Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Quinolinate synthase A (nadA)

What is Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni?

Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni is a pathogenic spirochete bacterium that serves as one of the most common causative agents of leptospirosis, a zoonotic disease affecting humans and animals worldwide. L. interrogans belongs to spirochetes, considered evolutionarily primitive prokaryotes characterized by their thin, coiled morphology with hook-like structures . This particular serogroup and serovar combination represents a specific antigenic variant within the L. interrogans species with significant clinical importance.

The identification and characterization of this specific serovar has been greatly enhanced by advanced molecular techniques. Researchers have successfully developed methods involving hybridization capture followed by Illumina sequencing, which achieved a remarkable 72 to 13,000-fold increase in pathogen reads compared to standard sequencing approaches . This significant technological advancement allows for direct genetic characterization of strains from clinical samples without requiring bacterial culture, which is particularly valuable since Leptospira is notoriously fastidious and challenging to isolate in laboratory settings .

In epidemiological studies, serovar copenhageni within the Icterohaemorrhagiae serogroup has been identified as one of the predominant strains circulating in multiple geographic regions, including documented cases throughout mainland France . The genomic analysis of clinical isolates has revealed distinctive genetic signatures that allow for precise identification of this serovar, contributing to our understanding of leptospirosis epidemiology and pathogen transmission dynamics.

What is Quinolinate synthase A (nadA) and what is its role in bacterial metabolism?

Quinolinate synthase A (nadA) is an iron-sulfur enzyme that plays a critical role in the biosynthetic pathway of nicotinamide adenine dinucleotide (NAD), a fundamental cofactor involved in numerous essential redox biological reactions across all living organisms . In prokaryotes like Leptospira, nadA functions in concert with L-aspartate oxidase (nadB) to synthesize quinolinic acid, which serves as the essential precursor for NAD biosynthesis .

The enzyme's structure and function are defined by its iron-sulfur cluster, which is absolutely required for catalytic activity. When isolated under anaerobic conditions, nadA contains approximately 3-3.5 iron and 3-3.5 sulfide atoms per polypeptide chain . Mössbauer spectroscopic analysis has revealed that the majority of this iron exists in the form of a (4Fe-4S)²⁺ cluster, which constitutes the reactive center of the enzyme . This cluster is highly sensitive to oxygen exposure, which leads to its degradation and subsequent inactivation of the enzyme, necessitating specialized handling under anaerobic conditions for experimental work .

The metabolic significance of nadA stems from the fundamental difference in NAD biosynthetic pathways between prokaryotes and eukaryotes. While eukaryotes produce quinolinic acid through the degradation of tryptophan, prokaryotes utilize the nadA/nadB pathway starting from L-aspartate and dihydroxyacetone phosphate . This distinct metabolic divergence makes nadA an attractive potential target for antimicrobial development, as inhibiting this enzyme would disrupt essential NAD biosynthesis in bacterial pathogens without interfering with the host's biosynthetic pathway .

Why is recombinant protein technology used to study Leptospira interrogans nadA?

Recombinant protein technology represents an indispensable approach for studying Leptospira interrogans nadA due to several significant advantages it offers researchers. The primary challenge with native protein isolation from Leptospira is the bacterium's fastidious nature and slow growth, making it exceptionally difficult to culture in sufficient quantities for protein purification . Recombinant expression systems, particularly in Escherichia coli, circumvent this limitation by enabling production of substantial amounts of target protein for comprehensive structural, functional, and immunological investigations.

The methodological advantages of recombinant expression for nadA study are multifaceted. This approach allows researchers to introduce specific modifications such as affinity tags for streamlined purification, site-directed mutations for structure-function relationship analysis, and isotope labeling for advanced structural studies using NMR spectroscopy or X-ray crystallography. Similar strategies have been successfully employed for other Leptospira proteins, including immunoglobulin-like proteins LigA and LigB, where recombinant fragments with glutathione-S-transferase (GST) fusion tags facilitated both purification and functional characterization .

For nadA specifically, recombinant expression must address the challenge of maintaining the oxygen-sensitive iron-sulfur cluster that is essential for enzymatic activity . This requires specialized anaerobic expression systems or post-purification reconstitution protocols to ensure the integrity of the cluster. The methodological approach demonstrated for E. coli nadA, involving anaerobic purification to maintain the iron-sulfur cluster, provides a valuable template for similar work with the Leptospira enzyme .

Additionally, recombinant nadA can serve as a platform for vaccine development or diagnostic tool creation, following the successful precedent set by recombinant LigA fragments as vaccine candidates against Leptospira infection . The production of purified, well-characterized recombinant nadA enables immunization studies to evaluate potential protective effects and the development of specific antibodies for diagnostic applications.

What experimental approaches can identify structural characteristics of nadA's iron-sulfur cluster in Leptospira?

The experimental characterization of the iron-sulfur cluster in Leptospira nadA requires a multifaceted approach employing specialized techniques designed to preserve and analyze this oxygen-sensitive structural feature. While the search results don't provide Leptospira-specific structural information, the methodological framework established for E. coli nadA offers valuable guidance .

Spectroscopic characterization represents the cornerstone of iron-sulfur cluster analysis. UV-visible spectroscopy can identify characteristic absorption features indicative of iron-sulfur clusters, while electron paramagnetic resonance (EPR) spectroscopy provides insights into the electronic structure and oxidation states of the iron centers. Most definitive is Mössbauer spectroscopy using ⁵⁷Fe-labeled protein, which can precisely determine the oxidation state and coordination environment of iron atoms within the cluster. In E. coli nadA, this technique confirmed the presence of a (4Fe-4S)²⁺ cluster as the predominant iron species .

Functional validation of the cluster's importance can be achieved through site-directed mutagenesis of predicted iron-coordinating residues, followed by activity assays to correlate structural integrity with enzymatic function. The development of a specific enzymatic assay measuring quinolinate formation, as established for E. coli nadA , would be critical for this functional correlation.

How does nadA expression vary between pathogenic and attenuated Leptospira strains?

The variation in nadA expression between pathogenic and attenuated Leptospira strains provides critical insights into the potential role of this enzyme in virulence. While the search results don't directly address nadA-specific expression differences, comparative genomic and proteomic analyses between virulent and attenuated strains offer valuable contextual information .

The comprehensive study comparing the virulence-attenuated L. interrogans serovar Lai strain IPAV with its pathogenic ancestor strain 56601 revealed that genetic variations between these strains affected 101 genes, with functional impacts on processes including signal transduction, stress response, transmembrane transport, and notably, nitrogen metabolism . Given nadA's role in nitrogen-containing compound biosynthesis (NAD), it potentially falls within this category of differentially regulated genes.

Methodologically, the identification of expression differences would involve several complementary approaches. Quantitative RT-PCR would provide a targeted assessment of nadA transcript levels across different strains under standardized growth conditions. More comprehensive transcriptomic analysis through RNA-Seq could place nadA expression changes within the broader context of global gene expression differences between pathogenic and attenuated strains.

Functional analysis comparing nadA enzymatic activity between pathogenic and attenuated strains would provide further insights. Additionally, examination of the nadA genomic sequence across strains might reveal mutations in regulatory regions or coding sequences that could affect expression levels or protein functionality, potentially contributing to the attenuated phenotype observed in laboratory-passaged strains.

What methods are most effective for expression and purification of recombinant nadA from Leptospira interrogans?

The expression and purification of recombinant nadA from Leptospira interrogans demands specialized methodologies that address the unique challenges posed by its oxygen-sensitive iron-sulfur cluster. Based on approaches used for analogous proteins, particularly E. coli nadA, an optimized protocol would incorporate several key elements and considerations .

For expression system design, E. coli BL21(DE3) strains or derivatives engineered for iron-sulfur cluster assembly offer the most promising foundation. The expression vector should incorporate a promoter system that permits tight regulation, allowing for controlled induction under optimized anaerobic conditions. Critical consideration must be given to the selection of fusion partners; while GST has been successfully used for other Leptospira proteins like LigA , smaller tags such as His6 or Strep-tag II may be preferable for nadA to minimize interference with iron-sulfur cluster formation.

Expression conditions require precise optimization with particular attention to oxygen limitation. A recommended approach involves growing cultures aerobically to mid-log phase, followed by transition to anaerobic conditions prior to induction. Media supplementation with iron sources (ferrous ammonium sulfate) and sulfur-containing amino acids (cysteine) supports iron-sulfur cluster assembly. Induction at reduced temperatures (16-18°C) for extended periods (16-24 hours) promotes proper folding and cluster incorporation while minimizing formation of inclusion bodies.

The purification strategy must maintain strictly anaerobic conditions throughout all steps. This necessitates conducting procedures in an anaerobic chamber or using thoroughly degassed buffers containing reducing agents such as dithiothreitol (1-5 mM) or β-mercaptoethanol. Affinity chromatography using immobilized metal affinity chromatography (IMAC) for His-tagged constructs provides an efficient initial purification step, followed by size-exclusion chromatography to achieve homogeneous protein preparations.

What techniques can determine the role of nadA in Leptospira interrogans virulence?

Elucidating the role of nadA in Leptospira interrogans virulence requires a multidisciplinary experimental approach integrating advanced molecular genetics, infection models, and systems biology. Building on methodologies used for characterizing other Leptospira virulence factors, a comprehensive investigative framework can be established.

Genetic manipulation strategies represent the foundation of this investigation. CRISPR-Cas9 gene editing or traditional homologous recombination can be employed to generate precise nadA knockout mutants or strains with specific mutations in the iron-sulfur cluster binding residues. These genetic constructs must be complemented with wild-type nadA to confirm that observed phenotypes are specifically attributable to nadA disruption rather than polar effects or secondary mutations. Additionally, conditional expression systems utilizing inducible promoters allow temporal control of nadA expression, enabling the study of its role during different infection stages.

Animal infection models provide the critical context for virulence assessment. The Golden Syrian hamster model has demonstrated particular utility for Leptospira virulence studies, as evidenced by successful vaccination experiments with recombinant Lig proteins . Challenge experiments comparing wild-type and nadA-modified strains would assess virulence parameters including survival rates, bacterial organ burden, and histopathological changes in target tissues such as kidneys, liver, and lungs. Complementary cell culture infection assays using relevant host cell types can elucidate specific host-pathogen interactions at the cellular level.

Comparative genomic and phenotypic analysis between naturally occurring strains offers another valuable approach. The methodology employed to compare the virulent L. interrogans serovar Lai strain 56601 with its attenuated derivative IPAV provides a valuable template . Analysis of nadA sequence, expression levels, and enzymatic activity across strains with different virulence profiles could reveal correlations between nadA characteristics and pathogenic potential. This approach can be extended to clinical isolates from varying disease severities, potentially linking nadA variants to patient outcomes.

Systems biology approaches provide a holistic perspective on nadA's role in virulence. Transcriptomic analysis comparing wild-type and nadA-deficient strains reveals downstream effects on global gene expression. Proteomic analysis using quantitative LC-MS/MS methods, as employed for comparing strains IPAV and 56601 , can identify changes in virulence factor expression and metabolic pathway components. Metabolomic profiling focusing on NAD levels and related metabolites provides functional validation of nadA's impact on bacterial metabolism during infection.

How can structural analysis of nadA inform potential antimicrobial development against Leptospira?

Structural analysis of nadA from Leptospira interrogans offers a powerful foundation for rational antimicrobial development, particularly given that nadA participates in a prokaryote-specific pathway for NAD biosynthesis that is absent in humans . This metabolic divergence creates an opportunity for selective targeting that could minimize host toxicity while effectively inhibiting bacterial growth and virulence.

Active site characterization through integrated structural and functional analyses provides essential insights for inhibitor design. Site-directed mutagenesis of putative catalytic residues, followed by enzymatic activity measurements, can identify critical amino acids involved in substrate binding and catalysis. Isothermal titration calorimetry measurements with substrates, products, and preliminary inhibitor candidates would provide thermodynamic parameters of binding interactions. Hydrogen-deuterium exchange mass spectrometry can further map protein dynamics upon ligand binding, identifying regions that undergo conformational changes during catalysis.

Comparative structural analysis significantly enhances the specificity of drug design efforts. Structural alignment of Leptospira nadA with homologous enzymes from other pathogens would identify conserved features for broad-spectrum targeting, while Leptospira-specific structural elements could be exploited for selective inhibition. Importantly, comparison with human enzymes involved in NAD biosynthesis would ensure that designed inhibitors do not cross-react with host proteins, minimizing potential side effects.

The iron-sulfur cluster presents both a challenge and an opportunity for inhibitor design. While its oxygen sensitivity complicates structural studies, the cluster's essential role in catalysis makes it an attractive target. Computational modeling using quantum mechanical calculations can simulate interactions between potential inhibitors and the unique electronic environment of the iron-sulfur cluster. Inhibitors specifically designed to disrupt cluster assembly or stability would represent a novel class of antimicrobials with a distinct mechanism of action.

Structure-guided inhibitor development follows an iterative optimization process. Initial virtual screening of compound libraries against the nadA structure identifies preliminary hits with predicted binding affinity. These candidates undergo biochemical validation through enzyme inhibition assays, followed by co-crystallization studies to confirm binding modes. Medicinal chemistry optimization guided by structure-activity relationships progressively enhances potency, selectivity, and pharmacokinetic properties, ultimately generating lead compounds for further preclinical development.

What bioinformatic approaches can analyze nadA conservation across Leptospira serovars?

Comprehensive bioinformatic analysis of nadA conservation across Leptospira serovars requires a multifaceted methodological approach that leverages genomic data, evolutionary analysis, and structural prediction. Building upon approaches used for Leptospira genome analysis in previous studies, a systematic framework can reveal patterns of conservation and variation with implications for both basic biology and applied research .

Multiple sequence alignment using progressive algorithms like MUSCLE or MAFFT reveals the pattern of conservation across the nadA coding sequence. Quantitative analysis through percent identity matrices and sliding window conservation plots identifies regions of high conservation that may be essential for function versus variable regions that might contribute to strain-specific properties. Particular attention should focus on residues predicted to coordinate the iron-sulfur cluster, as these are likely under strong selective pressure for conservation.

Phylogenetic analysis provides evolutionary context for nadA variation. Construction of maximum likelihood phylogenetic trees using models optimized for bacterial genes allows visualization of evolutionary relationships among nadA variants. Comparison of the nadA-based phylogeny with whole-genome phylogenies can reveal instances of horizontal gene transfer or recombination that might have contributed to nadA diversity. Molecular clock analysis can estimate when key divergence events occurred in the evolutionary history of this gene.

Structural context significantly enhances interpretation of sequence conservation. Homology modeling of nadA from different serovars, based on experimentally determined structures of related proteins, allows mapping of sequence conservation onto three-dimensional models. This approach identifies spatially clustered conserved residues that may form functional sites or structural cores. Particular emphasis should be placed on characterizing the iron-sulfur cluster binding environment, as this represents the catalytic heart of the enzyme.

Comparative genomic context analysis extends beyond the nadA coding sequence to examine its genomic neighborhood. Analysis of upstream regulatory regions may reveal conserved transcription factor binding sites indicative of common regulatory mechanisms or serovar-specific regulatory elements suggesting differential expression. Investigation of operonic structure and associated genes can identify functional relationships that provide context for nadA's role in Leptospira physiology and pathogenesis.

How can proteomics approaches elucidate nadA interaction networks in Leptospira?

Proteomics approaches offer powerful tools to elucidate the nadA interaction network in Leptospira interrogans, providing insights into its functional context within bacterial metabolism and potential role in virulence. Building on the comparative proteomic analysis approaches described for Leptospira strains , a comprehensive methodology can be established to map protein-protein interactions centered on nadA.

Affinity purification coupled with mass spectrometry (AP-MS) represents the cornerstone approach for interaction network mapping. This requires genetic modification of Leptospira to express nadA with an affinity tag (His, FLAG, or biotin acceptor peptide) that enables selective purification of nadA along with its interacting partners. To capture both stable and transient interactions, a combination of native purification and chemical crosslinking methods should be employed. Quantitative comparison of proteins co-purifying with nadA under different conditions (aerobic vs. anaerobic growth, virulent vs. attenuated strains) would reveal context-dependent interactions potentially relevant to pathogenesis.

The experimental design must incorporate rigorous controls to distinguish genuine interactions from background contamination. These include parallel purifications from strains expressing the tag alone or tagged unrelated proteins, alongside statistical filtering methods to identify high-confidence interactors. Critically, all procedures must be conducted under anaerobic conditions to maintain the integrity of the nadA iron-sulfur cluster, which is essential for its native conformation and potentially for specific protein-protein interactions .

Complementary to traditional AP-MS, proximity-based labeling techniques provide a powerful approach for mapping spatial relationships within the bacterial cell. Fusion of nadA with enzymes like BioID or APEX2, which catalyze biotinylation of proteins within a defined radius, allows identification of proximal proteins that may not form stable complexes detectable by AP-MS. This approach is particularly valuable for mapping the broader cellular neighborhood of nadA beyond direct binding partners.

Global proteome analysis comparing wild-type and nadA-deficient Leptospira provides functional context for the interaction network. Quantitative proteomics using stable isotope labeling or label-free approaches can identify proteins whose abundance changes when nadA is absent, indicating functional relationships even without direct physical interaction. Particular attention should focus on NAD-dependent enzymes that might be affected by alterations in NAD biosynthesis, as well as virulence factors whose expression might be linked to metabolic status.

The integration of proteomic data with other omics datasets creates a comprehensive systems biology view of nadA's role. Network analysis algorithms can identify functional modules within the interaction network and predict additional components based on known association patterns. Comparison with interaction networks from other bacterial species can distinguish conserved interactions involved in core metabolism from Leptospira-specific interactions potentially linked to this pathogen's unique biology or virulence mechanisms.