Interruption of the cydB Locus in Brucella abortus Attenuates Intracellular Survival and Virulence in the Mouse Model of Infection

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 by Brucella abortus to combat host defenses or to persist and replicate within host cells. Transposon (Tn5) mutagenesis of B. abortus and 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 in cydB, a gene that is part of the cydAB operon encoding cytochrome bd oxidase, which catalyzes an alternate terminal electron transport step in bacterial respiration. The elevated affinity for molecular oxygen of this enzyme in Escherichia coli has suggested that it is involved in the protection of sensitive enzymatic activities such as those of hydrogenases and nitrogenases from damage. B. abortus cydB::Tn5 strains 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 of cydAB or by B. abortus genes encoding the oxidative radical-scavenging enzyme Cu/Zn superoxide dismutase or catalase. These results suggest a bifunctional role for the products of the cydAB operon, 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 the cydAB operon are under investigation and may be linked to the acid sensitivity also observed in this strain.

Roles of Brucella abortus SpoT in morphological differentiation and intramacrophagic replication

“…A number of recent studies have reported the virulence genes of Brucella (Drazek et al, 1995;Endley et al, 2001;Eskra et al, 2001;Foulongne et al, 1999). The VirB type IV secretion system of Brucella has been identified recently Sieira et al, 2000).…”

Membrane topology and mutational analysis of Escherichia coli CydDC, an ABC-type cysteine exporter required for cytochrome assembly

“…Intriguingly, Lactococcus lactis exhibits the tra983B gene encoding the transposase of IS983B. Immediately upstream of the cydAB operon of Brucella melitensis biovar Abortus, two genes encoding ABC-type transporters of 331 and 322 residues were found (Endley et al, 2001). Tentatively, these genes were named cydC and cydD by the authors although no functional evidence was provided.…”

“…Thus, in Azotobacter vinelandii and Klebsiella pneumoniae, cytochrome bd deficiency severely impairs nitrogen-fixing ability under aerobic or microaerobic conditions (Kelly et al, 1990;Juty et al, 1997;Edwards et al, 2000). Furthermore, in Shigella flexneri and Brucella abortis, the Cyd 2 phenotype includes attenuation of intracellular survival and virulence (Way et al, 1999;Endley et al, 2001).…”

“…Thus, in Azotobacter vinelandii and Klebsiella pneumoniae, cytochrome bd deficiency severely impairs nitrogen-fixing ability under aerobic or microaerobic conditions (Kelly et al, 1990;Juty et al, 1997;Edwards et al, 2000). Furthermore, in Shigella flexneri and Brucella abortis, the Cyd 2 phenotype includes attenuation of intracellular survival and virulence (Way et al, 1999;Endley et al, 2001).In E. coli, two other genes, cydD and cydC, constituting an operon unlinked to cydAB, are necessary for cytochrome bd assembly (Georgiou et al, 1987;Poole et al, 1989 Poole et al, , 1993Bebbington & Williams, 1993;Delaney et al, 1993). These genes encode the strikingly similar, but distinct, components of an ATP-binding cassette (ABC)-type transporter (Poole et al, 1993), thought to be the first found in bacteria composed of only two non-identical subunits.…”

Gramicidin S identified as a potent inhibitor for cytochrome bd‐type quinol oxidase

“…Cytochrome bd also plays an important role in survival and adaptation of pathogenic bacteria that encounter host environments in which dioxygen is progressively limited [16][17][18]. Since cytochrome bd is totally absent in eukaryotic mitochondria, such alternative respiratory enzymes are promising targets for new chemotherapeutics.…”