Comparative Study of the Roles of AhpC and KatE as Respiratory Antioxidants inBrucella abortus2308

Abstract

Brucella strains are exposed to potentially toxic levels of H 2 O 2 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 H 2 O 2 -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 H 2 O 2 generated by aerobic metabolism. KatE, on the other hand, plays a major role in scavenging exogenous and supraphysiologic levels of H 2 O 2 , although this enzyme can play a supporting role in the detoxification of H 2 O 2 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 H 2 O 2 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 H 2 O 2 .

Brucella melitensis MucR, an Orthologue of Sinorhizobium meliloti MucR, Is Involved in Resistance to Oxidative, Detergent, and Saline Stresses and Cell Envelope Modifications

“…As previously demonstrated in a B. abortus hfq mutant, stationary-phase physiology plays an important role in the ability of brucellae to establish and maintain long-term intracellular residence in host macrophages (84) and probably also to cope with the stresses of the early, nonreplicative phase (the first 10 h) of intracellular infection (85). A common feature of all these conditions is exposure to oxidative stress either through the accumulation of endogenous oxygen radicals (86)(87)(88) or following the oxidative burst inside infected macrophages (89). Based on unpublished microarray analysis, the involvement of mucR in stress response mechanisms has been suggested for B. melitensis (41).…”

The Brucella abortus General Stress Response System Regulates Chronic Mammalian Infection and Is Controlled by Phosphorylation and Proteolysis

“…This suggests a model in which B. abortus PhyR, once activated by phosphorylation, would lock a cell into a state in which E1 is permanently active as a transcriptional regulator. The lifetime of this active state may be longer than the lifetime of the cell, which could provide B. abortus with a "memory" of prior stress encounters and thus preadapt the cell to future endogenous (38) or exogenous stresses encountered during host residence. However, PhyR dephosphorylation mechanisms may exist in B. abortus that switch the system off, as has been outlined in Sphingomonas sp.…”

SodA is a major metabolic antioxidant in Brucella abortus 2308 that plays a significant, but limited, role in the virulence of this strain in the mouse model

“…2 , but it is important to note that the attenuation exhibited by the B. abortus sodA mutant in the mouse model is much less than that displayed by an isogenic mutant that lacks both KatE and AhpC (cytoplasmic antioxidants that detoxify H 2 O 2 of endogenous origin) (Steele et al, 2010). Thus, it appears that there is still a lot to learn about the nature of the oxidative stresses brucella strains encounter in the host and how they protect themselves against these stresses.…”

“…Macrophages were activated 30 min prior to infection by the addition of 25 U interferon-c ml 21 (Steele et al, 2010). For inhibition of the NADPH oxidase, cultured macrophages were treated with apocynin (Sigma) at a final concentration of 500 mM per well (Hart & Simons, 1992) immediately following seeding and then once more approximately 6 h prior to the first time point after infection.…”