A conservative amino acid change alters the function of BosR, the redox regulator of Borrelia burgdorferi

Department of Medical Microbiology and Immunology, 407 Reynolds Medical Building, Texas A&M University Health Science Center, College Station, TX 77843, USA.
Molecular Microbiology (Impact Factor: 4.42). 01/2005; 54(5):1352-63. DOI: 10.1111/j.1365-2958.2004.04352.x
Source: PubMed


Borrelia burgdorferi, the aetiologic agent of Lyme disease, modulates gene expression in response to changes imposed by its arthropod vector and mammalian hosts. As reactive oxygen species (ROS) are known to vary in these environments, we asked how B. burgdorferi responds to oxidative stress. The B. burgdorferi genome encodes a PerR homologue (recently designated BosR) that represses the oxidative stress response in other bacteria, suggesting a similar function in B. burgdorferi. When we tested the sensitivity of B. burgdorferi to ROS, one clonal non-infectious B. burgdorferi isolate exhibited hypersensitivity to t-butyl hydroperoxide when compared with infectious B. burgdorferi and other non-infectious isolates. Sequence analysis indicated that the hypersensitive non-infectious isolates bosR allele contained a single nucleotide substitution, converting an arginine to a lysine (bosRR39K). Mutants in bosRR39K exhibited an increase in resistance to oxidative stressors when compared with the parental non-infectious strain, suggesting that BosRR39K functioned as a repressor. Complementation with bosRR39K and bosR resulted in differential sensitivity to t-butyl hydroperoxide, indicating that these alleles are functionally distinct. In contrast to BosR, BosRR39K did not activate transcription of a napA promoter-lacZ reporter in Escherichia coli nor bind the napA promoter/operator domain. However, we found that both BosR and BosRR39K bound to the putative promoter/operator region of superoxide dismutase (sodA). In addition, we determined that cells lacking BosRR39K synthesized fourfold greater levels of the decorin binding adhesin DbpA suggesting that BosRR39K regulates genes unrelated to oxidative stress. Based on these data, we propose that the single amino acid substitution, R39K, dramatically alters the activity of BosR by altering its ability to bind DNA at target regulatory sequences.


Available from: Jenny Hyde, Oct 06, 2014
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    • "The Hk2-Rrp2 TCS activates the expression of the stationary phase sigma factor RpoS synergistically with RpoN (Burtnick et al., 2007; Ouyang et al., 2008; Blevins et al., 2009), which, in turn, chiefly regulates plasmid-borne genes (Yang et al., 2003a,b; Caimano et al., 2007) and induces the expression of genes, such as ospC (Hübner et al., 2001), which are known to be important for mammalian infection (Caimano et al., 2004; Fisher et al., 2005; Caimano et al., 2007; Boardman et al., 2008; Ouyang et al., 2008; Dunham-Ems et al., 2012; Ouyang et al., 2012) as well as genes involved in chitobiose utilization, which has been shown to be important for colonization of the tick (Sze et al., 2013). The Hk1-Rrp1 TCS converges with the Hk2-Rrp2 TCS through the regulator, BosR—a Fur/Per-like transcription factor that has been demonstrated to be essential for expression of rpoS (Boylan et al., 2003; Katona et al., 2004; Seshu et al., 2004; Hyde et al., 2009; Ouyang et al., 2009, 2011; Hyde et al., 2010)—which primarily regulates core chromosome-encoded genes (Rogers et al., 2009; He et al., 2011, 2014) and is required for tick colonization (Caimano et al., 2011; He et al., 2011; Kostick et al., 2011). Interestingly, Rrp1, the response regulator, lacks a DNA-binding domain, but instead contains a GGDEF domain, which has been associated with diguanylate cyclase activity (cyclic di-GMP synthase) in B. burgdorferi (Ryjenkov et al., 2005). "
    [Show abstract] [Hide abstract] ABSTRACT: In nature, the Lyme disease spirochete Borrelia burgdorferi cycles between the unrelated environments of the Ixodes tick vector and mammalian host. In order to survive transmission between hosts, B. burgdorferi must be able to not only detect changes in its environment, but also rapidly and appropriately respond to these changes. One manner in which this obligate parasite regulates and adapts to its changing environment is through cyclic-di-GMP (c-di-GMP) signaling. c-di-GMP has been shown to be instrumental in orchestrating the adaptation of B. burgdorferi to the tick environment. B. burgdorferi possesses only one set of c-di-GMP-metabolizing genes (one diguanylate cyclase and two distinct phosphodiesterases) and one c-di-GMP-binding PilZ-domain protein designated as PlzA. While studies in the realm of c-di-GMP signaling in B. burgdorferi have exploded in the last few years, there are still many more questions than answers. Elucidation of the importance of c-di-GMP signaling to B. burgdorferi may lead to the identification of mechanisms that are critical for the survival of B. burgdorferi in the tick phase of the enzootic cycle as well as potentially delineate a role (if any) c-di-GMP may play in the transmission and virulence of B. burgdorferi during the enzootic cycle, thereby enabling the development of effective drugs for the prevention and/or treatment of Lyme disease.
    Full-text · Article · May 2014 · Frontiers in Cellular and Infection Microbiology
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    • "Cell pellets were washed twice in BSK-II medium containing 1,000 U ml 21 of catalase and resuspended in 1 ml of BSK-II medium. Quantification of viability was determined by limiting dilution in 96-well plates (Fisher Scientific, Pittsburgh, PA) following incubation at 37uC/5% CO 2 for 14 days [51]. Controls were incubated in BSK-II and BSK-II without pyruvate and treated with catalase for comparison with H 2 O 2 treated samples. "
    Full-text · Dataset · Jan 2014
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    • "Cell pellets were washed twice in BSK-II medium containing 1,000 U ml−1 of catalase and resuspended in 1 ml of BSK-II medium. Quantification of viability was determined by limiting dilution in 96-well plates (Fisher Scientific, Pittsburgh, PA) following incubation at 37°C/5% CO2 for 14 days [51]. Controls were incubated in BSK-II and BSK-II without pyruvate and treated with catalase for comparison with H2O2 treated samples. "
    [Show abstract] [Hide abstract] ABSTRACT: Pathogenic spirochetes cause clinically relevant diseases in humans and animals, such as Lyme disease and leptospirosis. The causative agent of Lyme disease, Borrelia burgdorferi, and the causative agent of leptospirosis, Leptospria interrogans, encounter reactive oxygen species (ROS) during their enzootic cycles. This report demonstrated that physiologically relevant concentrations of pyruvate, a potent H2O2 scavenger, and provided passive protection to B. burgdorferi and L. interrogans against H2O2. When extracellular pyruvate was absent, both spirochetes were sensitive to a low dose of H2O2 (≈0.6 µM per h) generated by glucose oxidase (GOX). Despite encoding a functional catalase, L. interrogans was more sensitive than B. burgdorferi to H2O2 generated by GOX, which may be due to the inherent resistance of B. burgdorferi because of the virtual absence of intracellular iron. In B. burgdorferi, the nucleotide excision repair (NER) and the DNA mismatch repair (MMR) pathways were important for survival during H2O2 challenge since deletion of the uvrB or the mutS genes enhanced its sensitivity to H2O2 killing; however, the presence of pyruvate fully protected ΔuvrB and ΔmutS from H2O2 killing further demonstrating the importance of pyruvate in protection. These findings demonstrated that pyruvate, in addition to its classical role in central carbon metabolism, serves as an important H2O2 scavenger for pathogenic spirochetes. Furthermore, pyruvate reduced ROS generated by human neutrophils in response to the Toll-like receptor 2 (TLR2) agonist zymosan. In addition, pyruvate reduced neutrophil-derived ROS in response to B. burgdorferi, which also activates host expression through TLR2 signaling. Thus, pathogenic spirochetes may exploit the metabolite pyruvate, present in blood and tissues, to survive H2O2 generated by the host antibacterial response generated during infection.
    Full-text · Article · Jan 2014 · PLoS ONE
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