Abstract/Summary

The mbfA (membrane bound ferritin A) gene is carried by all Brucella species and is widely conserved in α-Proteobacteria. mbfA encodes a novel iron-export protein comprising an N-terminal peroxide reductase (erythrin) domain and a C-terminal, membrane-embedded, iron-export domain (vacuolar iron transport, VIT). In this thesis, for the first time, the role of mbfA in Brucella suis 1330 and Brucella melitensis 16M was investigated, and the contributions of the two domains towards a novel mechanism of oxidative-stress resistance was considered. In addition, any part played by MbfA in enhancing Brucella survival within the macrophage ‘Brucella-containing vacuole’ (BCV) was investigated. The isolated erythrin domain was shown to bind two iron atoms (as expected) or zinc which stabilized the protein, but no substantive peroxidase activity was exhibited, presumably due to lack of a reduction partner. mbfA complemented an E. coli mutant that was devoid of catalases/alkyl-hydroperoxidases, enhanced growth of a fur mutant with deregulated iron uptake, and impaired growth of a mutant lacking iron-uptake capacity. These findings support a role for MbfA in peroxide resistance and iron export. MbfA was shown to export 55Fe when expressed in E. coli, and this activity was found to be O2 and H2O2 dependent. Similar results were obtained using a Burkholderia multivorans mbfA mutant. In addition, MbfA mediated the decomposition of exogenous H2O2, which was mainly achieved by degradation of exogenous peroxide by exported iron. MbfA provided peroxide resistance in wildtype E. coli but only when endogenous catalases/peroxidases were inhibited by NO. Thus, MbfA is not markedly NO inhibited. MbfA iron export was found to be partly dependent on the presence of iron stores, in the form of bacterioferritin. A naturally-occurring single-nucleotide substitution in the proximal coding region of the mbfA gene renders this gene cryptic in B. melitensis (although mbfA appears functional in all other Brucella spp.). Thus, a B. melitensis mbfA mutant exhibited no observable phenotype whereas a B. suis mbfA mutant displayed enhanced sensitivity to both Fe2+- and H2O2-mediated oxidative stress. It is hypothesized that MbfA exports ferric iron into the luminal space of the endosomal Brucella-containing vacuole (eBCV) to counteract both the respiratory burst generated by NADPH oxidase as well as the export of iron into the macrophage cytoplasm by NRAMP1. Exported iron causes disproportionation of peroxide externally, thus reducing the potential for harmful oxygen- and nitrogen-radical formation within the bacterial cell. Macrophage survival assays show that the absence of functional mbfA decreases intracellular survival during the early colonization phase in the eBCV, suggesting a reduced capacity to resist the redox stress of the endosomal and transient lysosomal compartments in the macrophage, which precede transition to the replicative BCV. Additionally, in vivo, mbfA contributes significantly to the maintenance of chronic infection within the murine liver and spleen. Further, MbfA provided a greater advantage in NRAMP1+ macrophages, than in NRAMP1- macrophages. This finding supports a role for MbfA in countering the iron export role of NRAMP1 within the phagolysosome, such that the purpose of this NRAMP1 actiy would be to preserve peroxide against iron-catalysed degradation. In summary, the results are therefore consistent with a role for mbfA in intracellular survival and redox stress resistance in Brucella.