Interruption of thecydBLocus inBrucella abortusAttenuates Intracellular Survival and Virulence in the Mouse Model of Infection

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Abstract
Brucellosis is characterized by abortion in ruminants and a protracted undulant fever in humans, which often results in severe pathological manifestations. Scant information exists about the molecular mechanisms employed byBrucella abortusto combat host defenses or to persist and replicate within host cells. Transposon (Tn5) mutagenesis ofB. abortusand the subsequent screening of mutants for sensitivity to killing in murine macrophages and in the mouse model led to the identification of mutants which were severely attenuated for intracellular survival. One group of mutants was interrupted incydB, a gene that is part of thecydABoperon encoding cytochromebdoxidase, which catalyzes an alternate terminal electron transport step in bacterial respiration. The elevated affinity for molecular oxygen of this enzyme inEscherichia colihas suggested that it is involved in the protection of sensitive enzymatic activities such as those of hydrogenases and nitrogenases from damage.B. abortus cydB::Tn5strains exhibited heightened sensitivity to the respiratory inhibitors zinc and azide, highly reactive oxygen species such as hydrogen peroxide, low pH, and attenuated virulence in the mouse model of infection. Virulence was restored by an intact copy ofcydABor byB. abortusgenes encoding the oxidative radical-scavenging enzyme Cu/Zn superoxide dismutase or catalase. These results suggest a bifunctional role for the products of thecydABoperon, both in preventing the buildup of oxidative free radicals and in detoxifying the intracellular compartment, thus indicating the importance of these products in preventing intracellular destruction. Intracellular conditions that favor expression of thecydABoperon are under investigation and may be linked to the acid sensitivity also observed in this strain.

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