title: A DNA-binding peroxiredoxin of Coxiella burnetii is involved in countering oxidative stress during exponential-phase growth.
abstract: Coxiella burnetii is a Gram-negative, obligate intracellular bacterial pathogen that resides within the harsh, acidic confines of a lysosome-like compartment of the host cell that is termed a parasitophorous vacuole. In this study, we characterized a thiol-specific peroxidase of C. burnetii that belongs to the atypical 2-cysteine subfamily of peroxiredoxins, commonly referred to as bacterioferritin comigratory proteins (BCPs). Coxiella BCP was initially identified as a potential DNA-binding protein by two-dimensional Southwestern (SW) blots of the pathogen's proteome, probed with biotinylated C. burnetii genomic DNA. Confirmation of the identity of the DNA-binding protein as BCP (CBU_0963) was established by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). Recombinant Coxiella BCP (rBCP) was generated, and its DNA binding was demonstrated by two independent methods, including SW blotting and electrophoretic mobility shift assays (EMSAs). rBCP also demonstrated peroxidase activity in vitro that required thioredoxin-thioredoxin reductase (Trx-TrxR). Both the DNA-binding and peroxidase activities of rBCP were lost upon heat denaturation (100 degrees C, 10 min). Functional expression of Coxiella bcp was demonstrated by trans-complementation of an Escherichia coli bcp mutant, as evidenced by the strain's ability to grow in an oxidative-stress growth medium containing tert-butyl hydroperoxide to levels that were indistinguishable from, or significantly greater than, those observed with its wild-type parental strain and significantly greater than bcp mutant levels (P < 0.05). rBCP was also found to protect supercoiled plasmid DNA from oxidative damage (i.e., nicking) in vitro. Maximal expression of the bcp gene coincided with the pathogen's early (day 2 to 3) exponential-growth phase in an experiment involving synchronized infection of an epithelial (Vero) host cell line. Taken as a whole, the results show that Coxiella BCP binds DNA and likely serves to detoxify endogenous hydroperoxide byproducts of Coxiella's metabolism during intracellular replication.
authors.affiliation: Division of Biological Sciences, The University of Montana, Missoula, MT 59812, USA.
authors.forename: Linda D
authors.initials: LD
authors.lastname: Hicks
authors.forename: Rahul
authors.initials: R
authors.lastname: Raghavan
authors.forename: James M
authors.initials: JM
authors.lastname: Battisti
authors.forename: Michael F
authors.initials: MF
authors.lastname: Minnick
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D026501:Blotting, Southwestern; D016997:Coxiella burnetii / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D005816:Genetic Complementation Test; D013058:Mass Spectrometry; D018384:Oxidative Stress / Q000235:genetics / Q000502:physiology*; D054464:Peroxiredoxins / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D054464:Peroxiredoxins