale deletion studies

  • Create bioinformatic frameworks to predict functional relationships based on sequence and structural similarities

• Experimental evolution strategies:

  • Subject TA system mutants to long-term evolution under relevant stress conditions

  • Analyze compensatory mutations that arise in response to TA system deletions

  • Perform suppressor screens to identify genetic interactions

  • Use transposon mutagenesis to map genetic networks associated with TA systems

The current understanding that VapBC35 is necessary for oxidative stress survival but dispensable for guinea pig infection suggests functional redundancy among TA systems in vivo. Future studies should aim to construct multiple deletion mutants to understand the cumulative role of TA systems in M. tuberculosis pathogenesis, as suggested by researchers in the field . This approach would help define the hierarchy and interrelationships among the extensive network of TA systems in this important pathogen.

What are promising research directions for therapeutic targeting of VapC35?

Exploring VapC35 as a potential therapeutic target offers several promising research directions that could lead to novel anti-tuberculosis strategies. Key areas for investigation include:

• Structure-based inhibitor design:

  • Determine high-resolution crystal structures of VapC35 alone and in complex with RNA substrates

  • Identify druggable pockets within the catalytic domain

  • Apply computational screening to identify compounds that could inhibit ribonuclease activity

  • Design transition-state analogs that mimic Mg²⁺-RNA interactions at the active site

• Antitoxin mimetics development:

  • Characterize the structural interface between VapC35 and VapB35

  • Identify minimal peptide motifs that mediate toxin neutralization

  • Design peptidomimetics that can penetrate mycobacterial cells

  • Explore non-peptide small molecules that mimic the VapB35 binding interface

• TA system network modulation:

  • Target regulatory elements controlling VapBC35 expression

  • Develop compounds that disrupt VapBC35 binding to operator DNA

  • Design strategies to manipulate antitoxin stability

  • Explore synergistic targeting of multiple TA systems based on cross-interaction networks

• Conditional suicide systems:

  • Engineer modified VapC35 variants responsive to specific triggers

  • Develop inducible systems that activate VapC35 under defined conditions

  • Create delivery systems for VapC35 protein or expression constructs

  • Design bacterial sensitization strategies combining VapC35 activation with conventional antibiotics

• Host-pathogen interface targeting:

  • Investigate how host stress conditions modulate VapBC35 activity

  • Identify host factors that influence TA system dynamics

  • Develop host-directed therapies that indirectly activate toxins

  • Exploit oxidative stress dependency of VapBC35 for therapeutic strategies

• Combination approaches:

  • Explore synergies between VapC35 targeting and conventional antibiotics

  • Investigate potential for decreased resistance development with TA-targeted therapies

  • Design multi-target approaches addressing several TA systems simultaneously

  • Develop antibiotic potentiators based on TA system modulation

Future studies should include identifying the residues involved in cognate (VapB35:VapC35) and non-cognate (VapB3:VapC35) interactions, which could provide crucial information for designing specific inhibitors or modulators. High-throughput display methods, screening mutagenesis libraries, flow cytometry, and next-generation sequencing could facilitate this understanding, as suggested by recent literature .

The therapeutic potential of targeting VapC35 is particularly promising given its role in oxidative stress adaptation, which is a key aspect of M. tuberculosis pathogenesis and persistence during infection.