Comparative Study of the Roles of AhpC and KatE as Respiratory Antioxidants in Brucella abortus 2308
Steele, Kendra H.; Baumgartner, John E.; Wright Valderas, Michelle; Roop II, R. Martin
Brucella strains are exposed to potentially toxic levels of H2O2 both as a consequence of their aerobic metabolism and through the respiratory burst of host phagocytes. To evaluate the relative contributions of the sole catalase KatE and the peroxiredoxin AhpC produced by these strains in defense against H2O2-mediated toxicity, isogenic katE, ahpC, and katE ahpC mutants were constructed and the phenotypic properties of these mutants compared with those of the virulent parental strain B. abortus 2308. The results of these studies indicate that AhpC is the primary detoxifier of endogenous H2O2 generated by aerobic metabolism. KatE, on the other hand, plays a major role in scavenging exogenous and supraphysiologic levels of H2O2, although this enzyme can play a supporting role in the detoxification of H2O2 of endogenous origin if AhpC is absent. B. abortus ahpC and katE mutants exhibit wild-type virulence in C57BL/6 and BALB/c mice, but the B. abortus ahpC katE double mutant is extremely attenuated, and this attenuation is not relieved in derivatives of C57BL/6 mice that lack NADPH oxidase (cybb) or inducible nitric oxide synthase (Nos2) activity. These experimental findings indicate that the generation of endogenous H2O2 represents a relevant environmental stress that B. abortus 2308 must deal with during its residence in the host and that AhpC and KatE perform compensatory roles in detoxifying this metabolic H2O2. Brucella abortus, a facultative intracellular pathogen, causes abortion and infertility in cattle. Humans can also be infected by ingesting contaminated dairy products, through inhalation of infectious aerosols, or via direct contact with an infected fetus (43). Human brucellosis causes flu-like symptoms with a relapsing fever, and this debilitating disease can persist for months or years without appropriate treatment. Although human brucellosis remains a significant zoonotic disease worldwide (47) and a potential bioterrorism threat (70), there is currently no vaccine to prevent human infection, and antibiotic treatment of these infections remains problematic (2). Prolonged survival and replication in host macrophages play a critical role in the virulence of the Brucella spp. (34, 57). Experimental evidence indicates that reactive oxygen species (ROS) such as superoxide (O2−) and hydrogen peroxide (H2O2) are important components of the brucellacidal activity of these phagocytes (31). Because the brucellae rely on respiratory metabolism for their energy production (52), these bacteria must also deal with endogenous ROS generated as a by-product of aerobic metabolism (27). Several enzymes that directly detoxify O2− and H2O2 have been identified in Brucella. SodC is a periplasmic Cu-Zn superoxide dismutase (6), and phenotypic evaluation of an isogenic sodC mutant indicates that this enzyme protects B. abortus 2308 from O2− generated by the oxidative burst of host macrophages (22). Brucella strains also produce a single monofunctional catalase that is a structural homolog of Escherichia coli KatE. Although this protein does not possess a standard export signal sequence (63) or a predicted twin arginine transport signal sequence (data not shown), cell fractionation studies with the appropriate controls indicate that this protein resides in the periplasmic compartment (63). B. abortus and Brucella melitensis katE mutants exhibit increased sensitivity to H2O2 compared to their parental strains in in vitro assays (21, 33). A B. melitensis katE mutant retains its virulence in experimentally infected goats (21), and B. abortus katE mutants display wild-type virulence in the mouse model (59). These experimental findings suggest that KatE does not play an indispensable role in protecting the brucellae from oxidative killing by host phagocytes. A gene (BAB1_0591) encoding a Mn superoxide dismutase (SodA) has also been identified in B. abortus 2308. SodA activity increases in a B. abortus sodC mutant, suggesting that SodA works in concert with SodC to protect B. abortus 2308 from oxidative damage (65), but the precise role that SodA plays in resistance to oxidative stress in this bacterium remains to be determined experimentally. The genes designated BAB2_0531 and BAB2_0532 in the B. abortus 2308 genome sequence are predicted to encode the components of an alkyl hydroperoxide reductase complex (AhpC and AhpD, respectively). Peroxiredoxins of the AhpC family detoxify H2O2, organic peroxides, and peroxynitrite (ONOO−) (9, 48). AhpD and AhpF are peroxiredoxin reductases that use reducing equivalents generated by cellular metabolism to restore the enzymatic activity of AhpC (10, 49). Studies performed with multiple bacterial species indicate that the AhpCD and AhpCF complexes serve as important antioxidants (4, 8, 11, 15, 16, 36, 37, 41, 44, 55, 66), and indeed, work in E. coli suggests that AhpC is the major scavenger of H2O2 generated in the cytoplasm of this bacterium as a by-product of aerobic metabolism (61). AhpC has also been shown to play a role in the virulence of several bacterial pathogens, including Helicobacter pylori (45), Mycobacterium bovis (72), and Staphylococcus aureus (15) but does not appear to be required for the virulence of Salmonella enterica serovar Typhimurium (68), Mycobacterium tuberculosis (64), Legionella pneumophila (51), or Porphyromonas gingivalis (32) in experimental models. In this report, we present evidence that AhpC is the primary antioxidant used by B. abortus 2308 to detoxify endogenous H2O2 generated by respiratory metabolism during routine aerobic cultivation. KatE, on the other hand, plays a major role in scavenging exogenous and supraphysiologic levels of H2O2, although this enzyme can play a supporting role in the detoxification of H2O2 of endogenous origin if AhpC is absent. Interestingly, AhpC and KatE appear to play complementary roles in protecting B. abortus 2308 from H2O2 of metabolic origin during residence in mice, and the presence of either AhpC or KatE alone is sufficient to allow this strain to maintain a chronic infection.
Steele, Kendra H., & Baumgartner, John E., & Wright Valderas, Michelle, & Roop II, R. Martin. (October 2010). Comparative Study of the Roles of AhpC and KatE as Respiratory Antioxidants in Brucella abortus 2308. Journal of Bacteriology, (192:19), p.4912-4922. Retrieved from http://hdl.handle.net/10342/8917
Steele, Kendra H., and Baumgartner, John E., and Wright Valderas, Michelle, and Roop II, R. Martin. "Comparative Study of the Roles of AhpC and KatE as Respiratory Antioxidants in Brucella abortus 2308". Journal of Bacteriology. 192:19. (4912-4922.), October 2010. July 31, 2021. http://hdl.handle.net/10342/8917.
Steele, Kendra H. and Baumgartner, John E. and Wright Valderas, Michelle and Roop II, R. Martin, "Comparative Study of the Roles of AhpC and KatE as Respiratory Antioxidants in Brucella abortus 2308," Journal of Bacteriology 192, no. 19 (October 2010), http://hdl.handle.net/10342/8917 (accessed July 31, 2021).
Steele, Kendra H., Baumgartner, John E., Wright Valderas, Michelle, Roop II, R. Martin. Comparative Study of the Roles of AhpC and KatE as Respiratory Antioxidants in Brucella abortus 2308. Journal of Bacteriology. October 2010; 192(19) 4912-4922. http://hdl.handle.net/10342/8917. Accessed July 31, 2021.