Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Phosphoribosylamine--glycine ligase (purD)

What is Leptospira interrogans and how is it classified?

Leptospira interrogans is a pathogenic spirochete bacterium responsible for leptospirosis, a globally significant zoonotic infection. The bacterium belongs to the genus Leptospira, which includes saprophytic, intermediate, and pathogenic species as determined by both serological and DNA-based classification systems . L. interrogans serogroup Icterohaemorrhagiae includes virulent serovars such as Lai and copenhageni that cause severe manifestations of leptospirosis including hemorrhage, jaundice, and in severe cases, massive pulmonary hemorrhages .

The genomic structure of L. interrogans comprises a 4.33-megabase large chromosome and a 359-kilobase small chromosome, with approximately 4,768 predicted genes . This genomic architecture differs significantly from other pathogenic spirochetes like Treponema pallidum and Borrelia burgdorferi, despite sharing certain morphological features.

What is the function of Phosphoribosylamine--glycine ligase (purD) in Leptospira metabolism?

Phosphoribosylamine--glycine ligase (purD) is a critical enzyme in purine biosynthesis pathways, catalyzing the ATP-dependent formation of N1-(5-phospho-D-ribosyl)glycinamide from 5-phospho-D-ribosylamine and glycine. In Leptospira interrogans, this enzyme forms part of the complete metabolic systems for nucleotide biosynthesis, which distinguishes it from obligate parasitic spirochetes that rely on host resources .

Unlike restricted-host pathogens such as T. pallidum and B. burgdorferi, L. interrogans maintains the genetic capacity for de novo purine synthesis because it must survive in diverse environmental conditions during its zoonotic lifecycle. This metabolic flexibility reflects its status as a facultatively parasitic zoonotic bacterium that encounters extremely diverse environmental situations .

How does the purD gene contribute to Leptospira pathogenesis?

While no direct evidence links purD specifically to virulence in the provided research, its role in purine biosynthesis is likely crucial for pathogen survival and replication during infection. The ability to synthesize purines de novo would provide a metabolic advantage during infection when the pathogen must compete with host cells for limited resources.

The genomic study of L. interrogans revealed numerous genes potentially related to adhesion, invasion, and the hematological changes characteristic of leptospirosis . Although purD was not specifically identified among these virulence factors, metabolic capabilities often indirectly support pathogenic processes by enabling bacterial persistence in host tissues.

What expression systems are most effective for recombinant Leptospira protein production?

Based on successful recombinant protein expression studies with Leptospira antigens, the pRSET plasmid system (Invitrogen) has proven effective for expressing leptospiral proteins as His₆ fusion proteins in E. coli . This system allows for affinity purification using metal chelation chromatography.

The methodology involves:

• PCR amplification of the target gene from Leptospira genomic DNA

• Ligation into the pRSET expression vector

• Transformation into an appropriate E. coli strain

• Induction of protein expression

• Purification by affinity chromatography

For purD specifically, this approach would need to be optimized for protein solubility and activity, as recombinant expression of enzymes sometimes requires modifications to maintain functionality.

How can recombinant Leptospira proteins be validated for structural and functional integrity?

Validation typically employs a multi-faceted approach:

• Immunological confirmation: Testing reactivity with sera from leptospirosis patients or animals, as demonstrated with other Leptospira recombinant proteins like LipL32 .

• Enzymatic activity assays: For purD, this would involve measuring the formation of N1-(5-phospho-D-ribosyl)glycinamide using chromatographic or spectrophotometric methods.

• Structural assessment: Methods include circular dichroism spectroscopy to analyze secondary structure content and thermal stability measurements to assess proper folding.

• Mass spectrometry: To confirm the exact molecular weight and potential post-translational modifications.

As shown with other Leptospira recombinant proteins, initial validation can include immunoblot analysis with pooled sera from leptospirosis cases to verify antigenicity .

What are the optimal conditions for purification of recombinant Leptospira proteins?

Based on successful approaches with other Leptospira recombinant proteins, the following purification strategy would be recommended for recombinant purD:

• Expression optimization: Determine optimal induction conditions (temperature, inducer concentration, duration) to maximize soluble protein yield.

• Cell lysis: Gentle lysis methods to preserve protein structure, typically using bacterial protein extraction reagents or sonication in appropriate buffer systems.

• Affinity chromatography: Metal affinity purification using Ni-NTA columns for His-tagged proteins, with optimization of imidazole concentrations in wash and elution buffers.

• Secondary purification: Size exclusion chromatography or ion exchange chromatography to achieve higher purity if needed.

• Quality control: SDS-PAGE analysis to confirm purity and immunoblotting to verify identity.

Previous studies with leptospiral recombinant proteins have successfully employed these methods to obtain purified proteins suitable for immunological and structural studies .

How can the structure-function relationship of purD be investigated in Leptospira?

Investigating structure-function relationships requires a comprehensive approach:

• Comparative sequence analysis: Aligning purD sequences across Leptospira species and other bacterial genera to identify conserved catalytic residues and species-specific variations.

• Domain mapping: Generating truncated variants to determine essential functional domains.

• Site-directed mutagenesis: Systematically altering predicted catalytic residues to assess their contribution to enzymatic activity.

• X-ray crystallography or cryo-EM: These methods can determine the three-dimensional structure at high resolution, although they may require extensive optimization.

• Small-angle X-ray scattering (SAXS): This approach has been successfully employed with other Leptospira proteins to determine extended low-resolution structures .

The SAXS methodology, as demonstrated with LigB protein, can be particularly useful for multi-domain proteins to understand domain organization and flexibility .

What computational approaches aid in predicting purD structure and function?

Several computational methods can provide valuable insights:

• Homology modeling: Using known structures of purD from other organisms as templates to predict the Leptospira purD structure.

• Molecular dynamics simulations: To understand protein flexibility, substrate binding, and conformational changes during catalysis.

• Protein-ligand docking: To predict interactions with substrates (phosphoribosylamine, glycine, ATP) and potential inhibitors.

• Sequence-based predictions: Tools that identify conserved motifs, secondary structure elements, and disordered regions.

These computational predictions can guide experimental design, particularly for mutagenesis studies targeting residues predicted to be important for substrate binding or catalysis.

How is purD expression regulated during different phases of Leptospira infection?

While specific data on purD regulation is not provided in the search results, approaches to study its expression would include:

• Quantitative RT-PCR: To measure purD transcript levels under different growth conditions and during infection.

• RNA-Seq analysis: To position purD within global transcriptional networks and identify co-regulated genes.

• Reporter gene fusions: Constructing fusions of the purD promoter with reporter genes to monitor expression in real-time.

• In vivo expression technology (IVET): To identify if purD is differentially expressed during infection compared to laboratory culture.

Research on other Leptospira genes has shown that expression can be significantly altered during infection. For example, LipL36 expression is downregulated during infection, resulting in minimal reactivity with patient sera .

What environmental factors influence purD expression in Leptospira?

Based on the general understanding of bacterial purine biosynthesis regulation and Leptospira biology, several factors likely influence purD expression:

• Purine availability: Expression is typically repressed when exogenous purines are abundant.

• Temperature shifts: Changes from environmental to host temperature (37°C) likely affect expression.

• pH variations: Different host environments have varying pH levels that could influence gene expression.

• Osmolarity changes: Transition between freshwater and host tissues involves osmolarity shifts that affect gene expression.

• Nutrient limitation: Starvation conditions often upregulate biosynthetic pathways.

Experimental approaches to test these factors would include culturing Leptospira under controlled conditions varying one parameter at a time, followed by quantitative analysis of purD expression using qRT-PCR or reporter systems.

How can recombinant purD be evaluated as a potential vaccine candidate?

Evaluation of recombinant purD as a vaccine candidate would follow this methodological framework:

• Immunogenicity assessment: Determine if purD elicits antibody responses in animal models (hamsters are commonly used for Leptospira studies ).

• Antibody functionality testing: Evaluate if anti-purD antibodies have bactericidal activity through complement-mediated killing assays, as demonstrated with anti-LigB antibodies .

• Protection studies: Challenge vaccinated animals with virulent Leptospira to assess survival rates and pathogen burden in tissues.

• Combination approaches: Test purD in combination with other protective antigens, as single-domain chimeric constructs have shown enhanced protection compared to individual domains .

• Adjuvant optimization: Test various adjuvant formulations, such as Alhydrogel which has been used successfully in Leptospira vaccine studies .

The table below illustrates a typical experimental design for vaccine evaluation based on methodology used for other Leptospira antigens:

What methods are most effective for analyzing immune responses to Leptospira antigens?

Based on successful approaches with other Leptospira antigens, the following methods are recommended:

• ELISA: For quantifying antibody titers in serum samples from vaccinated animals or infected patients. ELISAs using recombinant Leptospira antigens have demonstrated high specificity (90-97%) when tested against sera from healthy individuals and patients with other diseases .

• Flow cytometry: To detect antibody binding to the surface of intact Leptospira cells, as demonstrated with anti-LigB monoclonal antibodies .

• Bactericidal assays: To assess functional activity of antibodies, including:

  • Dark-field microscopy counting of motile bacteria after antibody and complement exposure

  • Luciferase-based high-throughput assays to measure bacterial viability

• Immunoblotting: To analyze antibody specificity and cross-reactivity with other Leptospira proteins.

• Cytokine profiling: To characterize cell-mediated immune responses following vaccination.

How can CRISPR-Cas9 technology be applied to study purD function in Leptospira?

While the search results don't specifically mention CRISPR-Cas9 applications in Leptospira, this technology could be adapted to study purD function through:

• Gene knockout: Creating purD-deficient strains to assess growth defects and virulence attenuation.

• Gene editing: Introducing specific mutations to study structure-function relationships.

• CRISPRi: Using catalytically inactive Cas9 to temporarily repress purD expression without permanent genetic changes.

• CRISPR screening: Systematic targeting of genes in purine biosynthesis pathways to identify synthetic lethal interactions with purD.

The implementation would require optimization of Cas9 delivery and expression in Leptospira, selection of appropriate guide RNAs targeting purD, and development of efficient transformation protocols.

How can systems biology approaches integrate purD function into global metabolic networks?

Systems biology approaches would include:

• Metabolic network reconstruction: Positioning purD within the complete purine biosynthesis pathway and broader metabolic network of Leptospira.

• Flux balance analysis: Predicting how alterations in purD activity would affect metabolic flux through connected pathways.

• Multi-omics integration: Combining transcriptomic, proteomic, and metabolomic data to understand how purD regulation fits within global cellular responses.

• Interactome analysis: Identifying protein-protein interactions involving purD to uncover unexpected functional connections.

L. interrogans possesses complete metabolic systems for amino acid and nucleotide biosynthesis, making it more metabolically versatile than obligate parasites like T. pallidum and B. burgdorferi . This metabolic capacity likely reflects its adaptation to diverse environmental conditions during its lifecycle.