Recombinant Salmonella heidelberg Queuine tRNA-ribosyltransferase (tgt)

What is the function of tRNA-ribosyltransferase (TGT) in Salmonella Heidelberg?

TGT catalyzes a unique base-for-base exchange reaction in which guanine at position 34 in the anticodon loop of tRNAs with G34U35N36 sequence is replaced by queuine. This modification is a hallmark of almost all eubacteria and eukarya, suggesting evolutionary conservation despite the fact that it has been shown to be non-essential in multiple organisms . In Salmonella, as in other bacteria, this modification potentially influences various aspects of cellular physiology including translation efficiency and fidelity, though the specific impacts in Salmonella Heidelberg require further investigation.

How does bacterial TGT differ structurally from eukaryotic TGT?

Bacterial TGT functions as a single protein subunit, while eukaryotic TGT requires two subunits - QTRT1 (catalytic subunit) and QTRT2 (accessory subunit). Research demonstrates that human TGT can be produced by co-expression of an N-terminal polyhistidine tagged QTRT1 and a C-terminal SUMO-StrepII tagged QTRT2 in BL21(DE3) tgt::Kmr cells . This structural difference has implications for enzyme function, substrate specificity, and potential targeting strategies for antimicrobial development.

Why is recombinant Salmonella Heidelberg TGT being investigated for vaccine development?

Salmonella is a leading bacterial cause of human foodborne illnesses worldwide, with poultry products being a major source of infection. Current live attenuated vaccines against Salmonella suffer from problems including persistence and shedding of the organism in and from vaccinated animals . Recombinant surface-exposed proteins of Salmonella, including potentially TGT, are being investigated as subunit vaccine candidates to overcome these limitations while providing protective immunity.

What are the established methods for assessing TGT enzymatic activity in vitro?

Several robustly validated methods exist for studying TGT activity:

tRNA-[14C] guanine displacement assay:

• Pre-label tRNA with [8-14C] guanine using E. coli TGT

• Incubate labeled tRNA with test enzyme and potential substrates

• Separate tRNA from free nucleobases using DEAE cellulose chromatography

• Measure displaced [14C] guanine by liquid scintillation counting

tRNA-[14C] guanine incorporation assay:
This complementary method assesses the reversibility of the modification by attempting to incorporate [14C] guanine into previously modified tRNA .

Both assays provide quantitative measures of TGT activity and can be adapted for studying Salmonella Heidelberg TGT specifically.

How can researchers produce recombinant Salmonella Heidelberg TGT proteins for immunological studies?

Based on published methodologies, researchers should consider:

• Gene cloning: Amplify the tgt gene from Salmonella Heidelberg genomic DNA and clone into an appropriate expression vector.

• Expression system selection: E. coli BL21(DE3) strains are commonly used, potentially with modifications to prevent interference from endogenous TGT (tgt::Kmr).

• Protein purification: Incorporate affinity tags (His-tag, SUMO-tag) for efficient purification.

• Quality control: Verify protein identity by mass spectrometry and purity by SDS-PAGE.

• Activity validation: Confirm enzymatic activity using established assays before immunological testing .

As demonstrated with other Salmonella Heidelberg proteins, surface-exposed proteins can be successfully produced as recombinant proteins and tested for immunogenicity in animal models .

What cellular incorporation assays can determine if TGT substrates are effectively modifying tRNAs in vivo?

Researchers have established cellular incorporation assays for tracking tRNA modification:

Cellular incorporation protocol:

• Culture cells in the presence of test compound (potential TGT substrate)

• Wash and add radiolabeled substrate (e.g., [3H] queuine)

• Harvest cells, precipitate macromolecules with TCA

• Collect precipitates by vacuum filtration

• Rinse with TCA and ethanol, then dry

• Measure radioactivity by scintillation counting

This approach has been used to demonstrate that both human MDA-MB-231 cells and immune cells can incorporate TGT substrates into RNA, and that initial incorporation of certain substrates (like NPPDAG) can block subsequent modification by queuine, confirming irreversibility .

What immune responses are elicited by recombinant Salmonella Heidelberg proteins when used as antigens?

Studies with recombinant Salmonella Heidelberg surface proteins have shown variable immunogenicity:

The immunoglobulin (Ig) G, IgM and IgA from most vaccinated chickens reacted strongly to the recombinant FliD and FlgK proteins, but not from unvaccinated chickens. These antibody studies suggest that recombinant FliD and FlgK have potential as targets for vaccine development .

Why might certain recombinant proteins fail to elicit strong immune responses?

The poor antigenicity of some proteins (like FimA and FimW) may be due to their "size, composition, and/or structural complexity" . Several factors can influence antigenicity:

• Protein folding and native conformation

• Presence of immunodominant epitopes

• Stability of the protein in vivo

• Processing efficiency by antigen-presenting cells

• Previous exposure and immune tolerance

For TGT specifically, its location (cytoplasmic vs. surface-exposed) and level of conservation with host TGT would influence its potential as a vaccine antigen.

How can researchers design a chimeric protein vaccine incorporating TGT epitopes?

Based on evidence from the study of recombinant Salmonella proteins, researchers might consider:

• Epitope mapping: Identify immunogenic regions of TGT through computational analysis and experimental validation.

• Carrier selection: Choose highly immunogenic proteins (like FliD or FlgK) as carriers for TGT epitopes.

• Linker design: Incorporate flexible linkers between protein domains to maintain proper folding.

• Expression optimization: Ensure the chimeric construct can be efficiently expressed in recombinant systems.

• Functional validation: Verify that chimeric proteins retain desired antigenic properties.

The search results specifically note that "Because of the importance of bacterial fimbriae in pathogenesis and for immunogenicity, a chimeric protein of the FimA and FimW proteins is needed" , suggesting a similar approach could be applied for TGT.

What is the mechanism of irreversibility in TGT-catalyzed tRNA modification?

Queuine modification of tRNA is an irreversible event that occurs through a unique base-for-base exchange reaction (guanine replacement by queuine) . This irreversibility has been demonstrated experimentally:

• When tRNA is modified with queuine, subsequent attempts to incorporate [14C] guanine fail.

• In cellular studies, pre-treatment with queuine or NPPDAG strongly suppresses subsequent modification by radiolabeled queuine.

The exact molecular mechanism underlying this irreversibility isn't fully elucidated in the provided literature, but likely involves:

• Highly favorable energetics for the queuine-incorporated state

• Structural changes in the tRNA after modification that prevent reverse reaction

• Possible additional enzyme interactions that stabilize the modified state

How does substrate specificity differ between bacterial TGT and eukaryotic TGT?

The literature indicates several important differences in substrate specificity:

• Nucleobase recognition: Both enzymes recognize 7-deazaguanine derivatives, but with potentially different affinities.

• tRNA specificity: Both target tRNAs with G34U35N36 sequence, but may have different preferences for specific tRNA species.

• Engineered substrates: Compounds like NPPDAG have been designed to be incorporated by eukaryotic TGT into tRNA in an irreversible manner .

These differences in specificity could be exploited for developing selective inhibitors or creating TGT-dependent therapeutic approaches that target either bacterial or eukaryotic systems.

What is the relationship between TGT activity and antimicrobial resistance in Salmonella Heidelberg?

While direct evidence linking TGT activity to antimicrobial resistance is limited, there are intriguing connections:

• Salmonella Heidelberg strains with different antimicrobial resistance (AMR) profiles show varying persistence in environmental conditions.

• The strain SH-AAFC harboring the antimicrobial resistance gene blaCMY-2 on an IncI1 plasmid survived longer in pine wood shavings than other strains .

• Persistent clones carried higher copy numbers of Col plasmids than their ancestors, suggesting plasmid dynamics play a role in survival .

The potential relationship between tRNA modification by TGT and plasmid-mediated antimicrobial resistance represents an interesting area for future investigation, particularly whether TGT activity influences expression of resistance genes or stress responses that contribute to persistence.

How might modified nucleobases incorporated by TGT be used therapeutically?

The literature describes an innovative therapeutic application using a modified nucleobase:

NPPDAG, a 7-deazaguanine derivative designed to be incorporated by TGT but not recognized by HPRT (hypoxanthine-guanine phosphoribosyltransferase), demonstrated remarkable therapeutic effects in an experimental autoimmune encephalomyelitis (EAE) model. Treatment with NPPDAG:

• "Dramatically arrested and reversed the disease course after only five daily doses (30 mg/Kg) to a state indistinguishable from non-diseased controls" .

• The effect was TGT-dependent, as NPPDAG had no effect on disease progression in TGT-deficient mice.

• The compound exhibited "a significant anti-proliferative effect on T cells that was dependent on the TGT enzyme" .

This suggests that TGT-mediated incorporation of engineered nucleobases could potentially be adapted for treating other conditions, possibly including Salmonella infections.

What strategies could target TGT for antimicrobial development against Salmonella Heidelberg?

Several approaches could be explored:

• Selective inhibitors: Develop compounds that selectively inhibit bacterial TGT while sparing the host enzyme.

• Substrate analogs: Design toxic substrate analogs that are incorporated by bacterial TGT into tRNA, disrupting translation.

• Structural targeting: Exploit structural differences between bacterial and eukaryotic TGT for selective binding.

• Combination approaches: Pair TGT inhibitors with conventional antibiotics to enhance efficacy or reduce resistance development.

• Vaccine development: Use recombinant TGT or TGT-derived peptides as antigens in subunit vaccines.

The unique mechanism and evolutionary conservation of TGT make it a promising target for novel antimicrobial strategies.

How can researchers investigate the contribution of TGT to Salmonella persistence in environmental conditions?

Building on methodologies from the literature, researchers should consider:

• Comparative persistence assays: Test wild-type and TGT-deficient Salmonella strains in relevant environments (e.g., pine wood shavings used as poultry bedding).

• Environmental parameter correlation: Measure factors like water activity, which has been shown to correlate with Salmonella survival .

• Genetic analysis: Use whole genome sequencing to track genetic changes during persistence, particularly focusing on TGT and related pathways.

• Plasmid dynamics: Investigate whether TGT activity influences plasmid maintenance or copy number, which appears important for persistence.

• Transcriptional profiling: Compare gene expression patterns between persistent and non-persistent strains to identify TGT-regulated survival mechanisms.

These approaches would help elucidate whether and how TGT contributes to the environmental persistence of Salmonella Heidelberg, which has important implications for controlling transmission in poultry production environments.