Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Gamma-glutamyl phosphate reductase (proA)

What is the enzymatic role of gamma-glutamyl phosphate reductase (ProA) in Leptospira interrogans?

ProA catalyzes the second step in proline biosynthesis, converting gamma-glutamyl phosphate to glutamate-5-semialdehyde (GSA), a critical precursor for proline production. Proline serves as an osmoregulator and stress protectant in Leptospira, enabling survival in diverse environments, including host tissues . Functional characterization in Ralstonia solanacearum (a model for bacterial proline metabolism) demonstrated that proA deletion mutants fail to grow in minimal media unless supplemented with proline, confirming its essentiality in biosynthesis . In Leptospira, ProA’s role extends beyond metabolism; it may indirectly influence virulence by modulating stress responses during host infection .

How is recombinant ProA expressed and purified for functional studies?

Recombinant ProA is typically expressed in Escherichia coli due to its well-characterized genetics and scalability. The proA gene is cloned into expression vectors (e.g., pUC13 or pET systems) under inducible promoters (e.g., T7 or lacZ). Post-induction, the protein is purified via affinity chromatography, often using tags like glutathione-S-transferase (GST) or polyhistidine . For example, MyBiosource’s recombinant ProA (MBS1067524) is expressed in E. coli with >90% purity, validated through SDS-PAGE and enzymatic assays . Critical quality control steps include:

• Activity assays: Measuring NADPH-dependent reduction of gamma-glutamyl phosphate .

• Complementation tests: Restoring proline auxotrophy in proA-deficient E. coli strains .

What experimental models are used to study ProA’s role in leptospiral pathogenesis?

In vitro models:

• Gene knockout/complementation: proA deletion in Leptospira or surrogate bacteria (e.g., E. coli) to assess metabolic and virulence defects .

• Host-cell interaction assays: Monitoring proline utilization during infection of mammalian cell lines (e.g., CHO cells) .

In vivo models:

• Hamster challenge: Immunization with recombinant ProA followed by lethal-dose challenge with L. interrogans to evaluate protective efficacy .

• Plant-pathogen systems: Studying homologous ProA function in R. solanacearum to infer leptospiral mechanisms .

How do structural variations in recombinant ProA affect enzymatic activity across Leptospira serovars?

ProA’s catalytic domain is highly conserved, but sequence divergence in non-critical regions may influence enzyme kinetics or stability. For example:

A 15 kb leuB-complementing fragment from L. interrogans serovar Icterohaemorrhagiae showed no cross-complementation with proA, highlighting functional specificity . Structural predictions using molecular docking suggest that adjuvant interactions (e.g., hGMCSF) may enhance ProA’s immunogenicity without altering its enzymatic core .

What methodological challenges arise when analyzing ProA’s role in host-pathogen interactions?

• Genetic manipulation: Leptospira’s slow growth and resistance to classical transformation methods complicate proA knockout studies .

• Redundancy: Proline uptake pathways may compensate for proA deletion, masking phenotypic effects .

• Cross-species extrapolation: Findings from R. solanacearum (e.g., T3SS regulation by ProA) require validation in Leptospira due to evolutionary divergence .

Solutions:

• Use conditional knockouts or CRISPR interference to bypass genetic limitations.

• Employ dual RNA-seq to track proline metabolism genes during infection .

How can conflicting data on ProA’s immunogenicity be resolved in vaccine studies?

Some studies report strong antibody responses to recombinant ProA , while others emphasize its limited protective efficacy compared to outer membrane proteins (e.g., LigA or LipL41) . Key methodological considerations:

To reconcile discrepancies:

• Standardize challenge models (e.g., uniform bacterial doses and serovars).

• Compare ProA’s immunogenicity head-to-head with other antigens (e.g., LipL41) using identical adjuvants .

What advanced techniques are used to characterize recombinant ProA’s interaction with host proteins?

• Surface plasmon resonance (SPR): Quantifies binding affinity between ProA and host proline transporters.

• Cryo-EM: Resolves ProA’s conformational changes during catalysis .

• Differential scanning calorimetry (DSC): Assesses thermal stability of recombinant ProA variants .

A recent docking study revealed that ProA binds hGMCSF with a binding energy of -7.2 kcal/mol, suggesting adjuvant synergy .

How does proline availability in the host environment influence ProA’s role in infection?

Leptospira may switch between endogenous proline synthesis (via ProA) and scavenging from host tissues. In proline-rich environments (e.g., renal tubules), ProA activity decreases, downregulating biosynthetic genes. Conversely, in nutrient-poor niches (e.g., blood), ProA becomes essential . This dual strategy complicates in vitro studies, as culture media proline levels must mimic host conditions to yield translational insights .

What bioinformatic tools are critical for optimizing recombinant ProA design?

• Phyre2: Predicts tertiary structure using homologous templates (e.g., E. coli ProA).

• Clustal Omega: Aligns proA sequences across serovars to identify conserved domains.

• AutoDock Vina: Screens for adjuvant-antigen interactions (e.g., hGMCSF-ProA docking) .

A multi-sequence alignment of 12 Leptospira serovars revealed 89% amino acid identity in ProA’s catalytic domain, supporting broad vaccine applicability .

What are the unresolved research questions regarding ProA’s multifunctional roles?

• Does ProA directly regulate virulence genes, or are its effects metabolism-mediated?

• Can ProA-based vaccines provide cross-serovar protection, given its conservation?

• How do post-translational modifications (e.g., phosphorylation) affect ProA activity in vivo?