Recombinant Listeria monocytogenes serovar 1/2a Thioredoxin reductase (trxB)

What is the role of TrxB in the thioredoxin system of Listeria monocytogenes?

TrxB (thioredoxin reductase) works in conjunction with TrxA (thioredoxin) to maintain a highly reducing environment in the bacterial cytosol. TrxB is annotated as a thioredoxin reductase that catalyzes the NADPH-dependent reduction of oxidized thioredoxin. This enzyme is crucial for the thioredoxin system to function properly, as it regenerates the reduced form of thioredoxin after it has participated in redox reactions. The system is particularly important in defending against oxidative stress and ensuring correct disulfide bonding for protein function in L. monocytogenes .

How is TrxB expression regulated in response to oxidative conditions?

Expression of the thioredoxin system components, including TrxB, is significantly induced in L. monocytogenes when exposed to thiol-specific oxidizing agents such as diamide. Experimental data shows that TrxB is slightly induced by paraquat and H₂O₂, but much more strongly induced by diamide, especially at 30 and 60 minutes post-exposure . The alternative sigma factor SigH has been identified as playing a critical role in regulating TrxB expression. EMSA assays demonstrate that purified recombinant SigH binds to the promoter region of trxB, suggesting direct transcriptional regulation .

What is the relationship between TrxA and TrxB in the redox homeostasis of L. monocytogenes?

TrxA and TrxB function as a coupled system in L. monocytogenes, with TrxB (thioredoxin reductase) serving to regenerate the reduced form of TrxA (thioredoxin). This relationship is critical because only the reduced form of TrxA can function effectively to:

• Maintain the reduced forms of key regulatory proteins like PrfA for activation

• Reduce intermolecular disulfide bonds in proteins like MogR to ensure correct dimerization

• Provide a highly reducing environment for proper protein folding and function

The experimental evidence shows that disruption of this system through deletion of TrxA leads to significant impairment in bacterial responses to thiol-specific oxidative stress, suggesting the interdependence of these components .

What expression systems are most effective for producing recombinant L. monocytogenes TrxB?

Based on research methodologies used for similar recombinant Listeria proteins, effective expression systems for L. monocytogenes TrxB include:

For functional studies, expressing TrxB under its native promoter in L. monocytogenes is crucial, as demonstrated in complementation experiments with TrxA, where expression under the native promoter fully restored function while constitutive overexpression showed partial effects .

How can researchers assess TrxB activity in experimental settings?

TrxB activity can be assessed through several complementary approaches:

What are effective approaches for generating and validating TrxB deletion mutants?

To generate and validate TrxB deletion mutants in L. monocytogenes serovar 1/2a, researchers should consider:

• Homologous recombination techniques, creating an in-frame deletion to avoid polar effects on downstream genes

• Confirmation of deletion through both PCR verification and RT-PCR to ensure absence of transcription

• Complementation tests using both native promoter expression (CΔtrxB_PtrxB) and constitutive promoter expression (CΔtrxB_Phelp)

• Phenotypic validation through oxidative stress challenges, particularly with thiol-specific oxidants like diamide

• Transcriptomic analysis to identify genes differentially expressed in the mutant versus wild-type strain

How does the thioredoxin system contribute to L. monocytogenes survival under oxidative stress?

The thioredoxin system plays a crucial role in L. monocytogenes resistance to oxidative stress, particularly thiol-specific oxidative stress. Experimental evidence shows:

• Deletion of TrxA significantly increases sensitivity to the thiol-specific oxidizing agent diamide, resulting in a longer lag phase compared to the wild-type strain

• Expression of both TrxA and TrxB is significantly induced by diamide exposure, particularly at 30 and 60 minutes post-exposure

• The thioredoxin system maintains the reducing environment necessary for proper protein function, including virulence factors and motility regulators

• Under oxidative stress conditions, SigH (an alternative sigma factor) is released from its anti-sigma factor (RshA) and activates transcription of the thioredoxin system components

These findings suggest that TrxB, as part of this system, is crucial for maintaining redox homeostasis under oxidative stress conditions.

What is the relationship between oxidative stress resistance and virulence in L. monocytogenes?

Research demonstrates a clear connection between oxidative stress resistance and virulence in L. monocytogenes:

• Stress-tolerant L. monocytogenes strains are generally more invasive in vitro and more virulent in vivo

• The thioredoxin system contributes to oxidative stress resistance and significantly impacts virulence gene expression

• Deletion of TrxA results in downregulation of multiple virulence factors, including plcA, mpl, hly, actA, and plcB

• The ability to maintain redox homeostasis is critical for the activation of PrfA, the master regulator of virulence genes

• TrxA deletion mutants show attenuated virulence in mouse infection models, suggesting thioredoxin system components are essential for full pathogenicity

This suggests that TrxB, as a key component of this system, likely contributes significantly to both stress resistance and virulence potential.

How can researchers differentiate L. monocytogenes serovar 1/2a strains at the molecular level?

L. monocytogenes serovar 1/2a strains can be differentiated using PCR combined with restriction enzyme analysis (PCR-REA). A specific approach involves:

• Amplification of a 2,916 bp segment containing:

  • The downstream end of the inlA gene (955 bp)

  • The intergenic space between inlA and inlB (85 bp)

  • 1,876 bp of the inlB gene

• Digestion of this PCR product with the restriction enzyme AluI

• Gel electrophoresis separation of the resulting fragments to identify distinct restriction profiles

This method has successfully divided 100 L. monocytogenes serovar 1/2a strains into two distinct groups with different restriction profiles, with 70 strains sharing one profile and 30 strains sharing another .

What are the challenges in characterizing genetic variability in the thioredoxin system genes?

Characterizing genetic variability in the thioredoxin system genes presents several challenges:

• The essential nature of these genes may constrain genetic variation

• The interconnected function of system components requires consideration of the entire system rather than individual genes

• Regulatory elements may show more variation than the coding sequences themselves

• Expression differences may be more significant than sequence differences

• Phenotypic expression of genetic variants may be context-dependent and influenced by environmental conditions

Research suggests that PCR-REA methods targeting the internalin locus can effectively differentiate serovar 1/2a strains , and similar approaches might be applicable to analyzing variation in the thioredoxin system genes.

How does TrxB contribute to the redox-dependent activation of virulence regulators?

TrxB likely contributes significantly to virulence regulator activation through its role in the thioredoxin system:

• Experimental evidence shows that TrxA exhibits strong binding to PrfA (the master virulence regulator) in its reduced form

• The oxidized form of TrxA shows almost no binding affinity to PrfA

• PrfA activation requires a reducing environment, with only reduced PrfA dimers being able to bind DNA and activate virulence gene transcription

• TrxB is necessary to regenerate reduced TrxA, maintaining the redox cycle that supports this activation

• Similar mechanisms may apply to other redox-sensitive transcriptional regulators

The model suggests that TrxB indirectly contributes to virulence by ensuring TrxA remains in its reduced form, enabling proper PrfA activation and subsequent virulence gene expression.

What approaches can be used to study the structural determinants of TrxB substrate specificity?

To investigate the structural determinants of TrxB substrate specificity, researchers should consider:

• X-ray crystallography or cryo-EM to determine the three-dimensional structure of TrxB

• Site-directed mutagenesis of predicted active site residues to identify those critical for substrate interaction

• Isothermal titration calorimetry (ITC) to measure binding affinity between TrxB and potential substrates (similar to methods used for TrxA-PrfA interaction studies)

• Molecular dynamics simulations to model substrate interactions and predict conformational changes

• Comparative studies between TrxB from different Listeria serovars to identify conserved and variable regions that might influence substrate recognition

Understanding these structural determinants could provide insights into how TrxB contributes to specific aspects of L. monocytogenes physiology and pathogenesis.

What should researchers consider when designing oxidative stress challenges for TrxB functional studies?

When designing oxidative stress challenges to study TrxB function, researchers should consider:

Research with TrxA has shown that diamide at 4 mM significantly induces expression of both TrxA and TrxB, while H₂O₂ and paraquat have less pronounced effects . Similar patterns would be expected when studying TrxB directly. Time-course experiments should include measurements at 30 and 60 minutes post-exposure, as these timepoints showed significant induction in previous studies.

How can researchers effectively study the interactions between TrxB and other components of the L. monocytogenes redox system?

To effectively study interactions between TrxB and other redox system components, researchers should employ:

• Co-immunoprecipitation studies to identify protein-protein interactions in vivo

• Isothermal titration calorimetry (ITC) to quantify binding affinities and thermodynamic parameters of interactions

• Bacterial two-hybrid or yeast two-hybrid screening to identify novel interaction partners

• Electrophoretic mobility shift assays (EMSA) to study DNA-protein interactions for regulatory elements

• Transcriptomic and proteomic analyses comparing wild-type, ΔtrxB mutant, and complemented strains to identify genes and proteins affected by TrxB

• Redox proteomics to identify specific proteins whose redox state is maintained by the TrxB/TrxA system

These approaches would provide a comprehensive understanding of how TrxB functions within the broader redox network of L. monocytogenes.