Irr regulates brucebactin and 2,3-dihydroxybenzoic acid biosynthesis, and is implicated in the oxidative stress resistance and intracellular survival of Brucella abortus

Abstract

Brucella abortus faces iron deprivation in both nature and the host. To overcome this limitation, Brucella secretes the siderophores 2,3-dihydroxybenzoic acid and brucebactin. A Fur-like protein named Irr has previously been characterized in B. abortus; this protein is present in the a-2 group of Proteobacteria only, where it negatively regulates haem biosynthesis when iron is scarce. Additional evidence that Irr also regulates the synthesis of both siderophores is presented here. Transcriptional lacZ fusion and chemical determinations revealed that Irr induced the transcription of the operon involved in the synthesis of the catecholic siderophores, which were consequently secreted under conditions of iron limitation. Irr was able to bind the upstream region of the operon, as shown by electrophoretic mobility shift assay. A B. abortus irr mutant showed higher intracellular haem content, catalase activity and resistance to hydrogen peroxide than the wild-type strain. The mutation also improved the replication and survival of iron-depleted bacteria within cultured mammalian cells. Although the pathogenesis of Brucella correlates with its ability to replicate intracellularly, pathogenicity was not attenuated when assayed in a murine model. INTRODUCTIONIron is an essential micronutrient for almost all living organisms. In nature, iron is mainly present in a ferric insoluble state, with reduced biological availability. One of the strategies developed by bacteria to acquire iron under restrictive conditions is the synthesis of low-molecular-mass iron chelators, known as siderophores, together with the receptors for the internalization of ferrisiderophores (Wandersman & Delepelaire, 2004). This iron acquisition mechanism is frequently linked to the virulence of bacteria because it contributes to the establishment of a successful infection (Litwin & Calderwood, 1993). In contrast, a high concentration of iron is toxic, due to its participation in reactions that generate reactive oxygen species, which can lead to cellular damage. In these conditions, bacteria usually induce the synthesis of storage proteins and oxidative stress enzymes that remove toxic molecules. According to this scenario, the tight regulation of iron homeostasis is fundamental for bacterial life.Brucella abortus is the aetiological agent of bovine brucellosis, which is an infection of humans and cattle.The organism lives mainly intracellularly, and its pathogenesis correlates with the ability to invade and replicate within professional and non-professional phagocytes (Smith & Ficht, 1990). During infection, iron availability is reduced as part of the host defence against micro-organisms, implying that B. abortus faces iron limitation in this process (Bullen & Griffiths, 1999). Under conditions of iron depletion, B. abortus secretes the two catecholic siderophores brucebactin and 2,3-dihydroxybenzoic acid (2,3-DHBA), which have been associated with virulence of the species in ruminants, suggesting the importance of this mechanism for iron acquisition i...

Antiparallel and Interlinked Control of Cellular Iron Levels by the Irr and RirA Regulators of Agrobacterium tumefaciens

“…We measured the sensitivity of our mutants to SNG and H 2 O 2 and found that the sensitivity profiles of the single mutants corresponded to the amount of uncomplexed iron as measured by EPR; decreased iron in the irr mutant correlated with increased SNG/H 2 O 2 resistance, and increased iron in the rirA mutant correlated with decreased resistance. Increased H 2 O 2 resistance in an irr mutant has also been observed in Brucella abortus (36). The ⌬irr ⌬rirA double mutant does not universally follow the rule of increased uncomplexed cellular iron resulting in increased sensitivity, as it has a slightly increased EPR-detectable iron concentration but low SNG sensitivity.…”

TheBradyrhizobium japonicumIrr Protein Is a Transcriptional Repressor with High-Affinity DNA-Binding Activity

“…However, the affinity of Irr for those DNA elements is very weak in vitro (21). In fact, in vitro binding studies of Irr from several organisms all require micromolar concentrations of protein to detect DNA binding (3,14,21,27). Herein, we show that Irr is a transcriptional repressor with high affinity for target DNA and that Irr occupies the promoters of target genes in vivo.…”

“…A role for Irr as both a positive and a negative global regulator is based on several observations. Irr was initially discovered as a positive effector of ferric iron transport as well as a negative regulator of heme biosynthesis (11), and similar roles have been described for Brucella abortus Irr (13,14). Microarray analysis of a B. japonicum irr mutant shows that Irr affects the expression of many iron-regulated genes, and a heme-defective strain cannot maintain normal iron homeostasis (27).…”

The Importance of Haem vs Non-Haem Iron in the Survival and Pathogenesis of Brucella abortus

“…(Anderson et al, 2011;Paulley et al, 2007). When a different B. abortus irr mutant was previously tested in Balb/C mice, at 7 and 21 days after inoculation, neither the increment in catalase activity and haem content, nor the decline in siderophores biosynthesis affected the wild type virulence (Martinez et al, 2006). Although a different mice strain was used, the attenuation in the brucella virulence observed after 4 weeks p.i.…”

“…However, experimental evidence on mutants unable to synthesize those molecules failed to demonstrate that their production is critical for bacterial replication inside macrophages (Bellaire et al, 1999;Jain et al, 2011). In addition, a mutant in the main iron-response regulator gene, with deficient synthesis of siderophores and increased intracellular haem content, showed a better replication pattern inside professional phagocytes than wild type (Martinez et al, 2006). On the other hand, a B.…”