Enhanced Silkworm Cecropin B Antimicrobial Activity against

May 2, 2019 - Both peptides showed a high antimicrobial and membranolytic activity against P. aeruginosa, with Q53 CecB displaying greater activity ...
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Article Cite This: ACS Infect. Dis. 2019, 5, 1200−1213

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Enhanced Silkworm Cecropin B Antimicrobial Activity against Pseudomonas aeruginosa from Single Amino Acid Variation Ottavia Romoli,†,¶ Shruti Mukherjee,‡,¶ Sk Abdul Mohid,‡ Arkajyoti Dutta,§ Aurora Montali,∥ Elisa Franzolin,† Daniel Brady,† Francesca Zito,⊥ Elisabetta Bergantino,† Chiara Rampazzo,† Gianluca Tettamanti,∥ Anirban Bhunia,*,‡ and Federica Sandrelli*,† †

Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, Italy Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), 700 054 Kolkata, India § Department of Chemistry, Bose Institute, 93/1 A P C Road, 700 009 Kolkata, India ∥ Department of Biotechnology and Life Sciences, University of Insubria, Via Jean Henry Dunant, 3, 21100 Varese, Italy ⊥ Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, Institut de Biologie Physico-Chimique, CNRS, UMR7099, University Paris Diderot, Sorbonne Paris Cité, Paris Sciences et Lettres Research University, F-75005 Paris, France

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‡

S Supporting Information *

ABSTRACT: Pseudomonas aeruginosa is an opportunistic bacterial pathogen causing severe infections in hospitalized and immunosuppressed patients, particularly individuals affected by cystic fibrosis. Several clinically isolated P. aeruginosa strains were found to be resistant to three or more antimicrobial classes indicating the importance of identifying new antimicrobials active against this pathogen. Here, we characterized the antimicrobial activity and the action mechanisms against P. aeruginosa of two natural isoforms of the antimicrobial peptide cecropin B, both isolated from the silkworm Bombyx mori. These cecropin B isoforms differ in a single amino acid substitution within the active portion of the peptide, so that the glutamic acid of the E53 CecB variant is replaced by a glutamine in the Q53 CecB isoform. Both peptides showed a high antimicrobial and membranolytic activity against P. aeruginosa, with Q53 CecB displaying greater activity compared with the E53 CecB isoform. Biophysical analyses, live-cell NMR, and moleculardynamic-simulation studies indicated that both peptides might act as membrane-interacting elements, which can disrupt outermembrane organization, facilitating their translocation toward the inner membrane of the bacterial cell. Our data also suggest that the amino acid variation of the Q53 CecB isoform represents a critical factor in stabilizing the hydrophobic segment that interacts with the bacterial membrane, determining the highest antimicrobial activity of the whole peptide. Its high stability to pH and temperature variations, tolerance to high salt concentrations, and low toxicity against human cells make Q53 CecB a promising candidate in the development of CecB-derived compounds against P. aeruginosa. KEYWORDS: antimicrobial peptides, cecropin B, Bombyx mori, NMR, Pseudomonas aeruginosa

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stomoxins, papiliocins, enbocins, and spodopsins isolated from different insect species and some synthetically derived variants.2−5 Structurally, Cec AMPs are linear, cationic peptides with variable lengths from 31 to 39 residues; they have random-coil structures in aqueous solutions but form amphipathic helical structures upon interaction with cell membranes.2,3 Within the Cec group, the CecB subtype is active against both Gram-positive and Gram-negative bacteria and displays antitumor properties.6−10 CecB antimicrobial activity is related to interactions with bacterial membranes and to pore

he extensive use and misuse of conventional antibiotics have led to the emergence of multidrug-resistant (MDR) pathogens, causing a dramatic health burden worldwide. As a result, many initiatives for the discovery and development of new drugs have been promoted.1 In the urgent need for new therapeutics, antimicrobial peptides (AMPs) are considered interesting candidates as potential alternatives to treat MDR bacterial infections.1 AMPs are molecules produced by virtually all organisms as effectors of the innate immune response, representing the first line of defense against infections. Insects possess numerous AMPs, which are classified on the basis of their amino acid (aa) sequences and structures.2,3 Among AMPs, the cecropin (Cec) group includes five subtypes (A−E) as well as other peptides indicated with different names, such as sarcotoxins, © 2019 American Chemical Society

Received: February 1, 2019 Published: May 2, 2019 1200

DOI: 10.1021/acsinfecdis.9b00042 ACS Infect. Dis. 2019, 5, 1200−1213

ACS Infectious Diseases

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Figure 1. Comparative activity of E53 CecB and Q53 CecB peptides against P. aeruginosa live cells, LUVs, and LPS bicelles mimicking bacterial membranes. (A) E53 CecB and Q53 CecB peptide sequences. The arrow indicates the polymorphic site. (B) Time-course of bactericidal activity for CecB isoforms on P. aeruginosa (mean percentage of dead cells ± SEM for two independent experiments), determined via flow-cytometry analysis. Blue bars: 4 μM E53 CecB, red bars: 2 μM Q53 CecB, red striped bars: 4 μM Q53 CecB. Q53 CecB showed significantly higher bactericidal activity compared with E53 CecB at 4 μM (two-way ANOVA: F2,18 = 6.107, p < 0.001). (C) Effects of the two CecB isoforms on a bacterial model membrane, determined via a CF-dye-leakage assay with a 3:1 POPE/POPG model membrane. (D) Membrane-destabilization properties of E53 and Q53 CecB peptides on P. aeruginosa LPS bicelles, determined via a DPH-fluorescence assay. Blue line: E53 CecB, red line: Q53 CecB, black line: negative control (DPH-supplemented bicelles only). (E) Kinetics of P. aeruginosa membrane permeabilization, measured via the uptake of PI fluorescent dye after the addition of CecB peptides. Blue line: E53 CecB, red line: Q53 CecB, black line: negative control. AU: arbitrary unit.

formation,11,12 although some studies have suggested that DNA might be an intracellular target.5 In addition, like other Cec AMPs, CecB molecules are amidated at their C-termini, and this modification is important for broadening and increasing the peptide’s antimicrobial activity.2,13,14 CecB was first identified in the giant moth Hyalophora cecropia in 1980,10,15 and since then, CecB peptides have been isolated from many other insects, including the silkworm Bombyx mori.16,17 In B. mori, CecB is produced as a 63-aa propeptide and matured via a proteolytic cleavage in a 35-aa active form (R27−I61).2,17,18 The silkworm CecB peptide has been proposed for several biotechnological applications, from the generation of disease-resistant crops19 to biomaterial functionalization20 and food preservation.5 At a genomic level, B. mori shows six cecB paralogue genes (cecB1−6) characterized by distinct nucleotide sequences but the same deduced amino acid sequence.4,18 We recently identified a European B. mori strain homozygous for a nonsynonymous substitution in the cecB6 gene.18 This modification seemed to explain the higher tolerance of the strain to infections with the pathogen Enterococcus mundtii. At a protein level, the nucleotide substitution determined an

amino acid variation in the active portion of the peptide, so that the glutamic acid (E) in position 53 was replaced with a glutamine (Q, Figure 1A). The E53Q substitution caused a modification in the peptide net charge from +6 to +7, with an increment in the predicted antimicrobial potential.18 Here, we provide a comparison of the antimicrobial activities and mechanisms of action of the two CecB6 isoforms generated by the E53Q modification, henceforth referred to as “E53 CecB” and “Q53 CecB”. It is important to note that the B. mori CecB1−6 peptides show the same deduced amino acidic sequence, but in other silkworm strains, the same modification might map to different cecB paralogues.4,18 We found that Q53 CecB was more active than E53 CecB against different bacteria. In particular, Q53 CecB showed higher antimicrobial activity against P. aeruginosa, an opportunistic pathogen that causes severe infections in hospitalized and immunosuppressed patients, particularly individuals affected by cystic fibrosis.21 We showed that both B. mori CecB isoforms mainly interacted with P. aeruginosa membranes, and DNA appeared to be less probable as a possible internal target. Additionally, the Q53 CecB isoform makes the peptide more 1201

DOI: 10.1021/acsinfecdis.9b00042 ACS Infect. Dis. 2019, 5, 1200−1213

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Table 1. E53 CecB and Q53 CecB Antimicrobial Activities against Different Microorganismsa E53 CecB

Q53 CecB

MIC (μM)

MIC (μM)

microorganism

pH 5

pH 7

pH 8

pH 10

MBC (μM)

pH 5

pH 7

pH 8

pH 10

MBC (μM)

E. coli ATCC 25922 S. aureus ATCC BAA-44 P. aeruginosa ATCC 27853 P. aeruginosa ATCC 25668 S. epidermidis ATCC 700565 C. albicans ATCC 1023

0.2 >20 4 4 10 >20

0.2 >20 4 4 10 >20

0.2 >20 4 4 10 >20

nd nd 4 4 nd nd

2.5 nd 20 20 25 nd

0.15 >20 2.2 2.2 8 >20

0.15 >20 2.2 2.2 8 >20

0.15 >20 2.2 2.2 8 >20

nd nd 2.2 2.2 nd nd

2.5 nd 11 11 20 nd

Bacteria were grown and tested in PCA-medium buffer at 30 °C. P. aeruginosa strains were also evaluated at 37 °C.

a

Bactericidal Activity of CecB Peptides against P. aeruginosa and Dye-Leakage Assay. We first compared the bactericidal-activity kinetics of the two CecB variants. P. aeruginosa cells were treated with E53 or Q53 CecB peptides at their respective MICs, and we determined the percentages of dead cells over time. Additionally, Q53 CecB was also evaluated at a concentration corresponding to the MIC of the E53 variant. At different time points, peptide-treated cells were incubated with propidium iodide (PI), a dye that binds to nucleic acids of dead cells, and SYTO 9, a dye that binds to nucleic acids from both intact and damaged cells, and the fluorescence of each sample was measured by flow-cytometry (Figure 1B). The percentage of cells positive for both dyes was considered representative of the percentage of dead cells in the sample.25 Untreated cells were analyzed in parallel and did not show any significant PI signal (data not shown). At their MICs, the two CecB isoforms showed a similar bactericidal kinetics against P. aeruginosa, with percentages of dead cells ranging from ∼15% after 10 min to ∼60% after 120 min of incubation (p = 0.28, not significant; Figure 1B). In contrast, the Q53 CecB isoform showed a significantly higher bactericidal effect on P. aeruginosa when evaluated at the MIC of E53 CecB, with dead-cell percentages ranging from ∼30% after 10 min to ∼70% after 120 min of incubation (p < 0.01, Figure 1B). We therefore investigated the impact of the two CecB isoforms on a model bacterial membrane, performing a 6carboxyfluorescein (CF)-dye-leakage assay on a 3:1 1palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) model membrane, which mimics the inner membrane of Gram-negative bacteria. 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) lipids were also used to model the outer leaflet of the mammalian membrane.26,27 The preparation of both 3:1 POPE/POPG and the POPC large unilamellar vesicles (LUVs) is described in the Supporting Information. Both CecB isoforms caused disruption of the model membrane as a consequence of peptide binding. Indeed, after 20 min of incubation, 5 μM E53 CecB caused ∼40% dye leakage, which reached ∼85% with a 25 μM concentration of the same peptide (Figure 1C). Furthermore, 5 μM Q53 CecB induced ∼97% dye leakage at the same incubation time (20 min), confirming the highest capability of the Q53 CecB variant to induce membrane disruption (Figure 1C). Because lipopolysaccharide (LPS) is a major constituent of the outer membrane of P. aeruginosa, we also compared the membrane destabilization properties of the two peptides on P. aeruginosa LPS bicelles. Increases in 1,6-diphenyl-1,3,5hexatriene (DPH) fluorescence in the presence of LPS bicelles upon peptide addition was taken as a measure of bicelle

attractive to the membrane and hence results in higher membranolytic activity. Our study also showed that Q53 CecB remained stable against pH variations, high-temperature treatments, and high salt concentrations but was sensitive to trypsin digestion. Furthermore, Q53 CecB exhibited low toxicity against human cells and maintained antimicrobial activity in 20% fetal-bovine serum, albeit at reduced levels. Therefore, we believe Q53 CecB represents a promising molecule for the development of CecB-derived compounds to treat Pseudomonas infections.

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RESULTS AND DISCUSSION Antimicrobial Activity of CecB Isoforms and the Effect of pH. Both B. mori CecB isoforms were effective against Escherichia coli, Staphylococcus epidermidis, and two strains of Pseudomonas aeruginosa (ATCC 27853 and ATCC 25668) but had weak antimicrobial activities against Staphylococcus aureus and Candida albicans (Table 1). Despite their similar antibacterial properties against E. coli, with minimuminhibitory-concentration (MIC) values at ∼0.2 μM for both peptides, Q53 CecB was more active than E53 CecB against S. epidermidis and P. aeruginosa, showing ∼20 and 50% lower MIC values, respectively (Table 1). Previous studies on B. mori CecB corresponding to the E53 variant detected MICs ranging from 0.63 to 2 μM for different E. coli strains and MICs of 0.63 and 8 μM for P. aeruginosa spp. but no activity against S. aureus.22,23 Higher antibacterial activities were reported for two chemically synthetized 37-aa CecXJ-37 variants (i.e., CecB forms carrying the E53 residue and with two additional amino acids at the C-terminus).5 These variations among laboratories are probably due to the use of different protocols or bacterial strains or the employment of CecB peptides variably amidated in their C-termini. In our study, the two C-terminus-amidated CecB variants were analyzed for antimicrobial activity simultaneously; therefore, their MIC values are fully comparable. Both isoforms showed unaltered antimicrobial activity against the different bacteria when evaluated at pHs 5 and 8 and against Pseudomonas strains at pH 10 (Table 1). The minimum-bactericidal-concentration (MBC) analysis indicated that Q53 CecB was more effective against S. epidermidis and P. aeruginosa compared with the E53 CecB variant (Table 1). In all the antimicrobial analyses, the two CecB peptides maintained the same differential activities against both P. aeruginosa strains. Given the clinical relevance of this opportunistic pathogen,21,24 using the ATCC 27853 strain, an extensive study was subsequently performed to understand the mechanisms of action of the two CecB isoforms against P. aeruginosa. 1202

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Figure 2. TEM micrograph of (A) control, (B,C) Q53 CecB treated, and (D,E) E53 CecB treated P. aeruginosa cells. In the bacteria incubated with 10 μM E53 CecB and 4 and 10 μM Q53 CecB, damage to the cell membranes and the release of cytoplasm contents are visible. The scale bars correspond to 1 μm (A) and 200 nm (B−E). Q53: Q53 CecB isoform, E53: E53 CecB isoform.

Table 2. Thermodynamic Parameters of E53 CecB and Q53 CecB Peptides in the Presence of LPSa peptide

n

KD (μM)

ΔH (kJ mol−1)

ΔS (J mol−1 K−1)

ΔG (kJ mol−1)

E53 CecB Q53 CecB

0.60 0.74

13.47 7.35

−81.34 ± 29.19 −51.81 ± 16.86

−179.6 −75.31

−27.82 −29.37

a

Conditions: 10 mM phosphate buffer with 150 mM NaCl (pH 7.4) at 298 K.

Interaction Study Using Fluorescence Spectroscopy and Isothermal-Titration Calorimetry. To clarify the interactions between CecB isoforms and P. aeruginosa cells, the intrinsic tryptophan (W) fluorescence of both peptides was monitored upon titration with increasing concentrations of bacterial suspensions or large unilamellar vesicles (LUVs). For both peptides, the emission maximum of W28 (the second residue in both E53 and Q53 CecB, Figure 1A) was ∼350 nm in aqueous solution. Upon stepwise addition of bacterial cells, the W28 emission maximum showed an enhancement in the fluorescence intensity and a shift toward the shorter wavelengths ∼329 and ∼334 nm for E53 CecB and Q53 CecB, respectively (Figure S1), suggesting the insertion of the W residues of both peptides into the hydrophobic environment of the cell membrane. Identical experiments were performed on bacterial cells in the absence of the peptide, and neither the increase in the fluorescence intensity nor the blue shift was observed (data not shown). However, blue shifts were also observed when both peptides were examined in the presence of 3:1 POPE/POPG LUVs (Figure S2). Possible effects of sample turbidity on the spectroscopic signal were checked by performing controlled measurements with increasing concentrations of LUVs in the absence of CecB peptides, which ruled out the presence of any relevant scattering-related artifacts (data not shown). We therefore calculated the bound fraction of each peptide in the 3:1 POPE/POPG LUVs (Figure S2). At 1× MICs, the E53 and Q53 CecB isoforms showed 67 and 98% membrane binding, respectively, reflecting the higher affinity of Q53 CecB toward the bacterial-membrane mimic (Figure S2B). In contrast, negligible dye leakage was observed with both CecB peptides in case of POPC LUV, which is eukaryotic-membrane model (Figure S3). Collectively, these data indicate that given their simplicity, liposomes are a good model for studying peptide association to bacterial lipids, because we obtained comparable results by evaluating the binding of both isoforms to live P. aeruginosa cells under similar experimental conditions.

disruption mediated by peptide anchoring and translocation.28−30 Both peptides exhibited time-dependent disruption of P. aeruginosa LPS bicelles. However, Q53 CecB showed an immediate increase in DPH fluorescence at 0.5× MIC (1.1 μM), which was equivalent to that caused by E53 CecB at 1× MIC (4 μM, data not shown). Figure 1D shows the linear increase in DPH fluorescence observed at 2× MIC for both peptides, with Q53 CecB (4.4 μM) displaying more LPS destabilization than the E53 isoform (8 μM). To confirm that both peptides disrupted bacterial-cellmembrane integrity, the kinetics of bacterial-membrane permeabilization was measured by evaluating the PI-dye uptake in P. aeruginosa cells in a 10 min time interval. Both peptides induced increases in PI fluorescence, reflecting cytoplasmic-membrane damage followed by interactions between PI and nucleic acids.31 Evaluated at the respective 1× MICs, Q53 CecB was more active and rapid in permeabilizing the inner membrane than the E53 CecB variant (Figure 1E). Further, P. aeruginosa cells were visualized using transmission electron microscopy (TEM) in the absence and presence of both peptides at 10 or 4 μM (corresponding to 2.5× and 1× MIC of the E53 CecB isoform, respectively). At 4 μM, Q53 CecB was able to damage bacterial membranes, whereas the E53 CecB isoform induced only a condensation of the cell content but not the dispersion of cytoplasm (Figure 2). In contrast, at 10 μM, both peptides could significantly damage the bacterial cells by leaking the cytoplasmic content (Figure 2). Taken together, these results suggest membranolytic activity for both CecB isoforms on the P. aeruginosa outer membrane, followed by permeabilization of the inner membrane, and the subsequent disintegration of both, causing leakage in cytoplasmic contents, as previously described for cationic amphiphilic AMPs.32 These data indicated that the membranedisruption activity of the Q53 CecB variant is more effective compared with that of the E53 CecB isoform. 1203

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Figure 3. CD spectra elucidating the secondary structures of (A) E53 CecB and (B) Q53 CecB when bound to live P. aeruginosa cells. In aqueous solution (black lines) both peptides adopted random-coil conformations, confirmed by the presence of strong negative minima at ∼200 nm. However, immediately after addition of P. aeruginosa cells, drastic peak shifts (green lines) were obtained, and after incubation of the samples for 2 h, peaks were observed at ∼208 and ∼225 nm (purple lines).

intensities of many proton resonances without any significant changes in chemical shifts, suggesting fast to intermediate exchanges between free and cell-bound CecB isoforms on the NMR time scale due to T2 relaxation. Q53 CecB isoform showed a higher rate of peak broadening across time compared with that of the E53 variant (Figures 4A,B and S5). In addition, a significant change was observed in the presence of cells for the indole-ring proton (NεH) of the W28 residue, which resonated at ∼10.16 ppm. A new set of NεH peaks appeared after a long incubation (∼8−10 h) of the peptides with the live cells (Figure 4A,B). These peaks did not appear in the absence of cells at the same time points, suggesting they were dependent on or catalyzed by the cell surface. Interestingly, the intensity of the new peak was more pronounced in the case of Q53 CecB than in the case of E53 CecB. Because no other new peaks appeared in the amide region of either peptide with cells, this data confirmed that such peaks were not the result of impurities or peptide degradation. Therefore, the new NεH peak had to arise from residues with high flexibility in the bound state, whose chemical shifts were different from those of their unbound state. These data were also consistent with a model of slow exchange of the entire peptide off the cell surface. Therefore, the chemical shift of the new peak was related to the average-time values for the on−off contacts with the cell. To identify the free and live P. aeruginosa cell-bound conformations of both peptides in solution, 2D 1H−1H totalcorrelation spectroscopy (TOCSY) and nuclear Overhausereffect spectroscopy (NOESY) were performed. The NOESY spectra of the free peptides were predominantly characterized by weak intraresidue NOEs and a few sequential NOEs (CαHi/ NHi+1) between backbone protons and side-chain protons, suggesting the free peptides were not adopting any stable conformations because of their flexible natures in aqueous solution (Figures S6 and S7). On the other hand, the cellbound transferred-NOESY (tr-NOESY) spectra of both peptides were characterized by the presence of more NOE cross-peaks, indicating the presence of cell-induced structural transitions in both peptides, from random-coil states to wellfolded conformations. However, severe signal overlap and the absence of unambiguous medium- and long-range tr-NOEs prevented us from determining the 3D bound conformations of the peptides. A few ambiguous cross peaks, such as F31 2H to I30/I34 CγH/CδH, were observed for Q53 CecB in the presence of cells, suggesting that the peptide could form

Next, isothermal-titration-calorimetry (ITC) experiments were done to determine the binding affinities of the CecB isoforms to LPS isolated from P. aeruginosa. Surprisingly, substantial differences were observed between the ΔH and ΔS values of the two CecB isoforms (Table 2 and Figure S4), giving a clear indication of the different modes of action employed by the two peptides. The ΔH and ΔS for the interactions of E53 and Q53 CecB peptides with LPS were found to be ΔH = −81.34 ± 29.19 and −51.81 ± 16.86 kJ mol−1 and ΔS = −179.6 and −75.31 J mol−1 K−1, respectively, suggesting that the reaction was enthalpy-driven and, hence, that van der Waals and hydrogen-bonding interactions may play a dominant role in the binding interaction.11 Apart from the thermodynamical parameters, the Q53 CecB−LPS binding reaction was more than twice as strong compared with the E53 CecB−LPS interaction, as evidenced from their KD values, 7.35 and 13.47 μM for Q53 and E53 CecB, respectively (Table 2 and Figure S4). Secondary Structures of CecB Isoforms When Bound to Live P. aeruginosa Cells. The circular-dichroism (CD) spectra of both peptides showed strong negative minima at ∼200 nm, implying random-coil structures for both isoforms in aqueous solution (Figure 3). For both peptides, CD spectra obtained immediately after mixing of the cell suspension with the peptide solution showed weaker signals when compared with those recorded after 2 h of incubation (Figure 3). Following subtraction of the CD spectra containing cells alone, peptide CD spectra showed two strong minima near ∼208 and ∼222 nm, indicating for both isoforms increases in helical propensity upon interaction with cells. These data suggest that the folding of these AMPs is a rather slow process; probably, both peptides first attach to the outer leaflets of bacterial cells and then fold into confined secondary structures, which in turn interact with the hydrophobic acyl chains of the bacterial membranes. NMR Studies of CecB Peptides in the Presence of Live P. aeruginosa Cells. To further understand the difference in biological activity for the E53 and Q53 CecB peptides, we chose to determine the bioactive conformations of both peptides in the cellular environment. 1D 1H NMR spectra of both isoforms showed distinct, sharp peaks in the aliphatic, aromatic, and amide regions (Figure S5) in aqueous environments. The addition of low concentrations of P. aeruginosa cells into the solutions of individual peptides resulted in concentration-dependent broadening and reduction in peak 1204

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Figure 4. STD NMR studies of the interactions of E53 CecB and Q53 CecB with P. aeruginosa cells. (A,B) 1D 1H NMR analysis of (A) E53 CecB and Q53 (B) CecB interactions with P. aeruginosa cells: (i) peptide alone and (ii,iii) peptide with increasing concentrations of bacteria. Dashed lines show the emergence of the second peaks in the W28 indole regions (NεH), indicating the presence of another conformation. (C,D) Reference and STD NMR spectra of the (C) E53 CecB and (D) Q53 CecB (1 mM) in the presence of P. aeruginosa cells (in 100% D2O, pH 6.5; stock: 108 cells/mL), showing nonexchangeable proton resonances. The light-blue stripes indicate the aromatic protons of F31. (E) E53 CecB (blue) and (F) Q53 CecB (red) models. Spheres of three different colors were used to reflect the vicinities of protons to the binding of live P. aeruginosa cells (white: 70−100%, yellow: 50−69%, green: