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 ionizationtandem 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°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.

A unique Coxiella burnetii lipoprotein involved in metal binding (LimB)

“…Anti-rLimB and anti-rCom1 polyclonal antibodies were generated in female New Zealand white rabbits, as described previously (Parrow et al, 2009), using 100 mg purified rLimB or rCom1 (Hicks et al, 2010) for both primary and booster injections. Rabbit anti-MBP polyclonal antibodies were purchased (NEB) and used as a negative control for the LimB fusion protein partner.…”

Identification of regulatory regions and regulatory protein complexes of theSpirulinadesDgene under temperature stress conditions: Role of thioredoxin as an inactivator of a transcriptional repressor GntR under low-temperature stress

“…First, ferredoxin might be involved as an electron transfer agent in biological redox reactions because BCP and Trx activities require NADPH, as discussed previously (Arias et al 2008;Clarke et al 2009;Hicks et al 2010). Second, adenylate kinase is well recognized to play a major role in ATP synthesis, suggesting that the DNA binding by the protein complex and its associated functions require energy (Nitschmann and Peschek 1986).…”

“…Moreover, in this study, the above mentioned protein domains were detected by LC-MS/MS, thus it is possible that Spirulina BCP contains these domains. Furthermore, Hicks et al (2010) reported that BCP is a DNA-binding protein that plays a critical role in detoxifying endogenous hydrogen peroxide byproducts generated from NADPH during the intracellular replication of Coxiella, thus protecting DNA from oxidative damage.…”

Strategies of Intracellular Pathogens for Obtaining Iron from the Environment

“…Reports have described the expression of a thiol-specific peroxidase (CBU0963) in C. burnetii that belongs to the atypical 2-cysteine subfamily of peroxiredoxins, also designated as bacterioferritin comigratory proteins (BCPs). The implication is that this protein might protect DNA from the Fenton reaction [ 60 ]. Comparison to L. pneumophila , a phylogenetic relative, revealed that C. burnetii rarely encodes any known Fe acquisition or storage proteins, aside from some Fe dependent pathways, as well as the heme biosynthesis pathway and proteins such as SodB.…”