SdrA, an NADP(H)-regenerating enzyme, is crucial for Coxiella burnetii to resist oxidative stress and replicate intracellularly
| Autor: | Nadeeka K. Wawegama, Mebratu A Bitew, Hayley J. Newton, David P De Souza, Janine Hofmann, Fiona M. Sansom |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Cytoplasm
Immunology Mutant Dehydrogenase Oxidative phosphorylation Microbiology 03 medical and health sciences Virology Humans Regeneration 030304 developmental biology chemistry.chemical_classification 0303 health sciences Short-chain dehydrogenase biology 030306 microbiology Macrophages Coxiella burnetii biology.organism_classification Molecular biology Oxidative Stress Enzyme chemistry Mutation Vacuoles NADP binding Q Fever Intracellular NADP HeLa Cells |
| Zdroj: | Cellular microbiologyREFERENCES. 22(5) |
| ISSN: | 1462-5822 |
| Popis: | Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is a Gram-negative bacterium that replicates inside macrophages within a highly oxidative vacuole. Screening of a transposon mutant library suggested that sdrA, which encodes a putative short-chain dehydrogenase, is required for intracellular replication. Short-chain dehydrogenases are NADP(H)-dependent oxidoreductases, and SdrA contains a predicted NADP+ binding site, suggesting it may facilitate NADP(H) regeneration by C. burnetii, a key process for surviving oxidative stress. Purified recombinant 6×His-SdrA was able to convert NADP+ to NADP(H) in vitro. Mutation to alanine of a conserved glycine residue at position 12 within the predicted NADP binding site abolished significant enzymatic activity. Complementation of the sdrA mutant (sdrA::Tn) with plasmid-expressed SdrA restored intracellular replication to wild-type levels, but expressing enzymatically inactive G12A_SdrA did not. The sdrA::Tn mutant was more susceptible in vitro to oxidative stress, and treating infected host cells with L-ascorbate, an anti-oxidant, partially rescued the intracellular growth defect of sdrA::Tn. Finally, stable isotope labelling studies demonstrated a shift in flux through metabolic pathways in sdrA::Tn consistent with the presence of increased oxidative stress, and host cells infected with sdrA::Tn had elevated levels of reactive oxygen species compared with C. burnetii NMII. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|