Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Phosphoribosylglycinamide formyltransferase 2 (purT)

What is the function of purT in Leptospira interrogans?

Phosphoribosylglycinamide formyltransferase 2 (purT) in L. interrogans functions in purine biosynthesis by catalyzing the formylation of glycinamide ribonucleotide (GAR). This catalytic mechanism specifically requires Mg(2+)ATP and formate as substrates. The enzyme belongs to the ATP-grasp superfamily of enzymes, characterized by three structural motifs referred to as the A-, B-, and C-domains . Unlike alternative purine biosynthetic pathways that utilize carbon from N10-formyltetrahydrofolate, the purT-catalyzed reaction directly incorporates formate, representing a critical pathway for nucleotide synthesis in the pathogen.

How does purT expression change under different environmental conditions?

L. interrogans undergoes complex changes in protein expression profiles in response to environmental shifts that mimic in vivo conditions. When exposed to iron-limited conditions with fetal bovine serum (−Fe/FBS media), several metabolic shifts occur in the bacterium. Proteins involved in protein synthesis are typically downregulated, reflecting a general trend toward reduced expression of proteins involved in energy production, metabolism, and regulation . While specific data on purT expression changes aren't directly presented in the search results, the global proteome analyses identified 563 proteins with altered expression of 65 proteins under in vivo-like conditions . Understanding these expression patterns is crucial for characterizing the metabolic adaptations of L. interrogans during infection.

How does purT from L. interrogans differ structurally from other bacterial formyltransferases?

The purT enzyme from L. interrogans belongs to the ATP-grasp superfamily, which shares common structural features across various organisms. One distinctive characteristic of purT transformylase is the conformation of the "T-loop," delineated by amino acid positions equivalent to Lys-155 to Gln-165 (based on E. coli studies). This T-loop is highly sensitive to the chemical identity of the nucleotide situated in the binding pocket . This sensitivity contrasts with "P-loop"-containing enzymes, where the conformation of the binding motif remains virtually unchanged regardless of nucleotide presence. In the ATP-grasp enzymes, adenosine nucleotide ligands are invariably wedged between the B- and C-domains, with the B-domains exhibiting significant movement upon nucleotide binding in some family members, such as biotin carboxylase and carbamoyl phosphate synthetase .

What are the optimal experimental conditions for expressing recombinant purT from L. interrogans?

Expressing recombinant purT from L. interrogans requires careful consideration of several experimental parameters. Based on proteome analysis methodologies, successful expression systems typically include:

• Expression Vector Selection: Vectors containing strong promoters compatible with the host expression system (commonly E. coli for initial characterization)

• Culture Conditions: Optimization of temperature (typically 25-30°C), induction timing, and media composition

• Protein Extraction Protocol: Use of pooled cultures to control for biological variation—studies have used 25-75 independent cultures pooled together to minimize variation

• Purification Strategy: Implementation of affinity tags (His-tag is common) followed by size exclusion chromatography

For verification of successful expression, a combination of SDS-PAGE, Western blotting, and activity assays should be employed. The experimental design should include appropriate controls for technical variation (replicate samples) and for experimental variation between independent experiments, as demonstrated in the iTRAQ experimental designs used for L. interrogans proteome studies .

How can purT activity be accurately measured in experimental settings?

Measuring purT activity requires assays that can detect the formation of formylglycinamide ribonucleotide (FGAR) from glycinamide ribonucleotide (GAR) in the presence of ATP, Mg2+, and formate. Several methodological approaches include:

• Spectrophotometric Assays: Monitoring ATP hydrolysis through coupled enzyme systems

• Radiometric Assays: Using 14C-labeled formate to track incorporation into FGAR

• HPLC-Based Approaches: Separation and quantification of reaction products

• Mass Spectrometry: For precise identification and quantification of reaction intermediates

When designing these assays, researchers should consider the following parameters:

• Optimal pH (typically 7.5-8.0)

• Mg2+ concentration (usually 5-10 mM)

• ATP concentration (1-5 mM)

• Temperature (typically 30-37°C for L. interrogans enzymes)

• Buffer composition (phosphate or Tris-based buffers)

The assay should include appropriate controls for spontaneous hydrolysis of ATP and background rates in the absence of one or more substrates.

What structural changes occur in purT upon substrate binding and how do they affect catalytic efficiency?

The structural dynamics of purT upon substrate binding reveal important insights into its catalytic mechanism. X-ray diffraction analyses have demonstrated that the conformation of the T-loop (Lys-155 to Gln-165) is highly sensitive to the chemical identity of the bound nucleotide . This conformational flexibility appears to be crucial for proper catalytic function.

When comparing purT bound to different adenosine nucleotides or analogs (Mg(2+)ATP, Mg(2+)-5'-adenylylimidodiphosphate, Mg(2+)-beta,gamma-methyleneadenosine 5'-triphosphate, Mg(2+)ATPgammaS, or Mg(2+)ADP), distinct conformational states of the T-loop can be observed . These structural changes likely facilitate:

• Proper orientation of the GAR substrate

• Activation of formate for nucleophilic attack

• Stabilization of the transition state

• Product release

The catalytic efficiency (kcat/Km) is directly influenced by these conformational changes, with substrate binding likely following an induced-fit mechanism rather than a lock-and-key model.

What are the most effective protocols for purifying recombinant purT while maintaining its activity?

The purification of recombinant purT from L. interrogans while preserving enzymatic activity requires careful consideration of buffer conditions, temperature, and purification steps. An effective protocol includes:

Extraction and Initial Purification:

• Cell lysis in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol, and protease inhibitors

• Clarification by centrifugation (20,000 × g, 30 min, 4°C)

• Affinity chromatography using Ni-NTA resin for His-tagged proteins

• Washing with increasing imidazole concentrations (20-40 mM)

• Elution with 250 mM imidazole

Secondary Purification:

• Size exclusion chromatography using a Superdex 200 column

• Buffer exchange to 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT

• Concentration using centrifugal filters (10 kDa cutoff)

Throughout the purification process, it is critical to maintain the protein at 4°C and include stabilizing agents such as glycerol (10%) in storage buffers. Activity assays should be performed at each purification step to track enzyme activity retention. When designing such protocols, researchers should control for technical variation through replicate samples, as demonstrated in proteome studies of L. interrogans where replicate samples (e.g., 2 × A1 and 2 × B1) were used to control for technical variation within experiments .

How can I design experiments to investigate purT expression during L. interrogans infection?

Investigating purT expression during L. interrogans infection requires specialized experimental approaches that can detect protein expression in host tissues. Based on published methodologies for L. interrogans virulence factors, the following experimental design is recommended:

In vitro expression under in vivo-like conditions:

• Culture L. interrogans under conditions mimicking the host environment, such as iron limitation combined with serum factors (−Fe/FBS media)

• Compare protein expression profiles between standard and in vivo-like conditions using quantitative proteomics approaches such as iTRAQ

• Include appropriate controls for biological variation (pooled cultures) and technical variation (replicate samples)

Detection of purT expression during infection:

• Generate specific antibodies against recombinant purT

• Collect serum samples from patients with leptospirosis or experimentally infected animals

• Perform immunoblot analyses to detect antibody reactivity against purT, indicating in vivo expression

• Use immunofluorescence microscopy on fixed tissue sections from infected animals to visualize purT expression in tissues

This experimental approach has been successfully applied to verify the expression of other L. interrogans proteins during infection, including putative catalase (LIC12032), ErpY-like lipoprotein (LIC11966), and putative coagulase (LIC13166) . The presence of antibodies against these proteins in serum samples from natural leptospirosis infections provides evidence of protein expression during infection.

What experimental approaches can determine if purT is essential for L. interrogans survival in different environments?

Determining whether purT is essential for L. interrogans survival across different environments requires multiple experimental approaches:

Gene Knockout/Knockdown Studies:

• Generate a purT knockout strain using homologous recombination or CRISPR-Cas9 techniques

• If lethal, create conditional knockdown systems using inducible promoters

• Compare growth rates of wild-type and mutant strains under various conditions

Environmental Survival Assays:

• Establish microcosm experiments simulating natural environments (soil, water, sewage)

• Inoculate with wild-type and purT-deficient strains at concentrations mimicking natural excretion (approximately 10^6 cells/ml or g)

• Monitor survival over time using multiple detection methods:

  • Culture-based recovery on selective media

  • Quantitative PCR (qPCR)

  • PMA-qPCR to distinguish viable cells

Table 1: Comparative survival of wild-type and purT-deficient L. interrogans in environmental microcosms

Based on previous environmental studies, L. interrogans cannot multiply in soil, water, or sewage but can survive for extended periods (days to weeks) . The hypothesis that L. interrogans serves as a temporary carrier rather than an environmental reservoir should be considered when designing experiments to test purT essentiality.

How should discrepancies between proteomics and transcriptomics data for purT expression be reconciled?

Discrepancies between proteomics and transcriptomics data for purT expression in L. interrogans are common and reflect the complex relationship between mRNA abundance and protein levels. To reconcile these differences:

• Consider post-transcriptional regulation: Analyze the 5' and 3' untranslated regions of purT mRNA for regulatory elements that may affect translation efficiency

• Examine protein turnover rates: Assess protein stability using pulse-chase experiments with labeled amino acids

• Investigate translational efficiency: Perform ribosome profiling to determine if purT mRNA is efficiently translated

• Account for technical limitations: Different sensitivities and dynamic ranges of proteomics and transcriptomics platforms can contribute to apparent discrepancies

When analyzing proteomics data, it's essential to control for biological variation (using pooled cultures) and technical variation (using replicate samples) as demonstrated in iTRAQ experiments with L. interrogans . Comparison of experimental replicates (e.g., A1 to A2 and B1 to B2) allows evaluation of variation introduced during protein extraction steps .

For robust data interpretation, integrate multiple layers of evidence and consider the environmental conditions being tested, as L. interrogans exhibits complex changes in protein expression in response to environmental shifts .

What statistical approaches are most appropriate for analyzing purT activity across different experimental conditions?

Analyzing purT activity across different experimental conditions requires robust statistical approaches that account for various sources of variation. The following statistical methods are recommended:

• Normality testing: Shapiro-Wilk or Kolmogorov-Smirnov tests to determine if data follow normal distribution

• Parametric tests (if normally distributed):

  • Student's t-test for comparing two conditions

  • ANOVA followed by post-hoc tests (Tukey's HSD or Bonferroni) for multiple comparisons

  • Repeated measures ANOVA for time-course experiments

• Non-parametric alternatives (if not normally distributed):

  • Mann-Whitney U test for two conditions

  • Kruskal-Wallis test for multiple conditions

  • Friedman test for repeated measures

Important statistical considerations:

• Use appropriate sample sizes (minimum n=3 for each condition, preferably n≥5)

• Control for multiple comparisons to prevent Type I errors

• Report effect sizes alongside p-values

• Analyze biological and technical replicates separately to assess different sources of variation

When designing experiments to examine purT activity under different conditions, researchers should follow established protocols that control for variation, such as those used in iTRAQ proteomics studies of L. interrogans where technical variation was controlled through replicate samples and experimental variation was controlled through independent experiments .

How can structural data of purT be integrated with functional analyses to guide drug development?

Integrating structural data of purT with functional analyses provides a powerful approach for structure-based drug design targeting L. interrogans. The following methodological framework enables this integration:

• Structural characterization:

  • X-ray crystallography of purT in multiple states (apo, substrate-bound, product-bound)

  • Analysis of the T-loop conformation (residues equivalent to Lys-155 to Gln-165) which is highly sensitive to nucleotide binding

  • Identification of catalytic residues and substrate binding pockets

• Functional mapping:

  • Site-directed mutagenesis of key residues

  • Kinetic analyses of mutants to correlate structure with function

  • Thermal shift assays to assess protein stability upon ligand binding

• Computational approaches:

  • Molecular dynamics simulations to examine conformational changes

  • Virtual screening of compound libraries against identified binding pockets

  • Docking studies to predict binding modes and affinities

• Experimental validation:

  • Enzyme inhibition assays with predicted compounds

  • Crystallography of enzyme-inhibitor complexes

  • Cellular assays to determine compound efficacy against L. interrogans

The unique sensitivity of the T-loop to nucleotide identity in purT transformylase provides a potential target for selective inhibition. By exploiting structural differences between bacterial and host enzymes, researchers can design compounds that specifically target the bacterial purT without affecting host purine metabolism.

How can recombinant purT be used to develop serological tests for leptospirosis?

Recombinant purT from L. interrogans has potential as an antigen for developing sensitive and specific serological tests for leptospirosis diagnosis. The methodological approach includes:

• Antigen preparation:

  • Express and purify recombinant purT with high purity (>95%)

  • Verify proper folding through circular dichroism and activity assays

  • Determine optimal coating concentration through titration experiments

• Assay development:

  • ELISA format: Optimize antigen coating, blocking, sample dilution, and detection parameters

  • Lateral flow format: Similar to the approach used for leucine-rich repeat proteins

  • Line blot assays: For multiplexed detection alongside other leptospiral antigens

• Validation strategy:

  • Test with sera from confirmed leptospirosis cases (culture-positive or PCR-positive)

  • Include appropriate controls (healthy individuals, patients with other febrile illnesses)

  • Calculate sensitivity, specificity, positive and negative predictive values

• Performance assessment:

  • Compare with existing tests (MAT, LipL32 ELISA)

  • Assess cross-reactivity with antibodies against other pathogens

  • Determine limit of detection and reproducibility

When developing such tests, it's important to consider the timing of antibody responses during infection. For example, studies with leucine-rich repeat proteins have shown good potential for detecting anti-leptospiral IgG in infected dogs, with sensitivity of 70.00%, specificity of 82.09%, and accuracy of 78.80% when compared with LipL32 ELISA .

What are the methodological considerations for using purT as a target for antimicrobial development?

Using purT as a target for antimicrobial development against L. interrogans requires several methodological considerations:

• Target validation:

  • Confirm essentiality of purT for bacterial survival through genetic approaches

  • Verify lack of functional redundancy with other formyltransferases

  • Demonstrate expression during infection using proteomics or immunological methods

• Assay development:

  • Establish high-throughput screening assays based on purT enzymatic activity

  • Develop counter-screens to eliminate compounds that affect host enzymes

  • Create cell-based assays to verify compound penetration and efficacy

• Structure-based design:

  • Exploit the unique T-loop conformation that is highly sensitive to nucleotide binding

  • Focus on differences between the ATP-binding pocket of purT and host enzymes

  • Design compounds that specifically interact with the bacterial enzyme

• Compound optimization:

  • Improve potency, selectivity, and physicochemical properties

  • Assess pharmacokinetics and toxicity profiles

  • Test efficacy in animal models of leptospirosis

• Resistance potential:

  • Evaluate frequency of resistance development

  • Characterize resistance mechanisms through whole-genome sequencing

  • Consider combination approaches to minimize resistance

The structural uniqueness of purT compared to "P-loop"-containing enzymes provides an opportunity for selective targeting . While P-loop enzymes show minimal conformational changes upon nucleotide binding, the T-loop in purT undergoes significant conformational changes, offering potential binding sites for selective inhibitors.

What are the most promising future research directions for L. interrogans purT studies?

Future research on L. interrogans purT should focus on several high-priority areas that build upon current knowledge and address significant knowledge gaps:

The advancement in global proteome analysis techniques for L. interrogans provides powerful tools to study purT expression under various conditions. The established protocols for studying L. interrogans survival in environmental microcosms offer methodologies to investigate purT's role in environmental persistence. Finally, the structural insights into the T-loop sensitivity to nucleotide binding provide a foundation for structure-based drug design targeting this enzyme.