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4.1.2.1 An introduction to metal stress

Metal ions of copper, iron, manganese and zinc are critical components of metalloenzymes, making them essential for the survival of both prokaryotic and eukaryotic cells (Agranoff and Krishna, 1998). One estimate approximates that half of all proteins contain metal ions in their structure and up to a third of enzymes require metals to function correctly (Thomson and Gray, 1998, Waldron and Robinson, 2009). Despite their necessity in biology, metals are toxic if intracellular levels are not strictly controlled. Ironically, the toxic effects of many metals result from the properties that make them so important. For example the redox cycling of Cu+ [Cu(I)] and Cu2+ [Cu(II)] ions allows this metal to be a critical component of proteins involved in numerous cellular processes i.e. denitrification, oxidative respiration, electron transport (Fraústo da Silva and Williams, 2001, Arguello et al., 2013). However, cellular imbalance of Cu+ ions can aid hydroxyl radical formation through reactions with H2O2 (via Fenton chemistry). This

results in nucleic acid, protein and membrane lipid damage, nitric oxide production through interactions with S-nitrosothiols and destabilisation of iron-sulphur cluster enzymes (Williams, 1999, Hiniker et al., 2005, Macomber and Imlay, 2009). In E. coli elevated intracellular copper does not cause significant DNA oxidative damage in vivo, suggesting copper toxicity occurs (in this instance) through a combination of the other processes described (Macomber et al., 2007). During metal deficiency, intracellular reserves are mobilised and transcription of metal carriers and influx systems are increased. The synthesis of storage proteins, an array of excretion systems, regulators and pumps (e.g. CopA, GesABC, ZntR, ZntAB, ZitB, YiiP, RcnA, Fur, Mur, MntR) help to neutralise the threats exhibited from elevated metal ion concentrations. For a review of metal sensing in Salmonella and the impact on pathogenicity see Osman and Cavet (2011).

4.1.2.2 Impact of copper on virulence of Salmonella

Elevated and depleted levels of metal ions in an intracellular environment can have a negative impact on virulence. Systems allowing iron, zinc, manganese, and copper uptake contribute significantly to virulence in pathogenic bacteria (Osman and Cavet, 2011, Guilhen et al., 2013, Crane et al., 2011, Eijkelkamp et al., 2015, Subashchandrabose and Mobley, 2015, Parrow et al., 2013). The importance of copper in bacterial pathogenicity and host immune systems is emphasised by the high infection rates experienced by patients suffering from the lethal Cu-deficiency disorder Menke’s disease (Uno and Arya, 1987, Gunn et al., 1984, Kreuder et al., 1993, Agertt et al., 2007). Farm animals with Cu-deficient diets also experience a marked increase in microbial infection occurrence and susceptibility (Samanovic et al., 2012).

During infection, Salmonella must overcome an array of stressful and diverse environments present within the host. This includes host-induced reductions in metal availability and metal-mediated toxicity. Although the levels of bioavailable iron are low in humans, neutrophils and macrophages further reduce availability at sites of infection through: iron uptake (Diaz-Ochoa et al., 2014), synthesis of the hormone hepcidin (Nicolas et al., 2001, Nicolas et al., 2002, Park et al., 2001, Nemeth et al., 2003) and iron storage proteins, e.g. transferrin (Nemeth et al., 2003, Armitage et al., 2011) and lactoferrin (Masson et al., 1969, Steinbakk et al., 1990, Goetz et al., 2002). Neutrophils and epithelial cells also release lipocalin-2, an antimicrobial protein that sequesters siderophores, including enterobactin, to limit the bacterial response to reduced iron (Aujla et al., 2008, Bachman et al., 2009, Raffatellu et al., 2009). However salmochelin, a derivative of enterobactin synthesised by Salmonella sp., Klebsiella sp. and UPEC, cannot be bound by lipocalin-2, making S. Typhimurium lipochalin-2 resistant (Hantke et al., 2003, Raffatellu et al., 2009, Bachman et al., 2011). Zinc and manganese are sequestered by more general metal ion binding proteins, such a calprotectin, an antimicrobial protein that comprises 50% of neutrophil cytosolic content (Hessian et al., 1993). Macrophages also utilise the toxic effects of copper during bacterial killing. The cytokine IFN-γ stimulates copper uptake in macrophages by

168 increasing expression of CTR1, a high affinity copper importer (White et al., 2009). This may have the added benefit of further reducing extracellular copper that is bioavailable. In addition, copper is exported from the Golgi to the phagosomal compartment through the ATP7A copper transporter, enhancing bactericidal activity of macrophages (White et al., 2009). White and colleagues (2009) showed that the copper transporting ATPase, CopA, helps E. coli overcome this copper-mediated killing. A copA deletion renders the bacteria hypersensitive to killing by murine macrophages, in an ATP7A dependant manner. These descriptions of mammalian host manipulations of metals during immune response to infection is by no means exhaustive; see Diaz-Ochoa et al., (2014) for a comprehensive review. However, this summary provides an insight into the onslaught pathogens such as

Salmonella must overcome when establishing an infection. 4.1.2.3 Salmonella response to copper

Salmonella has evolved multiple DNA-binding metal responsive transcription

factors: CueR, Fur, Zur, MntR, NikR, GolS and RcnR, allowing optimisation of metal acquisition while affording protection from metal-mediated toxicity inflicted upon it within host immune cells (for full review see Osman and Cavet (2011). In addition to these metal-specific regulatory networks,

Salmonella employs multiple global stress responses in response to excess

copper and zinc (Pontel et al., 2014). As a result of Cu+ ion induced membrane damage, both CpxAR and SoxRS stress responses are activated by high copper concentrations (Kershaw et al., 2005, Yamamoto and Ishihama, 2006). Of these, the Cpx response appears to be critically important for copper stress in E. coli. Double cpxAR deletion strains exhibit higher sensitivity to copper than their WT equivalent, and transcriptomic analysis of the Cpx response revealed CpxA/R dependent regulation of 27 transcriptional units following copper exposure (Yamamoto and Ishihama, 2006, Yamamoto and Ishihama, 2005).

Unlike E. coli, which has the cue- and cus- copper homeostatic systems,

Salmonella lack cusCFBA, encoding an RND-type copper-transporting efflux

concentrations than E. coli during anaerobic growth (Pontel and Soncini, 2009). Salmonella, and many other species that lack a functional cus- operon (i.e. Yersina and Erwinia) instead possess a novel copper binding protein, CueP (Pontel and Soncini, 2009). This periplasmic protein sequesters free intracellular copper ions to reduce toxicity and is required for

S. Typhimurium copper tolerance under anaerobic conditions (Osman et al.,

2010, Pontel and Soncini, 2009). The Cu+ inducible sensor/transcriptional regulator CueR regulates expression of the cueP gene (STM3650/SL3616) (Pontel and Soncini, 2009). However, transcriptome data presented in Chapter 3 of this thesis provided evidence to support further, positive regulation of cueP by CpxR. The Cpx system of Salmonella may therefore provide additional compensation for the absence of cus- through CueP activation, making this ESR system an even greater contributor to copper tolerance in Salmonella than E. coli.

S. Typhimurium also has a multi-copper oxidase, CueO (alias CuiD), which

converts Cu+ ions to the less reactive Cu2+. In the absence of CueO, S. Typhimurium is copper sensitive and exhibits significant attenuation in murine infection models (Achard et al., 2010). This attenuation is, however, confined to the liver and spleen, with no significant differences in ΔcueO or WT strains recovered from Peyer’s patches or mesenteric lymph nodes. S. Typhimurium ΔcueO strains therefore show some similarity to the phenotype of SPI-2 mutants, growth of which is confined to the Peyer’s patch (Cirillo et al., 1998).