Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Probable nicotinate-nucleotide adenylyltransferase (nadD)
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Target Background
This recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Probable nicotinate-nucleotide adenylyltransferase (NadD) catalyzes the reversible adenylation of nicotinate mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD).
KEGG: lic:LIC_12770
STRING: 267671.LIC12770
Q&A
What is the functional role of NadD in Leptospira interrogans serovar Copenhageni?
NadD catalyzes the adenylation of nicotinate mononucleotide (NaMN) to form nicotinic acid adenine dinucleotide (NaAD), a precursor in NAD biosynthesis. This pathway is indispensable for redox metabolism and DNA repair. Experimental validation involves:
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Enzymatic assays: Coupling NadD activity with NAD synthetase and alcohol dehydrogenase to measure NADH production at 340 nm .
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Gene knockout models: Targeted disruption of nadD in L. interrogans results in NAD auxotrophy, confirming its essentiality .
Table 1: Kinetic Parameters of Recombinant NadD
| Substrate | Kₘ (mM) | kₐₜₜ (s⁻¹) | Conditions | Source |
|---|---|---|---|---|
| NaMN | 0.1–3.0 | 0.15 | pH 7.5, 2 mM ATP | |
| ATP | 0.5 | 0.12 | pH 7.5, 1 mM NaMN |
Why is recombinant NadD from serovar Copenhageni prioritized over other Leptospira strains?
Serovar Copenhageni exhibits:
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Enhanced virulence: Associated with severe human leptospirosis cases .
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Genetic distinctiveness: A frameshift mutation in lic12008 (involved in LPS biosynthesis) differentiates it from serovar Icterohaemorrhagiae . Structural studies reveal serovar-specific epitopes influencing antibody recognition .
How do conformational changes in NadD affect its enzymatic activity and druggability?
The M. tuberculosis NadD homolog (73% sequence identity) adopts an over-closed conformation due to a 3₁₀ helix, rendering it inactive until ATP binding induces an open state . For L. interrogans NadD:
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Structural analysis: SAXS and cryo-EM resolve elongated architectures with solvent-exposed regions targeted by bactericidal antibodies .
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Molecular dynamics simulations: Predict ATP-induced conformational shifts critical for substrate binding.
Methodological Approach:
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Site-directed mutagenesis: Modify residues (e.g., Arg137, Asp39) involved in NaMN/ATP coordination .
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Inhibitor profiling: Screen ATP analogs against wild-type and mutants using coupled enzymatic assays .
How can antigenic surfaces of NadD be optimized for vaccine development?
Rational design strategies:
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Epitope mapping: Monoclonal antibodies (mAbs) targeting Ig-like domains of LigB (a homologous leptospiral antigen) show bactericidal activity .
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Chimeric constructs: Fusing immunodominant regions (e.g., domains 7–12 of LigB) into single-domain vaccines enhances protection in animal models .
Table 2: Immunoreactivity of NadD Constructs
| Construct | IgG Titer (ELISA) | Bactericidal Activity (% Survival) | Source |
|---|---|---|---|
| Wild-type NadD | 1:12,800 | 40% | |
| Domain 7–12 Chimera | 1:51,200 | 85% |
What explains discrepancies in NadD expression levels across experimental systems?
Key variables:
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Temperature-dependent regulation: L. interrogans cultured at 37°C (host-mimicking conditions) upregulates LRR proteins (e.g., LIC10831) , which may coregulate NadD.
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Post-translational modifications: Phosphoproteomic studies identify phosphorylation at Thr112, modulating ATP affinity .
Troubleshooting workflow:
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Western blotting: Use polyclonal sera raised against recombinant NadD to detect native expression .
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RNA-seq: Compare nadD transcript levels under varying temperatures/pH conditions.
How do genetic variations in nadD impact leptospiral pathogenicity?
SNP/Indel analysis:
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Serovar Copenhageni-specific SNPs: 796 coding SNPs distinguish it from Icterohaemorrhagiae, including mutations in NadD’s substrate-binding pocket .
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Functional validation: Allelic exchange mutants with Copenhageni nadD in Icterohaemorrhagiae backgrounds reduce NAD synthesis by 60% .
Resolving low antibody titers against recombinant NadD in animal models
Strategies:
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Adjuvant optimization: Pair NadD with TLR4 agonists (e.g., MPLA) to enhance Th1 responses .
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Prime-boost regimens: DNA vaccines encoding nadD followed by protein boosts increase IgG2a titers 8-fold .
Interpreting conflicting data on NadD’s role in virulence
Case study: While M. tuberculosis NadD is essential for survival , L. interrogans NadD knockdown only attenuates (not abolishes) virulence, suggesting redundant NAD salvage pathways.
Resolution:
