Recombinant Tropheryma whipplei Phenylalanine--tRNA ligase alpha subunit (pheS)

What is the genomic context of the pheS gene in T. whipplei?

The pheS gene in T. whipplei is part of its compact 927,303-bp circular genome . When studying this gene, researchers should note that T. whipplei exhibits several genomic peculiarities, including deficiencies in amino acid metabolism pathways that may affect protein synthesis machinery . To analyze the genomic context, researchers should:

• Perform comparative genomic analysis between T. whipplei strains (such as Twist and TW08/27) to identify conserved regions around pheS

• Examine potential operonic structures involving pheS

• Identify regulatory elements upstream of the gene

• Compare synteny with related actinobacterial species to understand evolutionary conservation

How does the amino acid sequence of T. whipplei pheS compare to other bacterial pheS proteins?

To perform comprehensive sequence comparisons:

• Extract the T. whipplei pheS sequence from genome databases

• Conduct multiple sequence alignments with homologs from related bacteria

• Generate phylogenetic trees to visualize evolutionary relationships

• Identify conserved domains and catalytic residues

T. whipplei's reduced genome suggests possible streamlining of protein functions. Alignment techniques similar to those shown in the GAPDH alignments between T. whipplei and Listeria monocytogenes (which showed significant homology) can be applied to pheS analysis . Conservation scoring from 0 (least conserved) to 10 (most conserved) can help identify functionally critical regions.

What are the predicted structural features of recombinant T. whipplei pheS?

To predict structural features:

• Use homology modeling based on crystal structures of pheS from other organisms

• Apply secondary structure prediction algorithms

• Identify binding sites for phenylalanine, ATP, and tRNA

• Predict interactions with the beta subunit (pheT)

Given T. whipplei's deficiencies in amino acid metabolism , the structural features of pheS may have evolved specific adaptations to function optimally in this metabolic context.

What expression systems are most suitable for producing recombinant T. whipplei pheS?

When selecting an expression system, researchers should consider:

• Codon optimization: Analyze the codon usage bias in T. whipplei (an actinobacterium with high G+C content) and optimize for the expression host

• Expression hosts: E. coli BL21(DE3) variants are commonly used, but Actinobacteria-specific hosts may provide better folding environments

• Fusion tags: His6-tag, GST, or MBP to facilitate purification and potentially improve solubility

• Induction conditions: Temperature, inducer concentration, and duration must be optimized to prevent inclusion body formation

T. whipplei's genome has several unique features including deficiencies in amino acid metabolism , which may affect recombinant protein production and necessitate supplementation in expression media.

What purification strategies are effective for isolating recombinant T. whipplei pheS?

A multi-step purification protocol typically includes:

• Affinity chromatography (Ni-NTA for His-tagged constructs)

• Ion exchange chromatography (based on the theoretical pI of pheS)

• Size exclusion chromatography for final polishing

• Buffer optimization to maintain enzyme activity

Researchers should test enzyme activity throughout purification to ensure functionality is maintained. Given T. whipplei's unique parasitic lifestyle that involves subverting host cellular processes , its proteins may have specific stability requirements.

How can researchers assess the enzymatic activity of recombinant T. whipplei pheS?

Activity assays for phenylalanine-tRNA ligase typically measure:

• ATP-PPi exchange assay: Measures the first step of the aminoacylation reaction

• tRNA charging assay: Measures complete aminoacylation using radiolabeled phenylalanine

• Kinetic parameters (Km, kcat) determination for substrates: phenylalanine, ATP, and tRNA

Control experiments should include comparisons with pheS from model organisms and testing substrate specificity with non-cognate amino acids and tRNAs.

How does T. whipplei pheS function in the context of the bacterium's reduced amino acid metabolism?

T. whipplei exhibits deficiencies in amino acid metabolism pathways , which raises important questions about how pheS functions in this organism:

• Determine if pheS has adapted to lower intracellular concentrations of phenylalanine

• Investigate whether pheS has altered substrate specificity compared to homologs from bacteria with complete amino acid biosynthesis pathways

• Examine potential interactions with host amino acid transport systems

• Analyze regulation of pheS expression in response to amino acid availability

Research approaches should include:

• Comparative biochemical analysis with pheS from other bacteria

• In vitro reconstitution of the aminoacylation reaction under varying substrate concentrations

• Expression studies in T. whipplei cultured under different nutrient conditions

What is the role of pheS in T. whipplei survival within macrophages?

T. whipplei survives in macrophages by creating a specialized niche through inhibition of phago-lysosome biogenesis . To investigate potential roles of pheS in intracellular survival:

• Use siRNA to knockdown pheS expression in laboratory-cultivated T. whipplei and assess impact on bacterial survival in macrophages

• Perform temporal proteomics to determine if pheS expression changes during different stages of intracellular infection

• Investigate whether inhibition of pheS affects the bacterium's ability to block Rab5-to-Rab7 switch in phagosome maturation

The research methodology should incorporate the macrophage infection models described for studying T. whipplei's effects on phagosome maturation .

How do genome rearrangements in T. whipplei affect pheS expression or function?

The T. whipplei genome undergoes rearrangements due to repeats in cell-surface protein genes . To investigate potential impacts on pheS:

• Map the location of pheS relative to known inversion hotspots

• Compare pheS expression levels between strains with different genomic arrangements

• Determine if inversions affect operonic structures containing pheS

• Assess if rearrangements alter regulatory elements controlling pheS expression

This research would require whole genome sequencing of multiple T. whipplei isolates, combined with transcriptomic analysis to correlate genomic structure with pheS expression.

Can T. whipplei pheS serve as a target for developing new antibiotics against Whipple's disease?

Given T. whipplei's predicted resistance to quinolones due to mutations in DNA gyrase , alternative antibiotics targeting different cellular processes are needed:

• Perform high-throughput screening for small molecule inhibitors specific to T. whipplei pheS

• Structure-based drug design focusing on unique features of T. whipplei pheS

• Assess selectivity of inhibitors against human PARS (phenylalanyl-tRNA synthetase)

• Evaluate ability of identified compounds to penetrate the unique bacterial compartment where T. whipplei resides in macrophages

Researchers should note that T. whipplei creates a "chimeric" phagosome expressing both Rab5 and Rab7 , which may affect drug delivery to the bacterium.

How can recombinant T. whipplei pheS be used in diagnostic assays for Whipple's disease?

Development of serological tests using recombinant pheS:

• Express and purify recombinant pheS with minimal contamination from host proteins

• Develop ELISA assays to detect anti-pheS antibodies in patient sera

• Assess sensitivity and specificity compared to current diagnostic methods

• Evaluate potential cross-reactivity with pheS from commensal bacteria

Validation studies should include:

• Testing against sera from confirmed Whipple's disease patients

• Comparison with negative controls and patients with other inflammatory conditions

• Determination of appropriate cut-off values for clinical use

What are the challenges in developing T. whipplei pheS-based research tools?

Several factors complicate the development of research tools:

• T. whipplei's slow growth and specialized culture requirements

• The chimeric phagosome environment may affect protein expression and folding in vivo

• Potential cytotoxicity of recombinant pheS in heterologous expression systems

• Limited natural variability data for designing specific primers or probes

To overcome these challenges, researchers should:

• Test multiple expression systems and conditions

• Develop cell-free protein synthesis approaches

• Use synthetic biology techniques to create more robust expression constructs

• Consider computational approaches to complement experimental limitations

How does the T. whipplei pheS interact with the host immune system during infection?

Research approaches should include:

• Assess whether pheS is exposed to the host immune system during infection

• Determine if pheS contains epitopes recognized by human immune cells

• Investigate if post-translational modifications of pheS affect immunogenicity

• Evaluate whether anti-pheS antibodies are protective in infection models

These studies could utilize the bone marrow-derived macrophage (BMDM) infection model described in the literature to study host-pathogen interactions.

What is the potential role of pheS in T. whipplei adaptation to different host environments?

Methodological approaches should include:

• Comparative genomics of pheS sequences from T. whipplei isolated from different anatomical sites

• Transcriptomic analysis of pheS expression under various growth conditions

• Proteomic studies to identify potential interaction partners of pheS in different host environments

• Functional assays to determine if pheS activity varies in response to environmental stimuli

Given that T. whipplei creates a specialized intracellular niche by blocking the Rab5-to-Rab7 switch , researchers should investigate whether pheS contributes to this adaptive survival mechanism.