Characterization of an OxyR-regulated alkyl hydroperoxide reductase (ahpC2D) operon in Legionella pneumophila.

摘要

Legionella pneumophila expresses two catalase-peroxidase enzymes that exhibit only weak hydrogen peroxide (H2O2)-scavenging activity, suggesting that other enzymes might decompose H2O 2. Likely candidates included two alkyl hydroperoxide reductases (AhpC) that were identified in the L. pneumophila genome. AhpC1 and AhpC2/AhpD (AhpC2D) show similarity to the peroxide scavenging systems found in Helicobacter pylori and Mycobacterium tuberculosis , respectively. Our results indicated that: (i) expression of L. pneumophila ahpC1 and ahpC2 restores H2 O2 resistance in a catalase/peroxidase-deficient mutant of Escherichia coli; (ii) both L. pneumophila ahpC1::km and ahpC2D::km mutants are more sensitive to various peroxides or reactive oxygen intermediate (ROI)-producing compounds than wild-type, a phenotype that could be alleviated by complementation; (iii) L. pneumophila ahpC mutants were not affected in their intracellular growth in macrophage-like cells; (iv) expression of ahpC1 appeared post-exponentially in broth culture, whereas ahpC2 was expressed during early exponential phase; (v) ahpC1 mRNA levels were consistently higher than those of ahpC2D; (vi) ahpC2D expression is significantly increased upon loss of AhpC1 function. To address whether the L. pneumophila OxyR homologue (LpOxyR) could function as a regulator of the oxidative stress response as seen in other organisms, experiments were performed to demonstrate that: (i) reduced and oxidized forms of LpOxyR can bind the promoter region of ahpC2D (Pahpc2); (ii) defective LpOxyR binding resulted in loss of ahpC2 transcriptional activity; (iii) reduced LpOxyR displayed an extended DNA footprint that overlaps with the putative -35 region of ahpC2 which was fully accessible to RNA polymerase with oxidized LpOxyR; (iv) expression of LpOxyR was partially able to restore peroxide resistance in an E. coli oxyR::km mutant. However, unlike E. coli OxyR, LpOxyR was unable to bind to its own promoter. Since LpOxyR expression was growth phase-dependent, attempts were also made to determine possible regulators of oxyR expression. An acrylamide capture of DNA-bound complexes technique was used in an attempt to identify transcriptional regulators with no success. In summary, this study reports that AhpC1 or AhpC2D provide an essential peroxide-scavenging function to L. pneumophila and that LpOxyR functions as a peroxide sensor/transcriptional regulator capable of activating transcription of ahpC2D in response to oxidative stress.