Production of the human lipocalin and its homologues found in egg white

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Sequestration of bacterial siderophores by the lipocalin Ex-FABP

Ex-FABP was first discovered as a fatty-acid-binding protein with a role in hen-embryo development. Cancedda et al. (1988) were the first to report and identify Ex-FABP (Ch21) as a protein expressed and secreted by in vitro differentiating hen chondrocytes at a late stage of development. Ex-FABP was later shown to be a 21 kDa protein in cartilage (Cancedda et al., 1988), muscle tissue (Gentili et al., 1998) and granulocytes (Dozin et al., 1992) of chicken embryos. This protein was classified as a member of the superfamily of lipocalins and thus was considered to have a likely role in the transport of small hydrophobic molecules (Cancedda et al., 1990). The protein was renamed (from Ch21) ‘extracellular fatty acid-binding protein’ because of its ability to selectively bind and transport fatty acids (i.e. oleic, linoleic, and arachidonic acids) in extracellular fluids and serum (Cancedda et al., 1996). It was shown to be expressed during muscle-fibre formation (Gentili et al., 1998) and later shown to be involved in endochondral-bone formation (Cermelli et al., 2000, Gentili et al., 2005). Transfection of proliferating chondrocytes and myoblasts with an expression vector expressing antisense Ex-FABP cDNA led to a decreased cell viability. Therefore, Ex-FABP seems to play a part in cell differentiation and cell survival (Di Marco et al., 2003; Gentili et al., 2005). It was more recently shown that Ex-FABP binds the C16 and C18 isoforms of lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol-3-phosphate) (Correnti et al., 2011). LPAs are phospholipids mediating differentiation, inflammation, immune function, oxidative stress, cell migration, smooth muscle contraction, apoptosis and development (Zhao & Natarajan, 2014). It is likely that the functions of Ex-FABP reported above depend on its role in sensing or transporting phospholipids (Sia et al., 2013).
More recent reports indicate that Ex-FABP functions in pathogen defence through an ability to bind siderophores, in a manner analogous to that of LCN2 (Correnti et al., 2011; Garénaux et al., 2013). This suggests that Ex-FABP may have two distinct purposes, one in fatty acid/LPA binding and another as a siderophore-binding factor. Work of Correnti et al. (2011) shows that Ex-FABP sequesters ferric-enterobactin, as well as its mono-glucosylated (Fe-MGEnt) form, with an equilibrium dissociation constant (KD) of 0.22 and 0.07 nM, respectively, but not its di-glucosylated form (Fe-DGEnt; KD > 600 nM; Table 1.5). Furthermore, Ex-FABP at 4 μM caused growth inhibition of both E. coli and Bacillus subtilis under iron-limited in vitro conditions. Growth was restored by supplementing the cultures with stoichiometric amounts of FeCl3 (Correnti et al., 2011). Thus, Ex-FABP might act to reduce bacterial growth in EW by enhancing iron restriction. Ex-FABP did not inhibit Pseudomonas aeruginosa growth under iron limitation, which correlates with the observation that Ex-FABP does not bind the corresponding siderophores. Indeed, both enterobactin and bacillibactin produced by E. coli and B. subtilis (respectively) were found to be sequestrated by Ex-FABP (KD of 0.5 and 30 nM, respectively; Table 1.5), while pyochelin and pyoverdine produced by P. aeruginosa were not (Correnti et al., 2011). These findings are also in accordance with those from Garénaux et al. (2013) showing that E. coli K-12 is subject to a 105-fold growth reduction when exposed to 2.5 μM Ex-FABP or LCN2. However, when transformed with a plasmid harbouring the iroBCDEN cluster, no growth defect was observed with 2.5 μM Ex-FABP or LCN2. Exposure of six poultry APEC isolates to 2.5 μM Ex-FABP or LCN2 inhibited the growth of strains producing enterobactin as sole siderophore, but not those producing additional siderophores (salmochelin, aerobactin and/or yersiniabactin) (Garénaux et al., 2013). Therefore, it can be concluded that Ex-FABP is an avian siderocalin-type lipocalin with a function similar to that of LCN2.

Alpha1-ovoglycoprotein and Cal-γ – their potential functions in EW

In EW, α1-ovoglycoprotein has an average molecular weight of 30 kDa, an isoelectric point of 4.37–4.51 and a sugar content of about 25% (Matsunaga et al., 2004). While little is known about its function, this ovoglycoprotein is often used for its chiral properties to separate drug enantiomers (Sadakane et al., 2002; Haginaka & Takehira, 1997). However, its biochemical, functional and biological properties in EW remain unknown.
Cal-γ is a lipocalin-like prostaglandin synthase (PTGDS). In EW, two isoforms, of 22 kDa, can be separated by 2-DE thanks to their different isoelectric points (pI of 5.6 and 6.0) (Guérin-Dubiard et al., 2006). Pagano et al. (2003) have shown that Cal-γ expression correlates with endochondral bone formation and the inflammatory response. As for Ex-FABP, Cal-γ mRNA is increasingly synthesized during chondrocyte differentiation both in vivo and in vitro. Although Ex-FABP and Cal-γ may both play a part in bone formation and the inflammatory response, any possible role for Cal-γ in siderophore sequestration remains to be explored.

Approaches set in the PhD proposal

The antibacterial iron-restriction activity of EW, as mediated by oTf, is well established and it is now apparent that the EW lipocalin, Ex-FABP, can inhibit bacterial growth via an enhanced iron-restriction effect that is mediated by its siderophore-binding capacity (i.e. Fe-enterobactin sequestration). However, the siderophore-sequestering activity of Ex-FABP has neither been studied in EW, nor with appropriate S. Enteritidis or hen infection models. Furthermore, the concentration that this protein is found in EW remains unknown. As of yet, it is unclear whether the other lipocalins of EW (Cal-γ and α1-ovoglycoprotein) might also sequester siderophores. Although many EW proteins have been shown to be components of the arsenal of defence factors within EW, the contribution (if any) of the three lipocalins as new EW defence factors remains an open question. As matters stand, it is unclear whether the capacity of salmochelin to assist S. Enteritidis virulence in mammalian models can be extended to include support of S. Enteritidis survival or growth in EW (Figure 1.11). Thus, there remains much scope for further understanding of the role of lipocalin proteins in the defence of EW against bacteria and more research is required to understand all the components involved.

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Oligonucleotides used for polymerase chain reaction (PCR)

PCR primers used in an attempt to generate pET21a clones via the Gibson method (Thomas et al., 2015) are summarised in Table 2.3. PCR primers used to generate Salmonella deletion mutants and for post-deletion confirmation are summarised in Tables 2.4 and 2.5, respectively. The nucleotide sequences amplified can be found in Appendices 1, 2 and 3.

In vitro DNA procedures

General procedures were as previously described (Sambrook & Russell, 2001).

Plasmid purification

Plasmids were purified using the GeneJet plasmid miniprep kit (Thermo Scientific, Waltham, USA) according to the manufacturer’s guidelines. The purified products were eluted in 30 μL of MilliQ water and stored at -20 °C.

Agarose gel electrophoresis

DNA fragments were analysed by electrophoresis using 0.7% agarose gels containing a final concentration of 1X GelRed™ nucleic acid gel stain. Samples (2-4 μL) were mixed with 2 μL loading dye (6X ADD; Thermo Scientific) and 6 μL MilliQ water prior to loading onto the gel. The gel was then electrophoresed in 0.5X TBE buffer (0.4 M Tris, 0.4 M boric acid, 1 mM ethylenediaminetetraacetic acid (EDTA) pH 8) at 60 V for 70 min with a 1 kb ladder as a size marker (Thermo Scientific). To reveal DNA fragments, the gel was placed under ultraviolet (UV) light using a G:BOX (Syngene, Bangalore, India). Images taken were further analysed with GeneSys image capture software (Syngene).

Restriction digestion

A range of 60 to 100 ng of plasmid DNA was digested with 0.5 μL (10 units) fast digestion enzyme (NEB, Ipswich, USA), and 1X fast digest buffer in a final volume of 20 μL. The mix was incubated for 7 min at 37 °C for an optimal digestion. Enzymes were then inactivated by 10 min exposure at 70 °C and left on ice. The digestion product was analysed by agarose electrophoresis (2.2.2).

Clean-up of restriction digestion reactions

The GeneJETTM gel extraction kit (Thermo Scientific) was used according to the manufacturer’s guidelines. The purified products were eluted in 30 μL of MilliQ water and stored at -20 °C.

Polymerase Chain Reaction (PCR)

A Thermo Scientific Phusion Hot Start II DNA Polymerase kit was used for DNA amplification. For every reaction, 10 ng of DNA template were mixed with 1X Fusion HF buffer, 0.5 μM of forward and reverse primers, 200 μM of each dNTP, 3% DMSO, 0.02 U/μL Phusion Hot Start II DNA Polymerase in a final volume of 20 μL. Cycling instructions used were as follows: initial denaturation for 3 min at 98 °C, followed by 34 cycles (30 s denaturation at 98 °C, 30 s annealing at average Tm of primers minus 3 °C , 1 min extension at 72 °C), and then a final extension for 5 min at 72 °C.

Table of contents :

Chapter 1. Bibliographic study
1.1 Salmonella Enteritidis in eggs, a matter of public health
1.2 The powerful antibacterial defence mechanisms of egg white
1.3 Iron in the fight between Salmonella Enteritidis and egg white
1.4 Approaches set in the PhD proposal
Chapter 2. Material and methods
2.1 Reagents and chemicals
2.2 In vitro DNA procedures
2.3 Bacterial transformation
2.4 Protein procedures
2.5 Production of the human lipocalin and its homologues found in egg white
2.6 Measure of biomolecular interactions between lipocalins and siderophores
2.7 Genetic engineering techniques
2.8 Siderophore detection assay
2.9 Measurement of Salmonella Enteritidis growth dynamics
2.10 Statistical analysis
Chapter 3. Isolation of recombinant egg-white lipocalins
3.1 Bioinformatic analysis of lipocalin-2 homologues found in egg white
3.2 Overproduction of the lipocalin-2 and egg white lipocalins
3.3 Purification and characterisation of the overproduced lipocalins
3.4 Conclusion and future work
Chapter 4. Quantification of egg-white lipocalins and determination of their siderophore-binding activity
4.1 Egg white contains micro-Molar levels of all three lipocalins
4.2 Ex-FABP binds enterobactin with high affinity and strong preference for the ferrated form
4.3 Conclusion and future work
Chapter 5. Determination of the role of egg-white lipocalins in inhibiting siderophore-dependent iron sequestration by Salmonella Enteritidis
5.1 Library construction of mutants deficient in iron acquisition systems
5.2 Provision of Ex-FABP inhibits growth of a salmochelin-deficient Salmonella Enteritidis mutant in standard growth media
5.3 The ability to synthesise siderophore does not support Salmonella Enteritidis persistence in egg-white media
5.4 Ex-FABP antibacterial activity (via its siderophore-binding capacity) observed in standard growth media is not observed in egg-white media
5.5 Conclusion and future work
Chapter 6. General discussion
6.1 Salmonella Enteritidis metal acquisition in egg white
6.2 Could Ex-FABP be a component of the hen immune defence?
6.3 Conclusion and future work


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