Synchrotron FTIR spectroscopy reveals molecular changes of Escherichia coli upon Cu2+exposure

NUCLEAR PHYSICS AND INTERDISCIPLINARY RESEARCH

Synchrotron FTIR spectroscopy reveals molecular changes of Escherichia coli upon Cu²⁺exposure

Xiao-Juan Hu，

Zhi-Xiao Liu，

Ya-Di Wang，

Xue-Ling Li，

Jun Hu，

Jun-Hong Lü

Nuclear Science and Techniques

Vol.27, No.3

Article number 56

Published in print 20 Jun 2016

Available online 09 May 2016

DOI：10.1007/s41365-016-0067-9

38700

Copper ions (e.g. Cu²⁺) have outstanding antibacterial properties, but the exact mechanism is rather complex and not fully understood. In this work, synchrotron Fourier transform infrared (FTIR) spectroscopy was used as an analytical tool to investigate the CuCl₂-induced biochemical changes in Escherichia coli. Our spectral measurements indicated that this technique issensitive enough to detect changes in membrane lipids,nucleic acids, peptidoglycans and proteins of Cu²⁺-treated bacteria. Interestingly, for short-timetreated cells, the effects on phospholipids composition were clearly shown while no significant alterationsof proteins, nucleic acids and peptidoglycans were found. Quantitative Nano-Mechanics (PF-QNM) mode atomic force microscopy (AFM) confirmedthe changes of the topography and mechanical properties of bacteria upon the Cu²⁺ exposure. This study demonstrated that FTIRspectroscopy combined with AFM can provide more comprehensive evaluation on the biochemical and mechanicalresponses of bacteria to copper.

copper ionsAntibacterial effectEscherichia coliSynchrotronFTIRspectroscopyAtomic force microscopy

1 Introduction

Copper，accredited to its antimicrobial properties，has been empirically utilized for more than 10,000 years and received renewed scientific interest in the last decades for its application potential in the modern healthcare setting[1]. However, the exact antibacterial mechanisms of copper are rather complex and not properly understood, althoughsome possible modes of action have been put forward[2].

Current biochemical and molecular evidence indicated that the action of copper ions on bacterial cells is multifactorial rather than aims at one target [3, 4]. Copper ions not only act as a catalyst to generate reactive oxygen species (ROS), which cause oxidative damage to proteins, lipids and nucleic acids [5-8], but also inactive proteins through replacing other metal ions binding sites on proteins or affect the metabolism by damaging Fe-S clusters in enzymes[9]. In addition, the membrane as a key target for the antimicrobial action of copper had been proposed [5, 10-13].Transmission electron microscopy (TEM) or AFM observation confirmed that copper can indeed induce the membrane surface damages.However, few studies wereperformed on the changes of cellular components and mechanical properties after bacteria were exposed to copper ions.

FTIR spectroscopy is a powerful tool to rapidly, non-destructively detect the chemical compositions of the biological samples[14]. Recently,it has been widely used in microbiology, particularly applied to study the molecular changes of microorganisms inthe stress conditions[15]. For instance, the responses of E. coli when subjected to heat, cold as well as ethanoland the interaction between bacteria and metals (e.g. Ag⁺, Zn²⁺, Fe₂O₃ and TiO₂) were investigated by ATR-sFTIR[16-18].

In this study, E. coli was chosen as the representative of Gram-negative bacterium to study the changes of cellular components along with time caused by copper ions through FTIR spectroscopy. To rule out the heterogeneity of bacterial cells among their biochemical compositions and their response to stress conditions[19], the average spectra of tens cells were measured and analyzed. In addition, the morphological and mechanical properties changes of E. coli induced by copper were simultaneously evaluated by using a novel mode of AFM based on PeakForce Quantitative Nano-Mechanics (PF-QNM)measurement [20].

2 Materials and methods

2.1 Bacterial strain and growth conditions

E.coli DH5 α strain was inoculated into 3mL Luria-Bertani broth, incubated overnight at 37 ^oC with continuous shaking (120 rpm, Qiangle HYL-C) until stationary growth phase. This preculture was then diluted to a concentration of approximately 10⁸ CFU/mL for the following experiments.

2.2 Copper ions solutions

CuCl₂ powder was dissolved into ultra-pure water (18.2 MΩ, USF-ELGA Maxima water purification system) to obtain a concentrated solution of copper ions (1 M) and then further diluted into the final concentration 6 mM[12].

2.3 Exposure of E.coli to copper ions

After exposed to 6 mM CuCl₂ for 0 min, 20 min, 40 min and 60 min, respectively, E. coli cells were collected by centrifugation (10,000 rpm, 1 min) and washed three times with sterile saline solution (0.9% NaCl). The bacterial number (CFU/mL) was counted after the harvested cells were plated on LB agar medium at 37 ^oC for 24 h.

2.4 Synchrotron FTIR spectroscopy of E.coli

FTIR spectroscopyexperiments were performed at the beamline BL01B1 of the Shanghai Synchrotron Radiation Facility (SSRF) as the following procedure: CuCl₂-exposed bacterial cells were harvested at 0 min, 20 min, 40min and 60min by centrifugation (10,000 rpm, 1 min) and washed five times to remove any medium. Prior to the FTIR measurement, one drop of harvested cells was deposited on the BaF₂ window and left to dry at room temperature. The absorbance spectra were collected by Nicolet 6700 FTIR spectrometer with Nicolet Continnum IR Microscope with an aperture of 20 × 20 μm. For each sample at different exposed time, 15 small clusters were measured within the range of 4000-800 cm^-1 with 256 co-added scans at 4 cm^-1 resolution at room temperature. Raw spectra were baseline corrected and smoothed by 13-point Savitzky-Golay method. The second-derivative was calculated to enhance the spectral resolution. Data were collected and analyzed by OMIC 9 (Thermo). To test the significance of the differences among the samples, Hypothesis Testing Two-Sample t-Test was performed on the groups. Analysis was made by OriginPro 8.

2.5 PeakForce Quantitative Nano-Mechanics (PF-QNM) measurements of bacterial cells

AFM measurements wereperformed at room temperature in deionized water using the PeakForce Quantitative Nano-Mechanics (PF-QNM) mode on a Bruker Multimode 8 SPM. Polyethyleneimine (PEI)-coated glasses, which provided positively chargesurfaces for bacterial adhesion, were used to immobilize cells [21, 22].V-shaped silicon nitride cantilevers with a spring constant of 0.35 N/m were used in all the measurement. All images were recorded at 256 pixels×256 pixels with an applied force of 6 nN, peak force frequency were set at 2 kHz and scan rate at 1 Hz. Image analysis was performed on NanoScopeoﬄine processing system.

3 Results and discussion

The survival curve of the E. coliexposed to copper ions (Fig. 1) indicated that 6 mM CuCl₂ has a significant effect on the bacterial survival.To investigate the changes of cellular components during this process, FTIR spectroscopy was performed on the cells with the exposure time 0 min, 20 min, 40 min and 60 min, respectively.

Fig. 1

E. coli survival curve following exposure to 6 mM CuCl₂ in LB medium.

A representative FTIR spectrum of E.coli in the region of 4000-800 cm^-1 was displayed in Fig. 2. According to the references, the regions 3000-2800 cm^-1 and 1480-1300 cm^-1 are assigned to fatty acids which mostly encountered in cell membranes. The regions 1760-1600 cm^-1 and 1600-1480 cm^-1 are representative of amide Iand amide IIbands, respectively. The amideIadsorption is mainly from C=O stretching of proteins, and amideIIadsorption is assigned to N-H bending vibration and C-N stretching vibration of proteins. The region from 1350 to 1000 cm^-1 represents PO₂ asymmetric stretching of mainly nucleic acidsand phospholipids [19] while sugarsexhibit strong bands in this region due to C–O–C and P–O–C vibrations[23].

Fig. 2

A representative FTIRabsorptionspectrum of E. coli in the region of 4000-800 cm^-1.

In our experiments, the changes of the spectra regions caused by copper treatment were analyzed. To eliminate the interference of baseline as well as the other background and to improve the sensitivity and resolution, 2^nd derivative spectra were used (Fig. S1, 2). As shown in Fig. 3 and Table 1, Cu²⁺ exposure caused kinds of shifts in the 2^nd derivative spectra. For example, in the fatty acid regions (3000-2800 cm^-1 and 1480-1340 cm^-1) (Fig. 3a), the wavenumbers 1,453 cm^-1 and 1,392 cm^-1 shifted to higher energies dramatically (p < 0.05) after 20 min’s exposure (Table 1), corresponding to the asymmetric CH₂ bending and the symmetric COO^-stretching[24-27], meaning that the conformation or structure of lipids in the cells had changed[28]. These results suggested that Cu²⁺hadinfluences on the structure of lipid moleculesin the early period of exposure, providing direct evidence that membrane lipids were targets of copper ions[3]. While in the amide Iand amideIIregions (1760-1480 cm^-1) (Fig. 3b), wavenumbers of 1,659, 1,639 and 1,547 cm^-1, corresponding to C=O stretching, N-H bending and C-N stretching of proteins[29-32], had no dramatically shift (p < 0.05) until 40 min exposure (Table 1).Generally, wavenumbers of 1,659 and 1,547 were assigned to α-helical conformation while wavenumber of 1,639 were attributed to β-sheet [33]. As seen from Table 1, copper treatment induced α-helical bands shift to higher frequencies. These shifts might result from the influences on H-bonding in amide groups and/or the metal binding with amide groups in cellular proteins[25]. These changes suggested that the protein secondary structure was damaged in 40 min after exposed to CuCl₂ [34]. In addition, in the 2^nd derivative spectra of nucleic acids and sugars regions (1350-1000 cm^-1) (Fig. 3c), wavenumbers of 1,242 and 1,082 cm^-1 were assigned to PO₂ asymmetric and symmetric stretching, which was mainly from nucleic acids, the contribution fromphospholipids was negligible[24]. Wavenumbers of 1,120 and 1,059 cm^-1 were assigned to symmetric CC stretching of ribose and symmetric C-O-C, P-O-C stretching of polysaccharides on capsule and peptidoglycan, respectively[30, 35-37]. They all shifted to lower frequencies dramatically (p < 0.05) after 40 min’s exposure (Table 1).The frequencies of PO₂ stretching of nucleic acids at 1,242 and 1082 cm^-1 were significantly lower; these changes could be the products of increased hydration of phosphate moieties, which may be due to metal binding. In previous study, it had been reported that Co²⁺ and Zn²⁺ could induce similar effect[25]. Changes of symmetric CC stretching of ribose might be the results of a modification affecting ribose[28]. Peptidoglycanlayer was generally considered toplay a dominant role for ionic exchanging and could absorb a lot of counter-ionsfrom the aqueous environment[38]. It had also been reported that the conductivity of periplasmic space reduced in the present of copper ions[39]. Our results showed that symmetric C-O-C, P-O-C stretching of peptidoglycan shifted to lower frequencies dramatically (p< 0.05), indicating an alteration in peptidoglycan structure. We supposed that the damage of peptidoglycan structure might play an important role in the conductivity reducing, which further resulted in the function disorder of E.coli. Overall, the FTIR results indicated that CuCl₂ could affect bacterial membrane lipids, proteins, DNA and peptidoglycan, supporting the hypothesis that the actions of copper ions are multifactorial.

The wavenumber values of the infrared bands of E. coli exposed to 6 mM CuCl₂ for 0 min, 20 min, 40 min and 60 min. The values are the mean ± S.D. for each group. Comparisons were done by Hypothesis Testing Two-Sample t-Test.

Functional groups	Wavenumber (cm^-1)
	0 min (n=15)	20 min (n=15)	40 min (n=15)	60 min (n=15)
Amide I	1659.15±0.28	1659.25±0.32↑	1660.42±0.9 *↑	1659.99±0.74*↑
	1639.37±0.26	1639.22±0.40 ↓	1638.75±0.18*↓	1638.55±0.22*↓
Amide II	1546.77±0.57	1546.55±0.38↓	1548.24±0.35*↑	1547.92±0.52*↑
CH₂ bending	1452.81±1.23	1454.47±0.40*↑	1453.38±0.27↑	1454.14±0.53*↑
COO^- stretching	1391.59±0.35	1392.78±0.82*↑	1392.13±0.36*↑	1392.69±0.76*↑
PO₂ asymmetric stretching	1241.68±1.28	1241.16±1.04 ↓	1240.59±0.56* ↓	1240.32±0.70*↓
PO₂ symmetric stretching	1082.44±0.57	1082.15±0.49↓	1082.05±0.36*↓	1081.74±0.35*↓
CC symmetric stretching	1119.68±0.65	1119.33±0.80↓	1119.13±0.39*↓	1118.88±0.70*↓
C-O-C, P-O-P stretching	1059.27±0.76	1059.11±0.96↓	1058.65±0.42*↓	1057.71±0.79*↓

* The degree of significance was denoted as: p < 0.05.

Fig. 3

2^nd derivative of FTIRspectra of E. coli exposed to 6 mM CuCl₂ for 0 min (1); 20 min (2); 40 min (3); and 60 min (4).The fatty acid regions (a) (3000-2800 cm^-1 and 1480-1340 cm^-1), the protein regions(b) (1760-1480 cm^-1) andthe nucleic acids and sugars regions (c) (1350-1000 cm^-1) were focused, respectively. The peaks were indicated by arrows.

It should be noted that copper ions seemed to affect the lipid components in bacteria first. Upon CuCl₂exposure, amide Iand amideII bands and peaks in nucleic acids as well as sugars regions didnot shift dramatically until 40 min, while the wavenumbers in lipid regions have already changed in 20 min. Based on the fact that a low rate of killing occurred in 20 min (Fig. 2), we assumedthe early killing events might be accompanied by the changes of lipids, then protein oxidization[40] and DNA degrade[11].

To further evaluate the effects of Cu²⁺ on bacterial cells, high resolution imaging and mechanical measurement were carried out using PF-QNM mode AFM (Fig. 4). The results showed that untreated E.coli cells had a smooth and featureless surface morphology (Fig. 4a, 4b) and the modulus varied from 0 to 120 MPa, with a peak value at 43.425 MPa(Fig. 4c).The adhesion forceand the stiffness value varied from 0 to 0.8 nN（peak value at 0.448 nN）and from 0.5 to 2.0 N.m^-1(peak value at 0.825 N.m^-1), respectively.

Fig. 4

AFM images of E. coli. (a) height image; (b) high-resolution Peak Force error image; (c) histogram of modulus; (d) histogram of adhesion; (e) histogram of stiffness. Histograms of c, d and e correspond to the zoom area of image b.

However, after exposed to 6 mM CuCl₂, the surface morphology of E.coli changed seriously (Fig. 5a and e), becoming uneven and exhibited protrusions along with time. The range of modulus, adhesion and stiffness of bacteria were shown in Fig. S3 and the peak values were shown in Fig. 6, respectively. The results showed that the CuCl₂ exposure obviously changed cells’modulus, adhesion force and stiffness. Both the modulus and stiffness of E.coli decreased along with time. We assumed that the cell softening after CuCl₂ exposure might be related to the changes of lipids as indicated in the FTIR results. Interestingly, the adhesion of cells surfaces increased first and then started to decrease after 20 min. Because the adhesion events were ascribed to the stretching of extracellular substances with the AFM tip[41], the adhesion force changes would be attributed to the changes of cell surface components. Although FTIR results showed that the lipids and polysaccharides have changed in the early period of exposure, whether and how the lipid changes resulted in the adhesion force increase and then polysaccharides led to adhesion force decrease still need more experiments.

Fig. 5

AFM height images (a, b, c, d) and high-resolution peak force error images (e, f, g, h) of E.coli exposed to 6 mM CuCl₂ for 0 min (a, e), 20 min (b, f), 40 min (c, g), 60 min (d, h).

Fig. 6

The peak values of mechanical properties of E.coli exposed to 6 mM CuCl₂at different time intervals. (a) Modulus; (b) adhesion; (c) stiffness. The error bars show the standard deviations of five cells in each condition.

4 Conclusion

This study demonstrated the effects of copper ions on the model bacterium E.coliusing synchrotron FTIRspectroscopy combined with PF-QNM mode AFM. FTIRspectroscopy revealed the changes of cellular components of bacteria induced by copper ions. The results provided evidences that copper ions targeted against several components of bacteria cells, including lipids, proteins, nucleic acids and peptidoglycans. Interestingly, spectral analysis further showed that the effects on phospholipids composition were clearly shown at the short-time treated cells while no significant alteration of proteins, nucleic acids and peptidoglycans were detected. Atomic force microscopy confirmedthe changes of that topography and mechanical properties upon the Cu²⁺ exposure. Thisstudy demonstratedthat the combination of FTIRspectroscopy and AFM images might provide more comprehensive technique to investigate the biochemical and mechanicalresponse of bacteria to copper.

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