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Seminal ESR research was conducted in non-pathogenic, laboratory strains of E. coli and transcriptomic analysis into the Cpx regulon of pathogenic strains and other species are relatively new. Five studies have been published to date utilising NlpE overexpression as a Cpx inducing cue in two species. Three of these studies identified Cpx regulon members in the model

E. coli strain MC4100 and EPEC strain E2348/69 (Price and Raivio, 2009,

Vogt et al., 2010, Raivio et al., 2013) and the two additional studies focused on the role of Cpx in the coccobacilli bacterium Haemophilus ducreyi (Gangaiah et al., 2013, Labandeira-Rey et al., 2010), the causative agent of the sexually transmitted disease chancroid. In addition to these broad-scale transcriptomic studies, De Wulf et al. (2002) used promoter recognition sites to identify CpxR-P target operons, Yamamoto and Ishihama (2005) identified the copper-inducible promoters for Cpx and Bury-Mone et al. (2009) compared the global transcriptional responses of the five main ESRs in

Figure 8:Overview of the Cpx two-component signal transduction system, components and inducing cues (adapted from Vogt et al.,

2013). Under non-inducing conditions (left) the RR, CpxR, is unphosphorylated and “switched off” due to phosphatase activity of the HK, CpxA. The CpxA inhibitory protein CpxP, in the presence of inducing cues (indicated in red), is titrated away from CpxA and degraded by HtrA.

116 parallel, providing additional insights into the Cpx response. All three of these latter studies used E. coli K-12 derivatives as genetic backgrounds, likely chosen as the Cpx response, and its first regulon members, were originally characterised in MC4100 (Cosma et al., 1995, Danese and Silhavy, 1997). No large-scale or transcriptomic analyses of the Cpx regulon in any

Salmonella serovar has been published to date. All known Cpx regulon

members identified from the current literature are summarised in Appendix G, Table G1 (see in table citations for references). In addition to descriptions of these Cpx regulated genes and their functions, information regarding the presence of a CpxR-P binding site, the in vivo effect of CpxR-P binding and if

in vitro binding of CpxR-P to these sites has been experimentally proven,

have been included where possible (Table G1).

Induction of Cpx by NlpE overexpression was utilised in the work presented here to allow the first transcriptomic analysis and comparison of S. Typhimurium SL1344 WT and cpxR deletion strains, through the use of microarrays. Price and Raivio (2009) showed that deletion of cpxR alone does not offer a strong (i.e. greater than 2-fold) change in gene expression for the majority of proposed Cpx-regulated genes. Other Cpx inducing cues, such as alkaline pH (Danese and Silhavy, 1997) or overproduction of P-pilus components: PapE, PapG and BfpA, (Jones et al., 1997, Nevesinjac and Raivio, 2005), are vastly more general and would result in activation of other stress responses, for example the σE _{pathway. Although NlpE}

overexpression produces a reduced phenotype (relatively) when compared to constitutive expression of cpxA (cpxA*) there are many pleiotropic effects as a result of the cpxA* mutation. Phenotypes associated with cpxA* in E.

coli and Salmonella strains include an increase in tolerance to

aminoglycoside antibiotics (amikacin and kanamycin), hydroxyurea (Rainwater and Silverman, 1990, Thorbjarnardottir et al., 1978, Mahoney and Silhavy, 2013, Humphreys et al., 2004), alkaline pH (Danese and Silhavy, 1998), copper (De Wulf et al., 1999) and the bactericidal toxins colicins A and K (Plate, 1976). Despite these increased tolerances, CpxA* strains are also more sensitive to high temperatures (McEwen and Silverman, 1980)

and SDS (Cosma et al., 1995), and present abnormal FtsZ formation, cell cleavage (McEwen and Silverman, 1982, McEwen et al., 1983, Pogliano et al., 1998) and swarming in E. coli (De Wulf et al., 1999). Specific changes to the bacterial envelope when cpxA is constitutively expressed include deficiency of OmpF and Braun lipoprotein in the OM (McEwen and Silverman, 1982, McEwen et al., 1983). When grown on succinate, L-lactose and L-proline, cpxA* strains present reduced growth (Rainwater and Silverman, 1990, Plate and Suit, 1981), but they are able to utilise L-serine as a sole carbon source (Morris and Newman, 1980, Newman et al., 1982, Su et al., 1989).

Due to the specific nature of the NlpE inducing cue, it can be proposed that any effects resulting from NlpE overexpression are due to activation of the Cpx 2CST system and subsequent downstream effects resulting from this, rather than interference of NlpE with other ESR systems. In combination, these findings make NlpE overexpression the most desirable Cpx induction method available.

Despite the use of NlpE overexpression in investigating Cpx-regulated genes in both model and clinically relevant strains of E. coli (EPEC, UPEC) and H.

ducreyi), the importance of elucidating Salmonella specific Cpx regulated

genes and further enhancing our understanding of the Cpx 2CST system in this organism cannot be understated. Significant differences within the ESRs of Salmonella and other organisms have been identified during the

Salmonella ESR studies already completed. The most noteworthy of these

differences is σE (RpoE), the extracytoplasmic function (ECF) sigma factor that is essential in E. coli (De Las Penas et al., 1997) (1.1.7). A viable rpoE deletion strain is producible in Salmonella spp. but ΔrpoE strains exhibit severe attenuation in murine models (Humphreys et al., 1999, Crouch et al., 2005, Testerman et al., 2002). A Salmonella rpoE deletion mutant is defective for survival and proliferation within epithelial and macrophage cell lines and is therefore of significant importance during Salmonella infection and as a potential target for the development of typhoid fever vaccines

118 (Humphreys et al., 1999, Kenyon et al., 2002, Testerman et al., 2002). Several periplasmic chaperones belonging to the σE and Cpx regulons, originally identified in model E. coli strains, also have key contributions to

Salmonella pathogenicity (e.g. HtrA [DegP], Skp, SurA) (see 1.2 for Salmonella pathogenicity review).

Through the identification of novel Cpx regulated genes in S. Typhimurium one can add to the broader knowledge collected in distinct and related species, in addition to identifying Salmonella specific Cpx regulated genes. These specific genes could provide greater insights into how Salmonella spp. responds to environmental and envelope stresses, how this organism responds to stresses inflicted upon it from the host, and how it establishes infection. This work ultimately aims to reveal new genes important for

Salmonella survival and pathogenesis, and consequently potential targets for

the treatment and prevention of Salmonella spp. infections.

3.2 Aim

The aim of this chapter was to 1) define the CpxR regulon in Salmonella Typhimurium to broaden our understanding of the physiological importance of Cpx and 2) to identify Salmonella specific, CpxR regulated genes.