Recombinant Mycobacterium ulcerans Nucleoside diphosphate kinase (ndk)

Biochemical Properties of Mycobacterial NDK

NDK catalyzes the transfer of phosphate groups between nucleoside diphosphates and triphosphates, maintaining cellular nucleotide pools. Key features include:

• Enzymatic Activity: Exhibits autophosphorylation and phosphotransferase activity, with a conserved catalytic histidine residue (His117 in M. tuberculosis) .

• Multifunctionality: Beyond nucleotide metabolism, NDK interacts with host GTPases (e.g., Rab5, Rab7, Rac1) to disrupt phagosome maturation and NADPH oxidase (NOX2) assembly .

• Structural Conservation: Shares >98% sequence identity with M. tuberculosis NDK, suggesting similar virulence mechanisms .

Phagosome Maturation Arrest

• GTPase Inactivation: Recombinant M. tuberculosis NDK acts as a GTPase-activating protein (GAP) for Rab5 and Rab7, preventing phagosome-lysosome fusion. This results in pathogen survival within macrophages .

  • In vitro assays show NDK-coated beads reduce lysosomal fusion by 40–90% compared to controls .

  • Knockdown of NDK in M. bovis BCG increases lysosomal fusion by ~40% and reduces bacterial survival .

Suppression of Oxidative Burst

• Rac1 Inhibition: NDK binds and inactivates Rac1, a GTPase essential for NOX2 assembly, reducing reactive oxygen species (ROS) production .

  • Macrophages infected with NDK-deficient M. tuberculosis mutants produce 3–5× higher ROS levels .

  • NDK-dependent ROS suppression correlates with reduced apoptosis, enhancing bacterial persistence .

DNase-like Activity

• DNA Cleavage: M. tuberculosis NDK exhibits sequence-specific nuclease activity, targeting regions like the c-myc promoter .

  • Kinetic parameters: $k_{cat}/K_M = 9.65 \times 10^6 \, \text{M}^{-1}\text{s}^{-1}$ for single-strand DNA cleavage .

  • GDP competitively inhibits nuclease activity ($K_I = 1.9 \, \text{mM}$) .

Implications for M. ulcerans Pathogenesis

While direct studies on M. ulcerans NDK are sparse, genomic analyses indicate:

• Genetic Conservation: M. ulcerans shares 98–99% genome identity with M. tuberculosis, including NDK homologs .

• Drug Resistance: Efflux pumps and mutations in genes like gyrA (DNA gyrase) contribute to intrinsic antibiotic resistance in M. ulcerans . NDK may indirectly modulate resistance via nucleotide pool regulation.

Research Gaps and Future Directions