Functional analysis of nucleoside diphosphate kinase from Mycobacterium avium subspecies paratuberculosis
Mycobacterium avium subspecies paratuberculosis (Map), the causative agent of Johne’s disease, is an intracellular pathogen capable of surviving and replicating within macrophages for extended periods with minimal activation of the innate immune response. Mycobacterial secreted protein, nucleoside diphosphate kinase (Ndk), altered signalling pathways for macrophage activation, subverting host innate immune responses, disrupting maturation of membrane-bound phagosomes, and enabling the intracellular survival of mycobacteria within macrophages. Rab small GTPase proteins play a major role in phagosome maturation, facilitating the transition from early phagosomes to phagolysosomes, where bacteria are degraded. Nucleoside diphosphate kinase (Ndk) from Mycobacterium tuberculosis inactivated Rab proteins, leading to evasion of innate immunity by interfering with phagosome maturation. Ndk is also a housekeeping enzyme, important in the maintenance of nucleoside triphosphate (NTP) pools, with histidine at position 117 (H117) involved in autophosphorylation and phosphotransfer activity, and synthesis of NTPs. In my thesis research, I have cloned and expressed putative recombinant Map Ndk (Map rNdk) in E. coli and generated anti-Ndk polyclonal antibodies in rats. Native Map Ndk was detected by immunoprecipitation using anti-Ndk antibodies. The interaction of Map rNdk with bovine macrophage proteins involved in phagosome maturation (Rab5 and Rab7) and other Map proteins, as well as its immunogenicity during natural infection were examined. In contrast to studies with M. tuberculosis, no interactions were observed between Map rNdk and Rab5 or Rab7. Map rNdk-H117 catalytic mutants were constructed and will be discussed.