Abstract/Summary

Bartonella henselae is a zoonotic human pathogen that must survive redox attack during its colonisation of both its invertebrate and mammalian hosts. However, its genome sequence is characterised by a paucity of genes responsible for redox stress resistance, especially hydrogen peroxide degradation systems such as catalases and alkyl hydroreductase. Here, the ability of B. henselae to resist two types of peroxide (hydrogen peroxide, tert-butylperoxide) showed that it displays resistance to high concentrations of both (at 400 and 1600 μM, respectively). The importance of an exogenous haem supply for B. henselae growth was also confirmed (B. henselae lacks a complete haem biosynthesis pathway). In addition, growth of B. henselae was shown to be greatly inhibited by high concentrations of the iron chelator, DTPA, suggesting a requirement for iron. To determine how B. henselae achieves resistance to H2O2 stress, the potential role of iron export was investigated. The results showed that B. henselae has the ability to export iron and that the iron export process is hydrogen peroxide dependent – export was inhibited by exogenous catalase and anaerobiosis whereas it was enhanced by provision of H2O2. In addition, the form of iron exported was largely ferric (rather than ferrous), indicating that iron is oxidised during the export process. Thus, the iron export activity shown resembles that mediated by the MbfA of Brucella suis, indicating that the MbfA B. henselae is responsible for the observed peroxide-induced iron export. In order to discover the impact of the iron export process on the resistance to H2O2, the degradation of H2O2 by B. henselae was assayed and the results showed that B. henselae mediates a rapid consumption of exogenously supplied H2O2. This degradation was entirely inhibited when iron chelators (DTPA and 2,3 dihydroxy benzoic acid) were included along with the H2O2 which strongly suggests that the observed degradation of H2O2 by B. henselae is dependent on the exported iron which, in free form, is well known to catalyse peroxide disproportionation through Fenton reactivity. Further work showed that preculturing under iron-restriction conditions resulted in a subsequent reduction in iron export capacity, presumably due to a lowered cellular iron content. It should also be noted that B. henselae lacks any apparent iron-storage system, so the source of exported iron remains unclear The resistance of B. henselae to NO was also tested since NO is generated by phagocytic host cells along with H2O2 and is suggested to potentiate the toxicity of H2O2 towards engulfed bacteria through inhibiting haem-dependent catalases and alkyl hydroperoxidases. NO was found to elicit a modest increase in sensitivity of B. henselae to hydrogen peroxide (an apparent additive effect) indicating that NO does not cause a marked potentiation of H2O2 activity, as seen in other haem-catalayse/peroxidase dependent bacteria (e.g., E. coli). Further, NO had only a modest impact on the ability of B. henselae to degrade exogenous H2O2. These observations indicate that MbfA of B. henselae is not subject to any major NO inhibition and thus suggests that MbfA may provide a particularly beneficial role in vivo. To determine whether the MbfA B. henselae is responsible for the observed peroxide-induced iron export, gene ‘knockdown’ was deployed. Thus, mbfA was expressed reverse orientation in plasmid pNSTrc such that the mbfA anti-sense strand was transcribed. The ability of bacteria to iron export and hydrogen peroxide degradation after knockdown were tested and results showed antisense mbfA lead to a significant decrease in exported iron when compare to iron export the vector control, supporting the role of MbfA in mediating iron export. However, antisense mbfA had little impact on H2O2 degradation. The contribution of superoxide dismutase to redox-stress resistance in B. henselae was also studied. Activity staining of native gels revealed two Sod species, as expected from the genome sequence, with one located in the periplasmic fraction (designated SodC) and the other in the cytosolic fraction (designated SodB). Activity staining indicated that these two enzymes are differentially regulated in response to H2O2 and high iron. To study the role of sodB and sodC in redox stress resistance through exposure bacteria to H2O2 and methyl viologen, the gene ‘knockdown’ approach was again utilised. The result showed suggested that antisense sodC decreased levels of SodC by ~50% when compared to the vector control and increased the sensitivity of this strain to hydrogen peroxide and methyl viologen. Also, antisense sodB deceased SodB activity, and inhibited growth curve and resistance to H2O2 and methyl viologen. The results therefore suggest that sodC and sodB play important roles in protect B. henselae against redox stress. In the second part of this work, the global regulatory response of Cutibacterium acnes to redox and iron stress was explored by RNA-seq. C. acnes is a commensal bacterium predominant in sebaceous sites that is essential in the regulation of skin homeostasis and protection of skin from colonisation by harmful pathogens. However, it can act as an opportunistic pathogen in acne vulgaris. It is subject to exposure to environmental stresses, such as reactive oxygen species (ROS) created from UV irradiated skin or from a phagocyte’s activation. Expression patterns were obtained that were indicative of an iron-restriction response (induction of four putative iron uptake/utilisation systems) in the presence of DTPA with respect to iron sulphate, with 64 genes showing more than fourfold differential expression. Of particular note was the high-iron induction of three genes encoding 50S ribosomal proteins (L28, L31 and L33) which have been reported to be subject to zinc dependent control in other bacteria. A set of specific sugar utilization pathways were subject to repression under high iron. Significant differential gene expression was also seen for H2O2 treatment with 59 genes showing a >fourfold significant change in expression. Five genes with roles in redox-stress resistance were induced whereas 20 ribosomal protein genes were down regulated which is indicative of a reduction on growth as a result of H2O2 treatment.