Metal complexation by histidine-rich peptides confers protective roles against cadmium stress in Escherichia coli as revealed by proteomics analysis

Bacterial strain and plasmids

E. coli strain TG1 (supE, hsdΔ5, thiΔ(lac-proAB), F’[traD36 proAB+ lacI^q lacZΔM15]) was used as host cell. To express cytoplasmic metal-binding motifs, a plasmid designated as pHis6GFPuv (Isarankura-Na-Ayudhya et al., 2005) that encodes a chimeric green fluorescent protein carrying hexahistidine was transformed and further expressed in host cells. Cells harboring pUC19 and pGFPuv (Clontech Laboratories, Mountain View, CA, USA) were used as controls. In parallel, a plasmid namely pEVZn (Mejare, Ljung & Bulow, 1998) that codes for the outer membrane protein A-polyhistidine fusion protein was expressed in order to generate cells expressing surface metal-binding motifs. For comparison, cells harboring pEV208 (Mejare, Ljung & Bulow, 1998) was used to over-express the outer membrane protein.

Growth patterns of cells in the presence of sub-lethal dose of cadmium ions

All of the aforementioned cells with the exception of E. coli host were grown in 5 ml Luria-Bertani (LB) broth (10 g/L tryptone, 5 g/L NaCl and 5 g/L yeast extract, pH 7.2) that is supplemented with 100 mg/L ampicillin at 37 °C, 150 rpm for overnight. The cultivation of the TG1 host was performed in a similar manner but without the addition of ampicillin. Then, 50 µl of overnight cultures were transferred into 5 ml broth and grown until OD₆₀₀ reached 0.5. Cells were adjusted to equal optical density at 0.05 in 5 ml LB broth supplemented with 100 mg/L ampicillin. Dose response assay was performed by addition of cadmium chloride stock solution to yield final concentrations of 0–1.0 mM. Cultures were further incubated at 37 °C, 150 rpm for 12 h. The growth rate was determined by monitoring the absorbance at 600 nm by spectrophotometer. It is noteworthy that the sub-lethal dose of 0.2 mM cadmium ions (exhibiting ∼50% growth inhibition of TG1 host) was selected as an effective dose for further experiments.

Two-dimensional gel electrophoresis (2-DE) of crude protein extracts

Overnight cultures of cells in 5 ml LB or LB/Amp were adjusted to OD₆₀₀ of 1.0. One milliliter of cell suspension was inoculated into 50 ml LB or LB/Amp. Cells were grown at 37 °C, 150 rpm for 3 h prior to addition of CdCl₂ to yield the final concentration of 0.2 mM. Cultivation was continued at 37 °C, 150 rpm for 15 h. Cells were collected by centrifugation at 4 °C, 6,000 rpm for 10 min. Crude protein extracts were prepared as previously described (Isarankura-Na-Ayudhya et al., 2010). The protein concentration was measured by Bradford’s method (Bio-Rad protein assay; Bio-Rad Laboratories, Hercules, CA, USA). These protein extracts were subjected to two-dimensional gel electrophoresis as follows. Three hundred micrograms of protein extract was mixed thoroughly with 250 µl of rehydration buffer (8 M urea, 4% CHAPS, 2 mM TBP, 0.001% bromphenol blue, 2.8 mg/ml dithiothreitol and 12 µl/ml destreak) containing 1% 3–10 IPG buffer and stored at room temperature for 10 min. Removal of insoluble material was further performed by spinning at 13,000 rpm for 10 min at 20 °C. The 13-cm IPG strips (pH range of 3–10) were placed in the IPGphor strip holder gel-side-down in rehydration solution containing sample proteins. These strips were then covered with mineral oil. Samples were run through steps of strip rehydration (30 V, 12 h) and isoelectric focusing (500 V for 1 h, 1,000 V for 1 h, and 8,000 V to reach 16,000 V h). The maximum current was maintained at 50 mA per strip. Once complete, the strip was equilibrated for two times (15 min each) in equilibration buffer (50 mM Tris pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.03% bromphenol blue) supplemented with 65 mM DTT and 135 mM iodoacetamide. The separation of protein in the second dimension was performed using Hoefer™ DALT on 12.5% SDS-polyacrylamide gels. Proteins were separated under applied voltage of 250 V, 10 mA per strip at 20 °C for 30 min following by 250 V, 20 mA per strip at 20 °C for 2.5 h until the bromphenol blue dye front reached 0.5 cm from the bottom of the gel. Gels were further stained with colloidal Coomassie brilliant blue G for overnight. Excess dye was removed by rinsing several times with deionized distilled water. Gels were scanned with the Canoscan LiDE20 scanner (Canon, Huntington, NY, USA). Intensity of protein spots was analyzed by the ImageJ software tool (Schneider, Rasband & Eliceiri, 2012).

Protein labeling and Two-Dimensional Difference in Gel Electrophoresis (2D-DIGE)

Peptide mass fingerprinting (PMF) analysis

Peptide mass fingerprinting (PMF) analysis of the protein separated by 2-DE was performed as previously described (Isarankura-Na-Ayudhya et al., 2010). The experiment was initiated by cutting spots of protein from gels and these spots were further transferred to 96-well microtitre plate and then soaked in 50% methanol and 5% acetic acid for overnight. In-gel digestion of protein was performed by the addition of sequencing grade of modified trypsin (Promega, Southampton, UK). Digested peptide fragments were extracted from gel segments on a Spot Handling Workstation (GE Healthcare, Chicago, IL, USA) using preset protocols from the manufacturer. Protein identification was further carried out using MALDI-TOF mass spectrometer (Model ReflexIV; Bruker Daltonics, Billerica, MA, USA) based on peptide fingerprint map. The tryptic digested peptide was mixed with a solution of 10 mg/ml α-cyano-4-hydroxycinnamic acid (LaserBio Labs, Sophia-Antipolis Cedex, France) in 66% acetonitrile and 0.1% trifluoroacetic acid (TFA) and further spotted onto a 96-well target plate. The acquisition of mass spectra was conducted in the positive ion reflector delayed extraction mode using approximately 200 laser shots. Creation of peak lists was performed using the XMASS software (Bruker Daltonics, FRG). These peaks were then queried using the MASCOT search engine (MatrixScience, http://www.matrixscience.com/) for protein identification. The reference database used in this study was NCBInr 20130802 (31350673 sequences; 10834990394 residues). The searching criteria were as follows: complete carbamidomethylation of cysteine and partial methionine oxidation; an initial mass tolerance of ±1.2 Da; the number of missed cleavage sites of up to 1. The accuracy of the experimental to theoretical pI as well as the molecular weight of proteins was carefully taken into consideration.

Protein network analysis

Identified proteins were queried using the STRING software version 9.05 (http://string-db.org/) in order to create functional protein association networks. This database weights and integrates direct (physical) and indirect (functional) associations from various sources, e.g., genetic context, high-throughput experiments, co-expression and previous knowledge.

Statistical analysis

For the 2D-DIGE gel analysis, only the spots with changes having an abundance ratio of 1.5 fold and P values < 0.05 (Student’s t-test) were marked and selected for further protein identification. For the PMF analysis, a search result score greater than 71 was considered to be of significant difference (p < 0.05).

Expression of green fluorescent protein of E. coli expressing cytoplasmic histidine-rich protein (chimeric His6GFP)

E. coli TG1 was applied as a host for the transformation of pUC19 (control plasmid), pGFPuv and pHis6GFPuv. Expression of the green fluorescent protein was confirmed by observing the bright greenish fluorescence of colonies of E. coli expressing native GFP or His6GFP under illumination with UV light (Figs. 1C and 1D). Meanwhile, no fluorescence was detected for TG1 host and TG1 carrying pUC19 (Figs. 1A and 1B).

Effect of cadmium stress on E. coli expressing cytoplasmic histidine-rich protein (chimeric His6GFP)

In the absence of cadmium ions, it seems that the TG1 carrying a control plasmid (Fig. 2A) growed up in the LB broth faster than the others. A long lag phase (∼3.5–4 h) could be detected in the cells expressing native GFP (Fig. 2B). The growth pattern of cells expressing His6GFP (Fig. 2C) resembled those in between the TG1 carrying pUC19 (Fig. 2A) and the native GFP-expressing cells (Fig. 2B).

In the presence of effective dose of cadmium, a growth arrest could be observed in cells expressing native GFP (Fig. 2B). However, this suppressing effect was more pronounced in cells carrying pUC19 (Fig. 2A). Interestingly, cells expressing the hexahistidine-GFP fusion protein displayed similar growth rates in the presence and absence of cadmium ions (Fig. 2C).

Proteomics profiling of E. coli expressing cytoplasmic histidine-rich protein (chimeric His6GFP)

Experimentation was initiated via the preparation of a reference map of the E. coli host proteome (Fig. 3). Approximately 200–250 spots of protein could be detected using a 13-cm IPG strip with pH range of 3–10. Some of these spots were picked up for further identification via peptide mass fingerprinting (http://www.matrixscience.com) as shown in Table 1. Most of the identified proteins pertained to energy metabolism, chaperones, heat shock proteins or stress proteins, transporters, protein synthesis machinery, and outer membrane proteins.

Further experiments were conducted to address the functional roles of cytoplasmic histidine-rich protein (His6GFP) on cellular adaptation against toxic cadmium ions. Therefore, protein expression profiles of E. coli expressing His6GFP grown in the absence or presence of 0.2 mM CdCl₂ (Fig. 4C) were compared with cells harboring pUC19 and cells expressing native GFP (as represented in Figs. 4A–4B, respectively). The effects of cadmium ions on the expression of various proteins were scrutinized as follows. Aconitase, an enzyme implicated in the Tricarboxylic acid (TCA) cycle, was down-regulated and disappeared in cells expressing His6GFP. Translation elongation factor-Ts was up-regulated in all cases (at 2.12, 1.84 and 1.35 fold for TG1/pUC19, TG1/pGFPuv and TG1/pHis6GFPuv, respectively) (Table 2). Glycerol kinase, an enzyme involved in glycerol uptake and lipolysis, was up-regulated in cells expressing His6GFP (+1.92 fold) and native GFP (+1.73 fold). Oligopeptide-binding protein was down-regulated at −1.65- and −1.34 fold for control cells and cells expressing His6GFP while up-regulation was found in the case of GFP (+1.37 fold). The H⁺-transporting ATPase was up-regulated at 2.69 and 1.30 fold in case of control cells and cells expressing His6GFP, respectively. More importantly, the presence of cytoplasmic histidine residue due to the expression of His6GFP was found to play imperative roles in stress defense mechanisms. Manganese-superoxide dismutase (MnSOD), an oxidative scavenging enzyme, was found to be down-regulated in cells expressing His6GFP (−1.65 fold). Alkyl hydroperoxide reductase, a thiol-specific antioxidant protein, was up-regulated in the case of native GFP-expressing cells (+1.71 fold). This is in contrast to those observed in the case of His6GFP (−1.10 fold). For membrane proteins, our results revealed the down-regulation of OmpC precursor (−1.15, −1.31 and −1.56 fold) and OmpF (−1.18 fold and absence) in all cases in response to cadmium ions.

Functional roles of histidine residue fused with outer membrane protein in E. coli

It has previously been proven that the presence of polyhistidine on the surface membrane of such engineered E. coli provided high binding affinity to zinc ions of up to 60 fold (Isarankura-Na-Ayudhya et al., 2005). Therefore, these cells were applied in the current study in order to reduce the cell permeability of cadmium ions. Figure 5 demonstrates the growth patterns of TG1/pEV208 and TG1/pEVZn in the presence and absence of cadmium ions. Our results revealed that the overexpression of the outer membrane protein of cells carrying plasmid pEV208 could protect the cell growth from the hazardous effect of cadmium ions. More importantly, the expression of polyhistidine-OmpA of cells harboring pEVZn could mediate a little bit higher degree of cell division than that of cells expressing the OmpA alone. Results from the proteomics analysis revealed that the enzyme glyceraldehyde-3-phosphate dehydrogenase form 1 was up-regulated in cells expressing OmpA (+2.69 fold) while the disapperance of this enzyme was observed in the case of cells expressing His-OmpA (Fig. 6 and Table 2). A diverse phenomenon was detected on the regulation of glyceraldehyde-3-phosphate dehydrogenase form 2 at −2.29 and +1.63 fold for TG1/OmpA and TG1/His-OmpA, respectively. Furthermore, H⁺-transporting ATPase was down-regulated in the case of the control (−2.64 fold) while no change was found in cells expressing His-OmpA. In addition, cadmium ions stimulated expression of MnSOD in cells expressing His-OmpA (+2.08 fold). It is noteworthy that an increased amount of the OmpC precursor was observed in cells expressing His-OmpA (+2.11 fold) as well as in cells expressing OmpA alone (+1.32 fold). Such a finding is in contrast to those observed in the case of cells expressing His6GFP and others as abovementioned.

Comparison of cadmium-induced protein alterations in engineered E. coli expressing cytoplasmic polyhistidine and polyhistidine- anchored surface membrane

Differentially expressed proteins of engineered E. coli upon exposure to cadmium ions were quantitatively identified as summarized in Table 2. For enzymes implicated in energy metabolism, glyceraldehyde-3-phosphate dehydrogenase form 1 was not detected in all cases except in cells expressing OmpA. The up-regulation of glyceraldehyde-3-phosphate dehydrogenase form 2 was found in TG1 carrying pUC19, cells expressing native GFP and cells expressing His-OmpA at 1.75, 1.65 and 1.63 fold, respectively. Aconitase was down-regulated in most cases. Meanwhile, the up-regulation of glycerol kinase was observed in the range of 1.3–1.9 fold in all cases except for the control. For the protein synthesis machinery, all cell types (with the exception of TG1 expressing OmpA) displayed approximately 1.3–2.1 fold increases. For transporters, oligopeptide-binding protein was decreased ∼1.3–1.6 fold only in the case of cells expressing His6GFP and control cells. Increased amounts of amino acid ABC transporter substrate-binding protein of 1.2–1.3 fold were found in TG1 expressing native GFP, His6GFP and His-OmpA while no change was observed in control cells. In regard to the H⁺-transporting ATPase, overexpressing of up to 2.7 fold was found in control cells. Up-regulation of approximately 1.9 fold of ATP synthase F1 (membrane-bound ATP synthase) was notified in cells expressing OmpA. For the stress defense mechanism, MnSOD is detected to be the most important antioxidative enzyme responsible for the toxic cadmium. Most cells enhanced the production of MnSOD in the range of 1.29–2.20 fold. Importantly, down-regulation of MnSOD (∼1.65 fold) was found only in cells expressing cytoplasmic polyhistidine. It should be noted that the up-regulation of AphC of up to 1.71 and 1.52 was found in TG1 expressing native GFP and cells expressing only outer membrane protein. For the ribosomal protein L9, up-regulation in the range of 1.3–2.1 fold was found in all cases with the exception of TG1 expressing His6GFP. Moreover, an increased expression of the OmpC precursor of approximately 1.3–2.1 fold as detected in cells expressing OmpA and His-OmpA. The disappearance of OmpF was observed in TG1 expressing native GFP, His6GFP and His-OmpA.

Confirmation of cadmium-induced protein alterations using two-dimensional difference in gel electrophoresis (2D-DIGE)

To further confirm the protective roles of metal complexation by histidine-rich peptides against cadmium stress, quantitative proteomics investigation using 2D-DIGE was employed as to provide high sensitivity and high discrimination power on protein expression. As shown in Fig. 7A, three different samples were individually labeled with three fluorescent dyes (Cy2, Cy3 and Cy5) and co-separated in one gel, which mediates spot matching and quantitation in a simpler and more accurate manner. An internal standard, as derived from a mixture of all samples pooled together and labeled with Cy2, was created to facilitate the normalization of each spot among all gels. This provides a reliable and reproducible detection by minimizing gel-to-gel variation and increasing experimental efficiency (Westermeier & Scheibe, 2008). Up- or down-regulation of proteins was compared via the detection of fluorescence intensities of Cy3 and Cy5 simultaneously at different wavelengths. Different expression of three key proteins (MnSOD, AhpC and Dps) involved in the stress defense mechanism was selected for further investigation (shown in Fig. 7B). Intracellular complexation of cadmium ions by His6GFP rendered cells to express minute amount of MnSOD (+1.65 fold) as compared to those of TG1 carrying pUC19 (+7.20 fold) and cells expressing native GFP (+3.08 fold). A similar observation was found with regards to the compensation of MnSOD expression by the His-OmpA (+3.31 fold) as compared to cells expressing only OmpA (+9.91 fold). The same phenomenon on the expression of AhpC in cells expressing native GFP and cells expressing OmpA was observed (Fig. 7B and Table 2). The high discriminative power of DIGE could quantify the up-regulation of Dps protein of up to 4.62, 1.82 and three fold in TG1 expressing native GFP, OmpA and His-OmpA, respectively. Moreover, the quantification of aconitase expression was obtained in a tangible manner. Down-regulation of aconitase of approximately 2.60 fold was detected in the case of TG1 expressing His-OmpA upon exposure to cadmium ions. Cadmium ions also induced the expression of ClpB of approximately 2.38 fold in TG1 expressing only OmpA. On the contrary, down-regulation of ClpB of up to 2.03 fold was observed when cells expressed polyhistidine on their surface membrane.

Interconnection between proteins implicated in cadmium stress

Analysis using the STRING software provided insights on the biological inference of proteins implicated in cadmium stress. As shown in Fig. 8, most of the important proteins as identified by 2-DE (Figs. 4, 6, 7 and Table 2) were mapped onto the protein network. MnSOD encoded by the sodA gene is known to be regulated by the SoxRS regulon (Fig. 9). Even though, alkyl hydroperoxide reductase (coded by AhpC gene) is under the control of the OxyR regulon, however, these two enzymes have some cross-functional roles and have also the same connection point as mediated by the trxA gene (coding for thioredoxin). In addition, these enzymes also function together with the F0F1 ATP synthase (an enzyme that regulates proton transportation across the membrane and ATP synthesis/hydrolysis) linked via the trxA gene. MnSOD was also found to share connectivity with aconitase enzyme (coded by AcnA and AcnB). Particularly, aconitases serve as protective buffers against oxidative stress. AcnA enhances the stability of the sodA transcript whereas AcnB lowers its stability (Han & Lee, 2006). The OxyR also plays an important role on the regulation of DNA starvation/stationary phase protection protein (Dps). The Dps unspecifically binds and protects DNA from oxidative damage mediated by hydrogen peroxide (Han & Lee, 2006). Moreover, ClpB, which is known as a stress-induced multi-chaperone system, was also found to possess linkages with these proteins and enzymes. Interconnections between these proteins/enzymes provide new knowledge on the cellular responses of bacteria in the presence of cadmium stress.