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

Synchrotron FTIR spectroscopy reveals molecular changes of Escherichia coli upon Cu 2+ exposure 1 2 first-author 1 2 1 2 3 1 1 corresp Correspondence address: lujunhong@sinap.ac.cn Correspondence address: lujunhong@sinap.ac.cn lujunhong@sinap.ac.cn Division of Physical Biology and CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China Division of Physical Biology and CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China University of Chinese Academy of Science,Beijing100049, China University of Chinese Academy of Science,Beijing100049, China Shanghai Center for Bioinformation Technology, Shanghai Academy of Science &Technology; Center for Clinical and translational medicine, Shanghai Industrial Technology Institute, Shanghai 201203, China Shanghai Center for Bioinformation Technology, Shanghai Academy of Science &Technology; Center for Clinical and translational medicine, Shanghai Industrial Technology Institute, Shanghai 201203, China Copper ions (e.g. Cu 2+ ) 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 CuCl 2 -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 Cu 2+ -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 Cu 2+ exposure. This study demonstrated that FTIRspectroscopy combined with AFM can provide more comprehensive evaluation on the biochemical and mechanicalresponses of bacteria to copper. copper ions Antibacterial effect Escherichia coli SynchrotronFTIRspectroscopy Atomic force microscopy 1 Introduction 1 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 1 ]. However, the exact antibacterial mechanisms of copper are rather c omplex and not properly understood, althoughsome possible modes of action have been put forward[ 2 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 3 , 4 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 5 - 8 8 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 9 ]. In addition, the membrane as a key target for the antimicrobial action of copper had been proposed [ 5 5 , 10 10 - 13 13 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 14 ]. Recently,it has been widely used in microbiology, particularly applied to study the molecular changes of microorganisms inthe stress conditions[ 15 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 + , Zn 2+ , Fe 2 O 3 and TiO 2 ) were investigated by ATR-sFTIR[ 16 16 - 18 18 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 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 20 ]. 2 Materials and methods 1 2.1 Bacterial strain and growth conditions 2 E.coli DH5 α strain was inoculated into 3mL Luria-Bertani broth, incubated overnight at 37 o C with continuous shaking (120 rpm, Qiangle HYL-C) until stationary growth phase. This preculture was then diluted to a concentration of approximately 10 8 CFU/mL for the following experiments. 2.2 Copper ions solutions 2 CuCl 2 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 12 ]. 2.3 Exposure of E.coli to copper ions 2 After exposed to 6 mM CuCl 2 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 o C for 24 h. 2.4 Synchrotron FTIR spectroscopy of E.coli 2 FTIR spectroscopyexperiments were performed at the beamline BL01B1 of the Shanghai Synchrotron Radiation Facility (SSRF) as the following procedure: CuCl 2 -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 BaF 2 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 2 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 21 , 22 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 1 The survival curve of the E. coli exposed to copper ions ( Fig. 1 Fig. 1 ) indicated that 6 mM CuCl 2 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. A representative FTIR spectrum of E.coli in the region of 4000-800 cm -1 was displayed in Fig. 2 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 PO 2 asymmetric stretching of mainly nucleic acidsand phospholipids [ 19 19 ] while sugarsexhibit strong bands in this region due to C–O–C and P–O–C vibrations[ 23 23 ]. 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 2 ). As shown in Fig. 3 Fig. 3 and Table 1 Table 1 , Cu 2+ 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 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 Table 1 ), corresponding to the asymmetric CH 2 bending and the symmetric COO - stretching[ 24 24 - 27 27 24-27 ], meaning that the conformation or structure of lipids in the cells had changed[ 28 28 ]. These results suggested that Cu 2+ hadinfluences on the structure of lipid moleculesin the early period of exposure, providing direct evidence that membrane lipids were targets of copper ions[ 3 3 ]. While in the amide Iand amideIIregions (1760-1480 cm -1 ) ( Fig. 3b 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 29 - 32 32 29-32 ], had no dramatically shift ( p < 0.05) until 40 min exposure ( Table 1 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 33 ]. As seen from Table 1 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 25 ]. These changes suggested that the protein secondary structure was damaged in 40 min after exposed to CuCl 2 [ 34 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 PO 2 asymmetric and symmetric stretching, which was mainly from nucleic acids, the contribution fromphospholipids was negligible[ 24 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 30 , 35 35 - 37 37 35-37 ]. They all shifted to lower frequencies dramatically ( p < 0.05) after 40 min’s exposure ( Table 1 Table 1 ).The frequencies of PO 2 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 Co 2+ and Zn 2+ could induce similar effect[ 25 25 ]. Changes of symmetric CC stretching of ribose might be the results of a modification affecting ribose[ 28 28 ]. Peptidoglycanlayer was generally considered toplay a dominant role for ionic exchanging and could absorb a lot of counter-ionsfrom the aqueous environment[ 38 38 ]. It had also been reported that the conductivity of periplasmic space reduced in the present of copper ions[ 39 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 CuCl 2 could affect bacterial membrane lipids, proteins, DNA and peptidoglycan, supporting the hypothesis that the actions of copper ions are multifactorial. * The degree of significance was denoted as: p < 0.05. 1 1 Functional groups 1 1 Wavenumber (cm -1 ) 1 1   1 1   1 1   1 1   1 1 0 min (n=15) 1 1 20 min (n=15) 1 1 40 min (n=15) 1 1 60 min (n=15) 1 1 Amide I 1 1 1659.15±0.28 1 1 1659.25±0.32↑ 1 1 1660.42±0.9 *↑ 1 1 1659.99±0.74*↑ 1 1   1 1 1639.37±0.26 1 1 1639.22±0.40  ↓  1 1 1638.75±0.18*↓ 1 1 1638.55±0.22*↓ 1 1 Amide II 1 1 1546.77±0.57 1 1 1546.55±0.38↓ 1 1 1548.24±0.35*↑ 1 1 1547.92±0.52*↑ 1 1 CH 2 bending 1 1 1452.81±1.23 1 1 1454.47±0.40*↑ 1 1 1453.38±0.27↑ 1 1 1454.14±0.53*↑ 1 1 COO - stretching 1 1 1391.59±0.35 1 1 1392.78±0.82*↑ 1 1 1392.13±0.36*↑ 1 1 1392.69±0.76*↑ 1 1 PO 2 asymmetric stretching 1 1 1241.68±1.28 1 1 1241.16±1.04  ↓  1 1 1240.59±0.56* ↓ 1 1 1240.32±0.70*↓ 1 1 PO 2 symmetric stretching 1 1 1082.44±0.57 1 1 1082.15±0.49↓ 1 1 1082.05±0.36*↓ 1 1 1081.74±0.35*↓ 1 1 CC symmetric stretching 1 1 1119.68±0.65 1 1 1119.33±0.80↓ 1 1 1119.13±0.39*↓ 1 1 1118.88±0.70*↓ 1 1 C-O-C, P-O-P stretching 1 1 1059.27±0.76 1 1 1059.11±0.96↓ 1 1 1058.65±0.42*↓ 1 1 1057.71±0.79*↓ It should be noted that copper ions seemed to affect the lipid components in bacteria first. Upon CuCl 2 exposure, 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 Fig. 2 ), we assumedthe early killing events might be accompanied by the changes of lipids, then protein oxidization[ 40 40 ] and DNA degrade[ 11 11 ]. To further evaluate the effects of Cu 2+ on bacterial cells, high resolution imaging and mechanical measurement were carried out using PF-QNM mode AFM ( Fig. 4 Fig. 4 ). The results showed that untreated E.coli cells had a smooth and featureless surface morphology ( Fig. 4a, 4b Fig. 4a, 4b ) and the modulus varied from 0 to 120 MPa, with a peak value at 43.425 MPa( Fig. 4c 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. However, after exposed to 6 mM CuCl 2 , the surface morphology of E.coli changed seriously ( Fig. 5a and e 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 Fig. 6 , respectively. The results showed that the CuCl 2 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 CuCl 2 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 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. 4 Conclusion 1 This study demonstrated the effects of copper ions on the model bacterium E.coli using 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. 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DOI: 10.1073/pnas.0501207102 http://doi.org/10.1073/pnas.0501207102 Nanoscale visualization and characterization of Myxococcus xanthus cells with atomic force microscopy We thank the staff of BL01B beamline at National Center for Protein Sciences Shanghai and Shanghai Synchrotron Radiation Facility for assistance during data collection. 10.1007/s41365-016-0067-9 Supported by National Natural Science Foundation of China (No.11474298), Shanghai Pujiang Program (No.13PJ1410500), Special Funds for Enterprise Independent Innovation of Shanghai (CXY-2013-58) and Hundred Talents Program of the Chinese Academy Sciences. 2015-12-27 2016-03-28 2016-04-01 2016-05-09 2016-06-20 2021-04-11 © China Science Publishing & Media Ltd. (Science Press), Shanghai Institute of Applied Physics, the Chinese Academy of Sciences, ChineseNuclear Society and Springer Nature Singapore Pte Ltd. 2016 This is a post-peer-review, pre-copyedit version of an article published in Nuclear Science and Techniques. The final authenticated version is available online at: http://dx.doi.org/10.1007/s41365-016-0067-9 http://dx.doi.org/10.1007/s41365-016-0067-9 . 2016 Nuclear Science and Techniques 03 27