Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2018

The role of nicotinamide adenine dinucleotide (NAD) synthesis in the pathogenesis of Coxiella burnetii (#161)

Mebratu A Asaye 1 , Chen Ai Khoo 2 , Nitika Neha 1 3 , Dedreia Tull 3 , David P De Souza 3 , Nadeeka K Wawegama 1 , Hayley J Newton 2 , Fiona M Sansom 1
  1. Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, The University of Melbourne, Parkville, VIC, Australia
  2. Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, VIC, Australia
  3. Metabolomics Australia, Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Parkville, VIC, Australia

Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is an intracellular Gram-negative bacterium. Although improved genetic tools and culturing techniques has recently advanced the study of Coxiella pathogenesis, the mechanisms that allow the bacterium to survive and replicate inside the hostile phagolysosome are not well understood. A recent screen of a Coxiella transposon mutant library for replication within HeLa cells identified a number of genes required for efficient intracellular replication, including nadB, which is predicted to encode L-aspartate oxidase, an enzyme required for de novo nicotinamide adenine dinucleotide (NAD) synthesis. To confirm the role of NadB in intracellular replication, complementation of the nadB mutant with a pJB:Kan-3xFLAG plasmid expressing 3xFLAG-NadB was performed. Quantitative and qualitative intracellular replication assays inside HeLa cells conclusively demonstrated that nadB is required for intracellular replication. This is in contrast to previous studies of the intracellular pathogen Mycobacterium tuberculosis, where the loss of nadB appears to be compensated for by salvage pathways, even in vivo. To functionally characterise NadB, we used an untargeted metabolomics approach to compare the metabolite profiles of wild-type, mutant and complemented strains. GC-MS and LC-MS analysis revealed key changes in the mutant compared to wild type, with an increase in key pathway metabolites preceding NadB, and a corresponding decrease in downstream metabolites. Bioinformatic analysis of the NadB amino acid sequence revealed the presence of a conserved arginine residue at position 275. Site-directed mutagenesis was performed to mutate this residue to a leucine, which abolishes activity of E. coli NadB, and both wildtype and R275L NadB-GST fusion proteins were expressed in and purified from E. coli JM109. Enzyme assays using recombinant wildtype NadB-GST demonstrated typical L-aspartate oxidase activity. Current work is focusing on functional characterization of R275L NadB-GST, to confirm loss of enzyme activity, and complementation of the nadB mutant with this mutant protein, to confirm the link between an intact de novo NAD synthesis pathway and intracellular replication.