Science Bite (3 min Oral Presentation) Lorne Infection and Immunity 2019

The Role of Metabolic Genes in the Pathogenesis of Coxiella burnetii, the Causative Agent of the Zoonotic Disease Q fever (#60)

Mebratu A. Bitew 1 , Chen Ai Khoo 2 , Nitika Neha 3 , David P. De Souza 3 , Dedreia Tull 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, Victoria, Australia
  2. Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, , The University of Melbourne, Parkville, Victoria, Australia
  3. Metabolomics Australia, Bio21 Institute of Molecular Science and Biotechnology, , The University of Melbourne, Parkville, Victoria, Australia

Coxiella burnetii, the causative agent of the neglected zoonotic disease Q fever, is an intracellular Gram-negative bacterium that replicates within a unique vacuole within the host cell. Previous screening of a transposon mutant library for replication within HeLa cells identified a number of genes required for efficient intracellular replication, including two genes encoding proteins with predicted metabolic functions, nadB and cbu_1276.

Quantitative and qualitative intracellular replication assays using transposon mutants and the corresponding complemented strains confirmed the requirement of each gene for wild type levels of replication within human cells. nadB encodes a putative L-aspartate oxidase, a key enzyme in de novo NAD synthesis, and GC-MS and LC-MS analysis revealed key changes in the nadB mutant compared to wild type, with an increase in key pathway metabolites preceding NadB, and a corresponding decrease in downstream metabolites.  Purification and assaying of recombinant GST-NadB confirmed its L-aspartate oxidase activity, and that a mutant derivative, R275L-GST-NadB, was inactive. Complementation of the C. burnetii nadB mutant with a plasmid expressing inactive R275L NadB failed to restore replication to wildtype levels, confirming the link between de novo NAD synthesis and intracellular replication of C. burnetii. This suggests that targeting this prokaryotic-specific pathway could advance the development of therapeutics to combat C. burnetii infections.

CBU_1276 is a putative short chain dehydrogenase. Assaying of recombinant 6xHis-CBU-1276 confirmed the function of this enzyme in reducing NAD(P) to NADP(H), which may explain the crucial role of this enzyme in replication, particularly as it is currently unclear how C. burnetii regenerates sufficient NADP(H)  in the absence of the oxidative branch of the pentose phosphate pathway. We are currently focusing on confirming the link between CBU_1276 enzymatic activity and intracellular replication.  Overall, this work demonstrates the importance of investigating the role of metabolic enzymes when studying bacterial pathogenesis.