Enhanced Silkworm Cecropin B Antimicrobial Activity against

Subscriber access provided by UNIV OF SOUTHERN INDIANA

Article

A Single Amino Acid Variation in the Silkworm Cecropin B Enhances the Antimicrobial Activity Against Pseudomonas aeruginosa 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 ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00042 • Publication Date (Web): 02 May 2019 Downloaded from http://pubs.acs.org on May 5, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Infectious Diseases

A Single Amino Acid Variation in the Silkworm Cecropin B Enhances the Antimicrobial Activity Against Pseudomonas aeruginosa

Ottavia Romoli1,#, Shruti Mukherjee2,#, Sk Abdul Mohid2, Arkajyoti Dutta3, Aurora Montali4, Elisa Franzolin1, Daniel Brady1, Francesca Zito5, Elisabetta Bergantino1, Chiara Rampazzo1, Gianluca Tettamanti4, Anirban Bhunia2,*, Federica Sandrelli1,*

1Department

of Biology, University of Padova, via U. Bassi 58/B, 35131 Padova, Italy of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), 700 054 Kolkata, India 3Department of Chemistry, Bose Institute, 93/1 A P C Road, Kolkata 700 009, India 4Department of Biotechnology and Life Sciences, University of Insubria, Via J.H. Dunant, 3, 21100 Varese, Italy 5Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, Institut de Biologie PhysicoChimique, CNRS, UMR7099, University Paris Diderot, Sorbonne Paris Cité, Paris Sciences et Lettres Research University, F-75005 Paris, France 2Department

#Equally contributing authors *Corresponding authors: Anirban Bhunia: email address: [email protected] Federica Sandrelli: email address: [email protected]

1 ACS Paragon Plus Environment

ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 43

Pseudomonas aeruginosa is an opportunistic bacterium causing severe infections in hospitalised 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 characterised 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 a 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 to the E53 CecB isoform. Biophysical analyses, live-cell NMR and molecular dynamic simulation studies indicated that both peptides might act as membrane interacting elements, which can disrupt the outer membrane organisation, facilitating their translocation towards 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 stabilising the hydrophobic segment that interacts with the bacterial membrane, determining the highest antimicrobial activity of the whole peptide. High stability to pH and temperature variations, the tolerance to high salt concentrations, and the low toxicity against human cells make Q53 CecB a promising candidate in the development of CecB-derived compounds against P. aeruginosa.

Key words: Antimicrobial peptides, Cecropin B, Bombyx mori, NMR, Pseudomonas aeruginosa


Page 3 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The extensive use and misuse of conventional antibiotics have led to the emergence of multi-drugresistant (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 of 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 and represent the first-line of defence against infections. Insects possess numerous AMPs, which are classified based on their amino acid (aa) sequence and structure.

2,3

Among them, the group of Cecropins

(Cec) includes five subtypes (A-E), as well as other peptides indicated with different names, as Sarcotoxins, Stomoxins, Papiliocins, Enbocins and Spodopsins isolated from different insect species, and some syntheticderived variants. 2-5 Structurally, Cec AMPs are linear, cationic peptides with a variable length from 31 to 39 residues, with a random-coil structure in aqueous solution but form amphipathic helical structures upon interaction with cell membranes. 2,3 Within the Cec group, CecB subtypes are active against both Gram-positive and Gram-negative bacteria, and display antitumor properties. bacterial membranes and pore formation, intracellular target.

5

6-10

Their antimicrobial activity is related to interaction with

11,12

although some studies suggest they might have DNA as

In addition, as other Cec AMPs, CecB are amidated at the C-terminus and this

modification is important to broaden and increase the peptide antimicrobial activity. 2,13,14 CecB was first identified in the giant moth Hyalophora cecropia in 1980

9,15,

and since then CecB

peptides were isolated from many other insects, including the silkworm Bombyx mori. 16, 17 In B. mori, CecB is produced as a 63- aa pro-peptide 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 crops 19, to biomaterial functionalization 20 and food preservation. 5 At a genomic level, B. mori shows six cecB paralogue genes (cecB 1-6) characterised by distinct nucleotide sequences but the same deduced aa sequence. 4,18 We recently identified a European B. mori strain homozygous for a non-synonymous 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 aa variation in the active portion of the peptide, so that the 3 ACS Paragon Plus Environment


Page 4 of 43

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 activity and mechanism 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 B. mori CecB 1-6 peptides show the same deduced amino acidic sequence but in other silkworm strains the same modification might map in different cecB paralogues.

4,18

We found

that Q53 CecB was more active than E53 CecB against different bacteria. In particular, Q53 CecB showed a higher antimicrobial activity against P. aeruginosa, an opportunistic pathogen causing severe infections in hospitalized and immunosuppressed patients, particularly individuals affected by cystic fibrosis.

21

We

showed both B. mori CecB isoforms mainly interacted with P. aeruginosa membranes, while DNA appeared less probable as a possible internal target. Additionally, Q53 CecB isoform makes the peptide more vulnerable to membrane and hence helps 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 an antimicrobial activity in 20% fetal bovine serum, even if at reduced levels. Therefore, we believe Q53 CecB represents a promising molecule in the development of CecB-derived compounds to treat Pseudomonas infections.


Page 5 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 with weak antimicrobial activities against Staphylococcus aureus and Candida albicans (Table 1). Despite their similar antibacterial properties against E. coli, with minimum inhibitory concentration (MIC) values at ~0.2 M 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 M for different E. coli strains and MICs of 0.63 and 8 µM for P. aeruginosa sp., while no activity against S. aureus.

22,23

Higher antibacterial activities were

reported for two chemically-synthetised 37-aa CecXJ-37 variants, CecB forms carrying the E53 residue, with two additional aa in the C-terminus.5 These variations among laboratories are probably due to the use of different protocols, bacterial strains and/or the employement of CecB peptides variably amidated in the Cterminus. In our study, the two C-terminus amidated CecB variants were analysed for the antimicrobial activity simultaneously, therefore their MIC values are fully comparable. Both isoforms showed an unaltered antimicrobial activity against the different bacteria when evaluated at pH 5 and 8 and against Pseudomonas strains at pH 10 (Table 1). The minimal bactericidal concentration (MBC) analysis indicated that Q53 CecB was more effective against S. epidermidis and P. aeruginosa compared to the E53 CecB variant (Table 1). In all the antimicrobial analyses, the two CecB peptides maintained the same differential activity against both P. aeruginosa strains. Given the clinical relevance of this opportunistic pathogen,

21,24

an extensive study was subsequently performed to understand

the action mechanism of the two CecB isoforms against P. aeruginosa, using the ATCC 27853 strain.

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 MIC, determining the percentages of dead cells over the time. Additionally, Q53 CecB was also evaluated at a concentration corresponding to the MIC 5 ACS Paragon Plus Environment


Page 6 of 43

of E53 variant. At different time points, peptide-treated cells were incubated with propidium iodide (PI) (a dye binding to nucleic acids of dead cells) and SYTO 9 (a dye binding 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 to both dyes was considered representative of the percentage of dead cells in the sample.

25

Untreated cells were analysed in parallel and did not show any significant PI signal (data not

shown). At their MIC, 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, Q53 CecB isoform showed a significantly higher bactericidal effect on P. aeruginosa, when evaluated at the 1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 bacterial model membrane, performing a 6-carboxyfluorescein (CF) dye leakage assay on a 3:1 POPE/POPG (1-Palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl--sn-glycero-3-phosphoglycerol) which mimics the inner membrane of Gram-negative bacteria.

26,27

model

membrane,

The preparation of 3:1 POPE/POPG and

POPC large unilamellar vesicles (LUVs) is described in Supporting Information. Both CecB isoforms caused a disruption of the model membrane as a consequence of peptide binding. Indeed, after 20 min of incubation, 5 µM of E53 CecB caused a ~40 % dye leakage, which reached a ~85 % with 25 µM of the same peptide (Figure 1C). Furthermore, 5 µM of Q53 CecB induced a ~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). Since lipopolysaccharide (LPS) is a major constituent of the outer membrane of P. aeruginosa, we also compared the membrane destabilisation property of the two peptides on P. aeruginosa LPS bicelles. Increment of 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence in the presence of LPS bicelles upon peptide addition was taken as a measure of bicelle disruption mediated by peptide anchoring and translocation.

28-30

Both peptides exhibited a time-dependent disruption of P. aeruginosa LPS bicelles. However, Q53 CecB showed an immediate increment in DPH fluorescence at 0.5MIC (1.1 M), which was equivalent to that caused by 1MIC (4 M) of E53 CecB (data not shown). Figure 1D shows the linear increase in DPH fluorescence observed at 2MIC for both peptides, with Q53 CecB (4.4 M) displaying a higher LPS destabilisation than E53 isoform (8 M). 6 ACS Paragon Plus Environment

Page 7 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


To confirm that both peptides disrupted bacterial cell membrane integrity, the kinetics of bacterial membrane permeabilisation was measured 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 interaction between PI and nucleic acids. 31 Evaluated at the respective 1MICs, Q53 CecB was more active and rapid in permeabilising the inner membrane than E53 CecB variant (Figure 1E). Further, P. aeruginosa cells were visualised using transmission electron microscopy (TEM) in the absence or presence of both peptides at 10 or 4 µM (corresponding to E53 CecB isoform 2.5 and 1MIC, respectively). At 4 µM, Q53 CecB was able to damage bacterial membranes, while 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 a membranolytic activity of both CecB isoforms on the P. aeruginosa outer membrane, followed by permeabilisation of the inner membrane and the subsequent disintegration of both, causing a leakage in cytoplasmic contents, as previously described for cationic amphiphilic AMPs. 32 These data indicated that the membrane disruption activity of the Q53 CecB variant is more effective compared to that of the E53 CecB isoform.

Interaction study using fluorescence spectroscopy and isothermal titration calorimetry To clarify the interaction 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 (second residue either in E53 or Q53 CecB variants; Figure 1A) was ~350 nm in aqueous solution. Upon stepwise addition of bacterial cells, the W28 emission maximum showed an enhancement in fluorescence intensity and a shift toward the shorter wavelength of ~329 and ~334 nm for E53 CecB and CecB, respectively (Figure S1), suggesting the insertion of the W residue 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 fluorescence intensity nor 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 7 ACS Paragon Plus Environment


Page 8 of 43

(Figure S2). Possible effects of sample turbidity on the spectroscopic signal were checked performing controlled measurements with increasing concentration of LUVs in absence of CecB peptides, ruling out the presence of any relevant scattering-related artefacts (data not shown). We, therefore, calculated the bound fraction of each peptide into the 3:1 POPE/POPG LUVs (Figure S2). At 1MICs, the E53 and Q53 CecB isoforms showed a 67 % and 98% of membrane binding, respectively, reflecting the highest affinity of Q53 CecB towards the bacterial membrane mimic (Figure S2B). In contrast, negligible dye leakage was observed by both the CecB peptides in case of POPC LUV, which mimic the eurokaryotic model membrane (Figure S3). Collectively, these data indicate that, given their simplicity, liposomes are a good model to study peptide association to bacterial lipids, since we obtained comparable results evaluating the binding of both isoforms to live P. aeruginosa cells under similar experimental conditions. Next, isothermal titration calorimetry (ITC) experiments were done to determine the binding affinity of CecB isoforms into LPS isolated from P. aeruginosa. Surprisingly, substantial differences were observed between the ΔH and ΔS values of the two CecB isoforms (Table 2; Figure S4), giving a clear indication of a different mechanism/mode of action employed by the two peptides. The ΔH and ΔS for E53 and Q53 CecB peptides with LPS interaction were found to be -81.34 ± 29.19 kJ mol-1, -179.6 J mol-1 K-1 and -51.81 ± 16.86 kJ/mol, -75.31 J mol-1 K-1, respectively, suggesting that the reaction was enthalpy driven and hence, the van der Waal, as well as hydrogen-bonding interactions may play a dominant role in the binding interaction.11 Apart from the thermodynamical parameters, Q53 CecB-LPS binding reaction was more than twice as strong compared to the E53 CecB-LPS interaction, as evident from their KD values, 7.35 M and 13.47 M for Q53- and E53 CecB, respectively (Table 2; Figure S4).

Secondary structure of CecB isoforms when bound to live P. aeruginosa cells The circular dichroism (CD) spectra of both peptides showed a strong negative minimum at ~200nm, implying a random coil structure for both isoforms in aqueous solution (Figure 3). For both peptides, CD spectra obtained immediately after mixing the cell suspension with the peptide solution showed weaker signals when compared to those recorded after 2 h of incubation (Figure 3). Following subtraction of the CD spectra containing cell alone, peptide CD spectra showed two strong minima near ~208 and ~222 nm, indicating for both isoforms an increase in helical propensity upon interaction with cells. These data suggest 8 ACS Paragon Plus Environment

Page 9 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


that the folding of these AMPs is a rather slow process; probably both peptides first attach to the outer leaflet of bacterial cells and then fold into a confined secondary structure, which in turn interacts with the hydrophobic acyl chains of the bacterial membrane.

NMR studies of CecB peptides in the presence of live P. aeruginosa cells To further understand the difference in biological activity for E53 and Q53 CecB peptides, we chose to determine the bioactive conformation of both peptides in the cellular environment. One-dimensional 1H NMR spectrum of both isoforms showed distinct sharp peaks in aliphatic, aromatic and amide regions (Figure S5) in an aqueous environment. Addition of low concentrations of P. aeruginosa cells into the solutions of individual peptides resulted in concentration-dependent broadening and reduction in peak intensities of many proton resonances without any significant change in chemical shifts, suggesting a fast to intermediate exchange between free and cell-bound CecB isoforms at NMR time scale due to T2 relaxation. Q53 CecB isoform showed a higher rate in peak broadening across time compared to the E53 variant (Figures 4A, B and S5). In addition, a significant change was observed for the indole ring proton (NH) of W28 residue, resonating at ~10.16 ppm, in the presence of cells. A new set of NH peaks appeared after long incubation (~8-10 h) of the peptides with the live cells (Figures 4A, B). These peaks did not appear from the peptides in the absence of cells at the same time points, suggesting they were dependent or catalysed by the cell surface. Interestingly, the intensity of the new peak was more pronounced in the case of Q53 than E53 CecB isoform. Since no other new peaks appeared in the amide region of both peptides in the context of cells, this data confirmed that such peaks were not the result of impurities or peptide degradation. Therefore, the new NH peak had to arise from residues with high flexibility in the bound state, whose chemical shift was different from that of its 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, two-dimensional 1H–1H total correlation spectroscopy (TOCSY) and nuclear overhauser effect spectroscopy (NOESY) were performed. The NOESY spectra of free peptides were predominantly characterised by weak intra-residue and few sequential NOEs (CαHi/NHi+1) between backbone proton and side chain proton, 9 ACS Paragon Plus Environment


Page 10 of 43

suggesting free peptides were not adopting any stable conformations due to their flexible nature in aqueous solution (Figures S6 and S7). On the other hand, the cell-bound transferred-NOESY (tr-NOESY) spectrum of both peptides was characterised by the presence of more NOE cross-peaks, indicating the presence of cell induced structural transition in both peptides, from random coil state to well-folded conformation. However, severe signal overlap and absence of unambiguous medium- and long-range tr-NOEs prevented to determine the three-dimensional bound conformation of the peptides. A few ambiguous cross peaks such as F31 2H to I30/I34 CγHs/CδHs were observed for Q53 CecB in the context of a cell, suggesting that the peptide could form a hydrophobic cluster after binding to a cell membrane, which in turn was responsible for the membranolytic activity for both peptides. The lack of NOEs between NεH proton of W28 and other residues indicated that the residue W28 was highly flexible.

Interactions between CecB isoforms and P. aeruginosa cells by Saturation-Transfer Difference -NMR Next, saturation transfer difference (STD) NMR experiments were performed to identify the residues of ligand molecules that are in close proximity to the high molecular weight receptors. Figures 4C and D respectively show the reference 1D 1H NMR spectrum of E53 and Q53 CecB isoforms in the presence of P. aeruginosa cells and the corresponding STD proton NMR spectrum, obtained after selective saturation of live cells. The STD NMR spectrum of both peptides clearly showed many proton resonances with different intensities indicating differential proximity between residues of CecB isoforms from live microbial cells. Figures 4E and F show that the maximum (100%) STD effects were observed for the CγH and CδH protons of I34/I45/I52 and F31 residues of Q53 and E53 CecB peptides, respectively. The aromatic protons (2H/6H) of F31 of Q53 CecB have also shown a 95% STD effect. This suggests that the side-chain aromatic proton of residue F31 of both peptides and the methyl protons of I34/I45/I52 of Q53 CecB are in close vicinity with bacterial cells. Relatively high STD effect could be unambiguously determined for the CβH of M37 which showed ~81% STD effect. Interestingly, STD effects appeared to be relatively low for all other residues in E53 CecB peptide. Also, all the alkyl side-chain groups of residues I34/I41/I45/I52 and V46/V54, resonating at ∼0.88 ppm accounted for a combined STD effect of ~70% for Q53 CecB and ~55% for E53 CecB isoforms, respectively (Table S1). This data suggests that the major hydrophobic residues of Q53 CecB are effectively bound on the surface of 10 ACS Paragon Plus Environment

Page 11 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


P. aeruginosa cells whereas E53 CecB shows a weaker interaction during docking onto the cell surface, in agreement with the higher membranolytic activity of the Q53 CecB isoform.

Comparison between the structures of CecB isoforms in the presence of model membrane through MD simulations The interaction between an AMP and lipid molecules is crucial to study the dynamics of the peptide within the membrane. Additionally, understanding the mechanism responsible for the interaction will provide greater insights into the mode of action for the two CecB isoforms. Most helical AMPs are known to be unstructured in aqueous media and acquire a helical form after membrane binding. 33 The presence of the membrane interface induces helicity in AMPs, which in turn influences the extent of binding to and perturbation of the membrane.

34

From the simulation trajectory it has been observed that the C-terminal

helix of E53 CecB started unfolding upon dissociation from the phosphate head groups due to charge repulsion (Figures 5 and S8). In contrast, the N-terminal helix is amphipathic and interacted with the membrane through electrostatic interaction between phosphate head groups of the outer lipid layer and positively charged residues like R27/K29/K32/K33/K36/R39 of CecB-E53. The substitution of E53 with a polar but uncharged residue helped to stabilize the conformation of the hydrophobic segment (A48GPAIQVLGSAKAI61) in the Q53 CecB isoform. The presence of both hydrogen bond donor (NH2) and acceptor (C=O) in Q53 side chain helped in forming inter-molecular (or inter-residue) hydrogen bonds which stabilised the helix-loop-helix structure as well as increased the membrane interacting surface area of the Q53 CecB peptide. Plot of Root Mean Square Deviation (RMSD) against simulation timescale (Figure 5C) clearly show that Q53 maintains its structure in the model membrane during the simulation. The higher fluctuation rate for E53 is mainly at the C-terminal region of helix-loop-helix structure. Additionally, it has been previously hypothesized that there is a threshold hydrophobicity to attain optimal antimicrobial activity; i.e., a decreased peptide hydrophobicity reduces the antimicrobial activity; in contrast, increasing peptide hydrophobicity to a certain extent will improve the antimicrobial activity.

32

The weaker antimicrobial

activity of E53 CecB compared to that of Q53 CecB is attributable to the lower overall hydrophobicity and helical propensity of E53 CecB in the membrane environment.



Page 12 of 43

Disruption of membrane integrity upon peptide binding The three-dimensional structure of CecB peptides could not be determined in live cells or in LPS, we, therefore, focused towards characterising the action mechanism of AMPs and membrane integrity upon peptide interaction using accelerated molecular dynamics (aMD) simulation (Table S2), an approach highly valuable to understand the action mechanism of AMPs at a molecular level. To this end, it is essential to study and characterise the various modulating effects on lipids caused by the interaction with the peptides. In this context, area per lipid, and membrane thickness are two fundamental parameters, which describe the distortion of membranes upon peptide interaction. Close inspection suggests that the average area per lipid of anionic POPG lipid moieties in 3:1 POPE/POPG model membrane increased up to 63.95Å in the presence of the Q53 CecB isoform, while the same parameter increased to 61.51 Å from 59.52 Å 35 in the presence of E53 CecB peptide (Figures 6A, B). This fluctuation was comparatively more elevated in the presence of Q53 CecB, spanning a range of approximately 25 Å, throughout the simulation timescale. These data also confirms the modification from the negatively charged E53 residue to the polar Q53 showed a substantial impact on the area per lipid of 3:1 POPE/POPG model membrane. Overall, the variations in the average area per lipid shed light on the increased permeability of the membrane, which is attributed to the presence of the AMP (Figures 6A, B). Additionally, membrane thickness, measured as a “phosphate-to-phosphate” distance, is another aggregated property, which is influenced by the acyl chain length, the tilt angle and the degree of unsaturation of its lipid components. Our data could distinctly identify a positive curvature induced local thinning of the membrane upon interaction with E53 or Q53 CecB isoforms. A heterogeneous 3:1 POPE/POPG bilayer exhibited an average membrane thickness of 41.65Å, which decreased in the presence of both CecB variants suggesting that the interaction between the cationic AMP and lipid head groups might be responsible for the thinning effect (Figure 6E). Peptide insertion into the lipid bilayer causes several structural and dynamical perturbations including a change in orientation and mobility of the C-H bond, measured by characterising the lipid order. Chapman et al. explained that biological membranes are extremely heterogeneous and display phase transition, with two different phases affecting the fluidity and permeability of the membrane, thereby changing its functions.

36

Thus, the order parameter (-SCD) helps in gaining insights into the state of the 12 ACS Paragon Plus Environment

Page 13 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


system, which correlates with its function and with the higher value corresponding to enhanced lipid order. Both the Cec B variants invoked a decaying pattern for -SCD (Figures 6C, D), with E53 CecB maintaining a more ordered orientation in a 3:1 POPE/POPG bilayer when compared to Q53 CecB, as expected by its lower interaction properties. This clearly indicates a strong association of Q53 CecB peptide with the membrane due to the interaction with the anionic POPG lipid moieties. Interestingly, the portion of the lipid chain attached to the phosphate head group maintained an ordered orientation, as suggested by the higher SCD values of the carbon atoms present in that region. Furthermore, the membrane insertion profile of E53 and Q53 CecB variants was probed by comparing the Solvent Accessible Surface Area (SASA) through the simulation timescale, calculated using visual molecular dynamics (VMD) (https://www.ks.uiuc.edu/Research/vmd/). Close inspection of the SASA for the two peptides depicted that Q53 CecB appeared more buried into the 3:1 POPE/POPG model membrane system and less exposed to the solvent than the E53 CecB variant (Figure S9). Collectively, the experimental results in conjunction with theoretical calculation substantially imply that the Q53 CecB variant exceeded the hydrophobic threshold for membrane insertion.

CecB isoforms did not target P. aeruginosa intracellular DNA in vivo In vitro studies suggested that in addition to membrane disrupting effects, silkworm CecB might also have DNA as a possible intracellular target.5 Therefore, we analysed whether the different antimicrobial activity of the two CecB isoforms could also be due to a diverse activity on nucleic acids. The in vitro DNA or RNA binding properties of both peptides were evaluated via a gel retardation assay. The Q53 CecB isoform interacted more efficiently with DNA compared to E53 CecB peptide, since it was found to halve the DNA band intensity at a DNA/peptide ratio of 2:1, while for E53 CecB isoform this reduction was observed at a 3.5:1 ratio (Figure S10A). Conversely, the two isoforms interacted similarly with RNA, since they halved the RNA band intensity at an RNA/peptide ratio of 1:1 (Figure S10B). To determine whether the highest Q53 CecB DNA affinity resulted in an inhibition of DNA transcription, we analysed the amount of RNA synthesised in vitro by the RNA polymerase holoenzyme in the presence of both peptides. In general, the amount of transcribed RNA decreased with higher CecB concentrations (Figure S10C), indicating that both peptides possessed an in vitro transcription inhibitory 13 ACS Paragon Plus Environment


Page 14 of 43

activity. However, E53 CecB isoform had a half maximal inhibitory concentration (IC50) of 175 µM, while Q53 CecB had an almost 10-fold lower IC50 value (18 µM), indicating a higher inhibition of transcription in vitro. To test whether the in vitro differences might contribute to the higher antimicrobial activity of Q53 CecB against P. aeruginosa in vivo, we performed peptide localisation experiments evaluating CF-labelled peptides using confocal microscopy. Bacterial cells were incubated with different concentrations of both labelled isoforms, and counterstained with DAPI (to detect DNA) and Nile Red (lipid/membrane staining). 37 CecB peptides showed a similar staining profile with Nile Red, and a weak or absent co-localisation with DAPI, suggesting that both variants mainly interacted with P. aeruginosa membranes (Figure 7). Similar indications were obtained when the peptide localisations were evaluated in presence of E. coli, and S. epidermidis (Figure S11), suggesting that at least with these three bacteria the interaction occurs mainly at the level of cell membrane. These data suggest that membranolytic activity is the primary mechanism of action for both CecB isoforms, although we might not exclude DNA as a target in other microorganisms. Several studies suggested in fact that the same AMP might possess different targets, when tested against different pathogens. 38,39

Towards biomedical applications: cytotoxic and haemolytic activity, effect of cations, serum and trypsin treatment on the anti-Pseudomonas activity of CecB isoforms CecB isoforms could represent good candidates for possible biomedical applications. We therefore investigated the cytotoxicity of both peptides against HeLa and human lung fibroblasts (CCD-Lu34) cell lines, and their haemolytic activity against human erythrocytes. Neither peptide exhibited significant cytotoxic or haemolytic effects at concentrations up to 200 µM (Figure 8A-C). P. aeruginosa is the most common pathogen causing pulmonary infections in cystic fibrosis patients, where airway surface fluids reach saline concentrations of 130 mM.

40

The antimicrobial activities of CecB

peptides were tested against both P. aeruginosa strains with increasing NaCl concentrations. Both isoforms showed a MIC greater than 6 µM at ≥ 300mM NaCl (Table S3). However, differently from E53, Q53 CecB isoform maintained its MIC at 2.2 µM, when evaluated up to 200 mM NaCl (Table S3). 14 ACS Paragon Plus Environment

Page 15 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The divalent cations Ca2+ and Mg2+ respectively show 1-2 and 0.5 mM concentrations in human saliva, and might inhibit AMP activity.

41,42

In 2 mM CaCl2, both isoforms retained an anti-Pseudomonas

activity, although at higher concentrations (8 and 6 M for E53 and Q53 CecB, respectively; Table S3). In contrast, in 1 mM MgCl2, both peptides maintained their MICs at 4 and 2.2 µM for E53 and Q53 CecB variants, respectively (Table S3). Next, we evaluated whether the peptides were effective in complex biological fluids, testing their activity in 20% fetal bovine serum (FBS). In these conditions, CecB peptides were active against P. aeruginosa, although both isoforms showed MICs 2.5-fold higher compared to those observed without FBS (Table S3). Other AMPs, proposed as possible antimicrobial compounds, showed an increment in effective concentrations, when tested in the presence of CaCl2 or serum, such as the human-derived MUC7 12-mer and frog-derived Esculentin peptides.42,43 Moreover, regarding CecB isoforms, the observed increments were relatively low considering their non-significant toxicity effects in human cells at concentrations up to 200 µM (Figure 8). AMPs proposed for biomedical applications should be thermostable to withstand high temperature treatments 44. Therefore, we pretreated CecB peptides at 100 °C for 5, 20 and 30 min and tested their activity against P. aeruginosa, showing no variations in the MIC values compared to non-heat-treated controls (Table S4). Finally, CecB isoforms were assessed for their resistance to degradation by trypsin. Following a 2h pretreatment of CecB with trypsin both peptides lost their anti-Pseudomonas activity when evaluated up to 22 µM, corresponding to the E53 CecB MBC (Table S4), and indicating CecB isoforms are targets for trypsin degradation. In similar conditions, sensitivity to trypsin was also detected for the CecXJ-37 variants, which however showed no or lower sensitivity to pepsin digestion. 5 Protease degradation represents one of the main limitations in the use of AMPs as antimicrobial drugs and several natural AMPs, as the human cathelicidin LL-37, frog Esculentin or H. cecropia CecB displayed similar sensitivities.45-47 However, the modification/deletion of specific aa or the substitution of some L-residues with their D-enantiomers within the peptide have been shown to overcome this drawback. 45-47 Similar strategies might be, therefore, applied to develop Q53 CecB-derived compounds active against Pseudomonas. 15 ACS Paragon Plus Environment


Page 16 of 43

CONCLUSIONS Structure-activity-relationship investigations are routinely performed to characterise AMPmembrane interactions, and their associated antimicrobial functions. However, relatively few studies can be found in the literature examining how specific peptide properties affect peptide-live cell/membrane interactions and their subsequent antimicrobial effects. Here, we analysed the antimicrobial activity and mechanism of action of E53 and Q53 CecB, two silkworm AMP natural variants, differing in only one amino acid residue. Our analyses suggest that the Q53 CecB isoform is a promising candidate for the development of Q53 CecB-derived anti-Pseudomonas drugs. Further studies are also required to determine the efficacy of this peptide on P. aeruginosa biofilms, alone or in combination with other AMPs or conventional antibiotics 48, and to develop suitable delivery systems for future applications in biomedicine.


Page 17 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


MATERIALS AND METHODS Peptide synthesis The CecB isoforms (Figure 1A) and their CF-labelled variants were synthesised in the Peptide Core Facility of the CRIBI (Biotechnology Centre, University of Padova, Italy), by solid phase peptide synthesis method using a multiple peptide synthesizer (SyroII, MultiSynTech GmbH). Labelled peptides were prepared incorporating 5,6-carboxyfluorescein during the last step of the synthesis. Peptide molecular masses were confirmed by MALDI TOF-TOF mass spectrometer (Model 4800–Applied Biosystems). The purity of the peptides was ≥ 95% as evaluated by analytical reverse phase HPLC. Each peptide carried a C-terminal amidation. The E/Q variation is at position 53 in the pre-peptide and position 27 in the active peptide. 1 mM stock solutions of each peptide were prepared by weighing the peptide either in autoclaved water or in 10 mM phosphate buffer (pH 7.4). P. aeruginosa serotype 10 LPS was obtained from Sigma-Aldrich (USA), while POPG [1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)], POPE (1palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), were from Avanti Polar Lipids (USA) and of > 99% purity. All other chemicals used were of analytical grade.

Microbial strains and culture conditions The following strains were used: E. coli ATCC 25922, S. aureus ATCC BAA-44, S. epidermidis ATCC 700565, P. aeruginosa ATCC 27853 and ATCC 25668. The reference P. aeruginosa ATCC 27853 strain was employed in all the analyses, while the clinically isolated ATCC 25668 was used for antimicrobial assays only. Bacterial strains were grown at 30 °C in Plate Count Agar (PCA) medium. P. aeruginosa strains were also cultured at 37 °C; C. albicans ATCC 10231 was grown at 35 °C in Sabouraud broth (pH 5.8).

MIC and MBC tests For each peptide, serial dilutions were prepared in ultrapure water. 10 µl of peptide solutions were tested in triplicate at final concetrations from 0.15 to 22 µM against 90 µl of microbial culture (5105 CFU/ml initial concentration) on sterile polypropylene 96-well plates (Costar). Microbial suspensions in growing medium (positive control) or with 50 µg/ml Ampicillin (negative control) were followed in parallel. 17 ACS Paragon Plus Environment


Page 18 of 43

Plates were incubated 24 h at 30 °C (35 °C for C. albicans), 50 rpm. P. aeruginosa strains were also tested at 37 °C. Delta optical density at 600 nm (ΔOD600) was calculated subtracting the OD600 recorded at time 0 to that obtained after 24 h. The MIC was considered the lowest concentration determining a ΔOD600 equal to 0 ± 0.05. To establish MBC, dilutions of each peptide (from 2.2 to 40 µM) were incubated in triplicate with 200 µl of microbial culture 24 h at 30 °C (30 and 37 °C for P. aeruginosa). Samples were plated on PCA and colonies were counted after 24-48 h. MBC was considered the peptide concentration able to inhibit 99.9% of the bacterial growth. Effects on CecB activity of (i) pH, (ii) NaCl, (iii) CaCl2, (iv) MgCl2, and (v) 20% FBS were investigated performing MIC tests with (i) bacterial cultures grown in PCA medium buffered at pH 5, 8 or 10; (ii-iv) in the presence of 100, 200, 300, 400, 500 mM NaCl,or 2mM CaCl2, or 1mM MgCl2; (v) adding inactivated FBS (Gibco, ThermoFisher Scientific, USA) at a final concentration of 20%. To evaluate CecB stability to high temperature, 600 M CecB solutions were pretreated at 100 °C for 0, 5, 10 and 30 min. MICs were determined as described above. Sensitivity to trypsin digestion was performed incubating each peptide (600 µM final concentration) with trypsin (Sigma-Aldrich, USA) 2h at 37 °C in 100 mM Tris-HCl pH8 (peptide-to-enzyme ratio 20:1), followed by 10 min at 100°C to inactivate trypsin. Trypsin-treated peptides were tested against P. aureuginosa as described above.

Flow cytometry P. aeruginosa cells at mid-exponential growth phase were diluted to 5106 CFU/ml in 0.9% NaCl and treated with E53 CecB(4 µM) or Q53 CecB (2.2. or 4 µM). At specific time points (0, 5, 10, 15, 30, 120 min), 100 µl of treated bacteria were mixed with 100 µl of 1.9 mM PI (Sigma-Aldrich, USA) and 0.5 mM SYTO 9 (Thermo Fisher Scientific, USA) in 0.9% NaCl, and incubated at dark for at least 15 min. Live and dead P. aeruginosa cells were prepared in parallel and stained with single and both dyes as controls. Data were acquired with a BD FACS Canto II cytometer (BD Biosciences, USA) using a 488 nm emitting laser and analysed with the FACSDiva software (BD Biosciences, USA). The experiment was performed twice.

TEM analysis


Page 19 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


P. aeruginosa cells at mid-exponential growth phase were diluted to 5107 CFU/ml in the salt-free Luria-Bertani (LB) medium. Bacteria were treated 30 min at 30 °C with each peptide at 4 or 10 µM. Negative controls were prepared in parallel. Cell pellets were obtained from 2 ml suspensions and fixed in 2% glutaraldehyde and HEPES 0.05 M, pH 7.4 for 4 h at room temperature. Samples were post-fixed 30 min in 1% osmium tetroxide in HEPES 0.05 M, dehydrated in an ethanol series and embedded in an Epon/Araldite 812 mixture. Thin sections were stained with lead citrate and uranyl acetate and observed with a JEM-1010 transmission electron microscope (Jeol, Japan).

Fluorescence spectroscopy Overnight-cultured P. aeruginosa cells were resuspended in 10 mM sodium phosphate buffer, pH 6.5 at a final cell concentration of 108 CFU/ml and kept on ice to prevent any change in the cell density. LUVs (please see Supporting Information for a detailed protocol) were prepared mimicking either bacterial or eukaryotic membrane in similar buffer with a final concentration of 2.5M. The intrinsic W fluorescence of the peptide was monitored upon titration with increasing concentration of P. aeruginosa cells and LUVs, separately. The initial concentrations of E53 and Q53 peptides were 4 and 2.2 M respectively in 10 mM phosphate buffer, pH 6.5. After each addition, samples were incubated a considerable amount of time, and fluorescence spectra were repeatedly recorded until no further changes in fluorescence intensity were observed. Fluorescence experiments were performed at 25°C using a Hitachi F-7000 FL spectrometer with a 0.1 cm path length quartz cuvette, using an excitation wavelength 280 nm and a 300-800 nm emission range. Excitation and emission slits were set to 2.5 nm. The bound fraction was calculated using phenomenological hill equation (Supporting Information).

Dye Leakage, Inner membrane permeabilisation, LPS Bicelle disruption assays Detailed descriptions are reported in Supporting information.

ITC analysis 19 ACS Paragon Plus Environment


Page 20 of 43

ITC analysis was performed using a TA-affinity ITC (TA Instruments, New Castle, DE). All chemicals were dissolved in 10 mM PBS, pH 7.4, which were filtered and degassed before use. A cell sample (182 μL) containing 0.025 mM P. aeruginosa LPS were titrated against 0.25 mM of each peptide at 298 K. Of note, the LPS was used above its critical micelle concentration (~1.3 M).49 A total of 20 injections were performed at an interval of 3 min with 2 μl of peptide per injection and a 75 rpm stirring speed. Raw data were plotted using NanoAnalyze 3.7.5 software. Each plot was fitted using a nonlinear equation, and an independent binding site model was employed to analyse the thermodynamic parameters. Dissociation constant (KD), change in the heat of enthalpy of reaction (ΔH), free energy of binding (ΔG) and entropy (ΔS) were evaluated using the equations ΔG = RT ln KD and ΔG = ΔH - TΔS, respectively.

CD spectroscopy A JASCO 815 spectrometer was used for CD spectroscopy experiments. P. aeruginosa cells were collected, washed and resuspended in 10 mM phosphate buffer, pH 7.4. 12-48 µl of cells were added (stock: 108 CFU/ml) to peptide solutions (300 µl), and CD spectra were recorded using a 1 cm quartz cell and a 260190 nm measurement range, 100 nm/min scanning speed, 2 nm bandwidth, 4 s response time and 1.0 nm data pitch after 2h incubation at 25 ºC. Similarly, the CD spectra for P. aeruginosa-only cells were measured keeping concentration and all other parameters constant. The spectrum for P. aeruginosa-only cell was subtracted from the cell and peptide mixture to interpret the change in secondary structure of the peptide in presence of live P. aeruginosa cells. Peptide concentration was 25µM in 10 mM phosphate buffer, pH 7.4. The variation in peptide secondary structure with time was also monitored by titrating increasing concentration of P. aeruginosa LPS (12.5 -50 µM).

NMR spectroscopy NMR experiments were performed at 298K either on Bruker AVANCE III 500 MHz (with SMART probe) or 700 MHz NMR spectrometer (with cryoprobe). Each peptide was prepared in 10 mM phosphate buffer, pH 6.5 containing 90 % water and 10% D2O. Trimethylsilylpropanoic acid (TSP) was used as an internal chemical shift standard. A series of one-dimensional (1D) 1H proton NMR spectra for both the E53 and Q53 CecB isoforms (0.5 mM) in free solution as well as in the presence of P. aeruginosa cells were 20 ACS Paragon Plus Environment

Page 21 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


recorded with an excitation-sculpting scheme for water suppression. A series of titrations were carried out with living P. aeruginosa cells resuspended in a phosphate buffer of similar pH until broadening of the 1D spectra of the peptide was observed. Two-dimensional TOCSY (80 ms mixing time, 16 scans) and NOESY experiments (100 and 150 ms mixing times with 32 scans) were set up with a 12 ppm spectral width in both dimensions. For live cell STD experiments, 0.5 mM peptide solution was prepared in 10 mM deuterated phosphate buffer (pH* 6.5). A suspension of P. aeruginosa cells (108 CFU/ml final concentration) was prepared in the same buffer. The on – and off– resonance frequencies were -1 ppm and 40 ppm, respectively. An STD experiment was run in three sets viz. (i) peptide alone; (ii) cells alone; and (iii) peptide in the presence of cells. Total saturation time was 2 s (40 Gaussian-shaped pulses of 49 ms and 1 ms delay between the pulses); 512 and 1024 scans were maintained for reference and STD spectra, respectively.

MD study The homology modelling of E53 or Q53 CecB was performed using I-Tasser web server

50

keeping NMR

structure of papiliocin isolated from the swallowtail butterfly, Papilio xuthus as a template (PDB acquisition code: 2la2.pdb). Next, we prepared the peptide-embedded membrane system using CHARMM-GUI server. The membrane geometry was fixed at 50 Å along both the X and Y-axis. A rectangular box with a water (TIP3P) thickness of ~20 Å on both sides of the lipid bilayer leaflet was used.51 Sodium ions were added to neutralise the system. The 3:1 POPE/POPG lipid moieties in both upper and lower leaflets were used for MD simulation. The detailed MD protocol is described in Supporting Information.

DNA/RNA binding assay and fluorescence-based in vitro transcription inhibition assay Detailed descriptions are reported in Supporting Information.

Confocal microscopy Experiments were performed as in

37

with minor modifications, using increasing concentrations of

CF-labelled CecB peptides and are described in Supporting Information.



Page 22 of 43

Haemolytic and Cytotoxic activity Detailed descriptions are reported in Supporting Information.

Number of experiments and statistical analysis Unless specified otherwise, all biological experiments were repeated at least three times, with three biological replicates each. SigmaPlot v12.0 (Systat Software, Inc) or Prism GraphPad were used for data analysis.


Page 23 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ANCILLARY INFORMATION

Supporting Information Supporting Materials and Methods on Calculation of Bound Fraction; LUV preparation and Dye Leakage assay; Inner membrane permeabilization assay; LPS Bicelle disruption assay; Molecular dynamics simulation; DNA/RNA binding assay; Fluorescence-based in vitro transcription inhibition assay; Confocal microscopy; Haemolytic activity; Cytotoxic activity. Intrinsic Tryptophan fluorescence of E53 CecB and Q53 CecB peptides monitored upon titration with increasing concentrations of bacterial suspensions (Figure S1) and of LUVs (Figure S2); Dye leakage assay in the presence of POPC LUV (Figure S3); ITC thermograms and thermodynamical parameters for LPS-peptide interaction (Figure S4); 1D NMR analysis of E53 and Q53 CecB isoform interactions with P. aeruginosa cells and upon titration with P. aeruginosa cells and LPS (Figure S5); Two-dimensional 1H-1H NOESY spectra of the E53 CecB isoform (Figure S6); Twodimensional 1H-1H NOESY spectra of the Q53 CecB isoform (Figure S7); Structural models indicating the changes of E53 and Q53 CecB isoforms in 3:1 POPE/POPG model membrane system from 50 ns accelerated molecular dynamic simulation (Figure S8); Calculated Solvent Accessible Surface Area for E53 CecB and Q53 CecB (Figure S9); In vitro interactions between nucleic acids and CecB peptides (Figure S10); Localisation of CF-labelled E53 or Q53 CecB peptides in E. coli and S. epidermidis (Figure S11); Relative STD percentage of CecB isoforms bound to P. aeruginosa cells (Table S1); Details of aMD parameters in the presence of E53 or Q53 CecB isoforms in a 3:1 POPE/POPG model system mimicking bacterial membrane (Table S2); MICs of the two CecB isoforms against P. aeruginosa ATCC 25668 and ATCC 27853 strains, evaluated at different cation concentrations and in 20% FBS (Table S3); MICs of the two CecB isoforms against P. aeruginosa ATCC 25668 and ATCC 27853 strains, evaluated after pretreatment at 100 °C or with trypsin (Table S4) .

Author information Corresponding authors: Anirban Bhunia: email address: [email protected] Federica Sandrelli: email address: [email protected] 23 ACS Paragon Plus Environment


Page 24 of 43

Ottavia Romoli’s present address: Microbiota of Insect Vectors Group, Institut Pasteur de la Guyane, 23 Avenue Pasteur, 97306 Cayenne, French Guiana, France

Author contribution statement AB, FS, GT: Conceptualization, Methodology, Data analysis, Investigation, Funding acquisition, Writing – original draft, review and editing; OR, SM: Conceptualization, Data analysis, Investigation, Methodology, Writing – original draft, review and editing; FZ, EB: Conceptualization, Writing –review and editing; SAM, AD, AM, EF, CR, DB: Investigation, Data Analysis.

Declarations of interest The authors declare no competing financial interest.

Acknowledgements The authors acknowledge CARIPARO (Progetti di Eccellenza 2011/12) for support to FS and GT; FS also thanks a grant from Università degli Studi di Padova (CPDA154301). This research was partly supported by Science and Engineering Research Board (File No. EMR/2017/003457 to AB), Government of India, and partly by “Indo-Sweden” research collaboration fund (DST/INT/SWD/VR/P-02/2016 to AB). AB would like to thank Prof Jayanta Mukhopadhyay, Bose Institute Kolkata for help in in vitro transcription inhibition assay. Central Instrument Facility (CIF) of Bose Institute is greatly acknowledged. Authors acknowledge Viviana Orlandi (University of Insubria, IT) for kindly providing P. aeruginosa ATCC 27853 strain, Elena Reddi (University of Padova, IT) for the S. epidermidis ATCC 700565, S. aureus ATCC BAA-44 and E. coli ATCC 25922 strains and Paola Venier (University of Padova, IT) for P. aeruginosa ATCC 25668 and C. albicans ATCC 1023 strains. AM is a PhD student of the “Life Science and Biotechnology” course at University of Insubria.


Page 25 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


References (1) Rabanal, F., and Cajal, Y. (2016) Therapeutic Potential of Antimicrobial Peptides. In: Villa T., Vinas M. (eds) New Weapons to Control Bacterial Growth. pp 433-451, Springer, Cham DOI 10.1007/978-3-319-28368-5_16 (2) Yi, H.Y., Chowdhury, M., Huang, Y.D., and Yu, X.Q. (2014) Insect antimicrobial peptides and their applications. Appl Microbiol Biotechnol 98, 5807-5822. DOI: 10.1007/s00253-014-5792-6 (3) Wu, Q., Patočka, J., and Kuča, K. (2018) Insect Antimicrobial Peptides, a Mini Review. Toxins (Basel). 10, pii: E461. DOI: 10.3390/toxins10110461 (4) Ponnuvel, K.M., Subhasri, N., Sirigineedi, S., Murthy, G.N., and Vijayaprakash, N.B. (2010) Molecular evolution of the cecropin multigene family in silkworm Bombyx mori. Bioinformation 5, 97-103. DOI: NOT FOUND (5) Liu, D., Liu, J., Li, J., Xia, L., Yang, J., Sun, S., Ma, J., and Zhang F. (2017) A potential food biopreservative, CecXJ-37N, non-covalently intercalates into the nucleotides of bacterial genomic DNA

beyond

membrane

attack.

J

Food

Chemistry

217,

576-584.

DOI:

10.1016/j.foodchem.2016.09.033 (6) Gazit, E., Lee, W.J., Brey, P.T., and Shau, Y. (1994) Mode of action of the antibacterial Cecropin B2: a spectrofluorometric study. Biochem 33, 10681-10692. DOI: 10.1021/bi00201a016 (7) Moore, A.J., Beazley, W.D., Bibby, M.C., and Devine, D.A. (1996) Antimicrobial activity of cecropins. J Antimicrob Chemother 37, 1077-1089. DOI: 10.1093/jac/37.6.1077 (8) Moore, A.J., Devine, D.A., and Bibby, M.C. (1994) Preliminary experimental anticancer activity of cecropins. Pept Res 7, 265-269. (9) Suttmann, H., Retz, M., Paulsen, F., Harder, J., Zwergel, U., Kamradt, J., Wullich, B., Unteregger, G., Stöckle, M., and Lehmann, J. (2008) Antimicrobial peptides of the Cecropin-family show potent antitumor activity against bladder cancer cells. BMC Urol 8, 5. DOI:10.1186/1471-2490-8-5 (10)

Steiner, H., Hultmark, D., Engström, A., Bennich, H., and Boman, H.G. (1981) Sequence

and specificity of two antibacterial proteins involved in insect immunity. Nature 292, 246-248. DOI:10.1038/292246a0 (11)

Christensen, B., Fink, J., Merrifield, R.B., and Mauzerall, D. (1988) Channel-forming

properties of cecropins and related model compounds incorporated into planar lipid membranes. Proc Natl Acad Sci USA 85, 5072-5076. DOI:10.1073/pnas.85.14.5072



(12)

Page 26 of 43

Efimova, S.S., Schagina, L.V., and Ostroumova O.S. (2014) Channel-forming activity of

Cecropins in lipid bilayers: effect of agents modifying the membrane dipole potential. Langmuir 30, 7884-7892. DOI: 10.1021/la501549v (13)

Callaway, J.E., Lai, J., Haselbeck, B., Baltain, M., Bonnesen, S.P., Weickmann, J., Wilcox,

G., and Lei, S.P. (1993) Modification of the C terminus of cecropin is essential for broad-spectrum antimicrobial activity. Antimicrob Agents Chemother. 37, 1614-1619. DOI: 10.1128/AAC.37.8.1614 (14)

Li, Z.Q., Merrifield, R.B., Boman, I.A., and Boman, H.G. (1988) Effects on electrophoretic

mobility and antibacterial spectrum of removal of two residues from synthetic sarcotoxin IA and addition of the same residues to cecropin B. FEBS Lett. 231,299-302. DOI NOT FOUND (15)

Hultmark, D., Steiner, H., Rasmuson, T., and Boman H.G. (1980) Insect immunity.

Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem, 106:7-16. DOI: 10.1111/j.1432-1033.1980.tb05991.x (16)

Teshima, T., Ueki, Y., Nakai, T., Shiba, T., and Kikuchi M. (1986) Structure determination

of lepidopteran, self-defense substance produced by silkworm. Tetrahedron 42, 829-834. DOI: 10.1016/S0040-4020(01)87488-3 (17)

Morishima, I., Suginaka, S., Ueno, T., and Hirano, H. (1990) Isolation and structure of

cecropins, inducible antibacterial peptides, from the silkworm, Bombyx mori. Comp Biochem Physiol B 95, 551-554. DOI: 10.1016/0305-0491(90)90019-P (18)

Romoli, O., Saviane, A., Bozzato, A., D’Antona, P., Tettamanti, G., Squartini, A.,

Cappellozza, S., and Sandrelli, F. (2017) Differential sensitivity to infections and antimicrobial peptide-mediated immune response in four silkworm strains with different geographical origin. Sci Rep. 7, 1048 DOI:10.1038/s41598-017-01162-z (19)

Sharma, A., Sharma, R., Imamura, M., Yamakawa, M., and Machii, H. (2000) Transgenic

expression of cecropin B, an antibacterial peptide from Bombyx mori, confers enhanced resistance to bacterial leaf blight in rice. FEBS Lett 484, 7-11. DOI: 10.1016/S0014-5793(00)02106-2 (20)

Saviane, A., Romoli, O., Bozzato, A., Freddi, G., Cappelletti, C., Rosini, E., Cappellozza, S.,

Tettamanti, G., and Sandrelli, F. (2018) Intrinsic antimicrobial properties of silk spun by genetically modiﬁed silkworm strains. Transgenic Res 27, 87-101. DOI: 10.1007/s11248-018-0059-0 (21)

Driscoll, J.A., Brody, S.L., and Kollef, M.H. (2007) The epidemiology, pathogenesis and

treatment of Pseudomonas aeruginosa infections. Drugs 67, 351–368. DOI: 10.2165/00003495200767030-00003 (22)

Yang, W., Cheng, T., Ye, M., Deng, X., Yi, H., Huang, Y., Tan, X., Han, D., Wang, B.,

Xiang, Z., Cao, Y., and Xia, Q. (2011) Functional divergence among silkworm antimicrobial peptide 26 ACS Paragon Plus Environment

Page 27 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


paralogs by the activities of recombinant proteins and the induced expression profiles. PLoS One. 6, e18109. DOI: 10.1371/journal.pone.0018109. (23)

Hongbiao, W., Baolong, N., Mengkui, X., Lihua, H., Weifeng, S., and Zhiqi, M. (2005)

Biological activities of cecropin B-thanatin hybrid peptides. J Pept Res. 66, 382-386. DOI:10.1111/j.1399-3011.2005.00299.x (24)

WHO

2017:

http://www.who.int/medicines/publications/global-priority-list-antibiotic-

resistant-bacteria/en/ (25)

Stiefel, P., Schmidt-Emrich, S., Maniura-Weber, K., Ren, Q. (2015) Critical aspects of using

bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide. BMC Microbiol. 15: 36. DOI: 10.1186/s12866-015-0376-x (26)

Lee, J., Jung, S. W., and Cho, A. E. (2016) Molecular Insights into the Adsorption

Mechanism of Human β-Defensin-3 on Bacterial Membranes. Langmuir 32, 1782-1790. DOI: 10.1021/acs.langmuir.5b04113 (27)

Amos, S. T., Vermeer, L. S., Ferguson, P. M., Kozlowska, J., Davy, M., Bui, T. T., Drake,

A. F., Lorenz, C. D., and Mason, A. J. (2016) Antimicrobial Peptide Potency is Facilitated by Greater Conformational Flexibility when Binding to Gram-negative Bacterial Inner Membranes. Sci Rep 6, 37639. DOI: 10.1038/srep37639 (28)

Fiorini, R., Valentino, M., Wang, S., Glaser, M., and Gratton, E. J. B. (1987) Fluorescence

lifetime distributions of 1, 6-diphenyl-1, 3, 5-hexatriene in phospholipid vesicles. Biochemistry 26, 3864-3870. DOI: 10.1021/bi00387a019 (29)

Lentz, B. R. (1989) Membrane “fluidity” as detected by diphenylhexatriene probes.

Chemistry and Physics of Lipids 50, 171-190. DOI: 10.1016/0009-3084(89)90049-2 (30)

Lentz, B. R., Wu, J. R., Zheng, L., and Prevrátil, J. (1996) The interfacial region of

dipalmitoylphosphatidylcholine bilayers is perturbed by fusogenic amphipaths. Biophys J 71, 33023310. DOI: 10.1016/S0006-3495(96)79522-X. (31)

Shi, L., Günther, S., Hübschmann, T., Wick, L. Y., Harms, H., and Müller, S. (2007) Limits

of propidium iodide as a cell viability indicator for environmental bacteria. Cytometry A 71, 592-8. DOI: 10.1002/cyto.a.20402. (32)

Chen, Y., Guarnieri, M. T., Vasil, A. I., Vasil, M. L., Mant, C. T., and Hodges, R. S. (2007)

Role of peptide hydrophobicity in the mechanism of action of alpha-helical antimicrobial peptides. Antimicrob Agents Chemother 51, 1398-1406. DOI: 10.1128/AAC.00925-06 (33)

Sani, M.A., and Separovic F. (2016) How Membrane-Active Peptides Get into Lipid

Membranes. Acc. Chem. Res. 49, 1130–1138. DOI: 10.1021/acs.accounts.6b00074 27 ACS Paragon Plus Environment


(34)

Page 28 of 43

Kalfa, V. C., Jia, H. P., Kunkle, R. A., McCray, P. B., Tack, B. F., and Brogden, K. A.

(2001) Congeners of SMAP29 kill ovine pathogens and induce ultrastructural damage in bacterial cells. Antimicrob Agents Chemother 45, 3256-3261. DOI: 10.1128/AAC.45.11.3256-3261.2001 (35)

Mukherjee, S., Kar, R. K., Nanga, R. P. R., Mroue, K. H., Ramamoorthy, A., and Bhunia, A.

(2017) Accelerated molecular dynamics simulation analysis of MSI-594 in a lipid bilayer. Phys Chem Chem Phys 19, 19289-19299. DOI: 10.1039/c7cp01941f (36)

Chapman, D., and Urbina, J.J. (1974) Biomembrane phase transitions. Studies of lipid-water

systems using differential scanning calorimetry. Biol Chem. 249, 2512-2521. (37)

Nagarajan, D., Nagarajan, T., Roy, N., Kulkarni, O., Ravichandran, S., Mishra, M.,

Chakravortty, D., and Chandra N. (2018) Computational Antimicrobial Peptide Design and Evaluation Against Multidrug-Resistant Clinical Isolates of Bacteria. J Biol Chem 293: 3492-3509 DOI: 10.1074/jbc.M117.805499 (38)

Farkas, A., Maróti, G., Durgő, H., Györgypál, Z., Lima, R.M., Medzihradszky, K.F.,

Kereszt, A., Mergaert, P., and Kondorosi, É. (2014) Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms. Proc Natl Acad Sci USA. 111, 5183-5188. doi: 10.1073/pnas.1404169111 (39)

Farkas, A., Maróti, G., Kereszt, A., and Kondorosi É. (2017) Comparative Analysis of the

Bacterial Membrane Disruption Effect of Two Natural Plant Antimicrobial Peptides. Front Microbiol. 8, 51. DOI: 10.3389/fmicb.2017.00051. (40)

Joris, L., Dab, I., and Quinton, P.M. (1993) Elemental composition of human airway surface

fluid in healthy and diseased airways. Am Rev Respir Dis 148: 1633-1637. DOI: 10.1164/ajrccm/148.6_Pt_1.1633 (41)

Rajesh, K.S., Zareena, Hegde, S., Arun Kumar, M.S. (2015) Assessment of salivary calcium,

phosphate, magnesium, pH, and flow rate in healthy subjects, periodontitis, and dental caries. Contemp Clin Dent. 6, 461–465. DOI: 10.4103/0976-237X.169846 (42)

Wei, G.X., Campagna, A.N., Bobek, L.A. (2007) Factors affecting antimicrobial activity of

MUC7 12-mer, a human salivary mucin-derived peptide. Ann Clin Microbiol Antimicrob. 6, 14. DOI: 10.1186/1476-0711-6-14 (43)

Mangoni, M.L., Maisetta, G., Di Luca, M., Gaddi, L.M., Esin, S., Florio, W., Brancatisano,

F.L., Barra, D., Campa, M., and Batoni, G. (2008) Comparative analysis of the bactericidal activities of amphibian peptide analogues against multidrug-resistant nosocomial bacterial strains. Antimicrob Agents Chemother. 52, 85-91. DOI: 10.1128/AAC.00796-07 28 ACS Paragon Plus Environment

Page 29 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(44)

Biswaro, L.S., da Costa Sousa, M.G., Rezende, T.M.B., Dias, S.C. and Franco, O.L. (2018)

Antimicrobial Peptides and Nanotechnology, Recent Advances and Challenges. Front. Microbiol. 9, 855. DOI: 10.3389/fmicb.2018.00855 (45)

Cappiello, F., Di Grazia, A., Segev-Zarko, L.A., Scali, S., Ferrera, L., Galietta, L., Pini, A.,

Shai, Y., Di, Y.P., and Mangoni, M.L. (2016) Esculentin-1a-Derived Peptides Promote Clearance of Pseudomonas aeruginosa Internalized in Bronchial Cells of Cystic Fibrosis Patients and Lung Cell Migration: Biochemical Properties and a Plausible Mode of Action. Antimicrob Agents Chemother. 60, 7252-7262. DOI:10.1128/AAC.00904-16 (46)

De Lucca, A.J., Bland, J.M., Vigo, C.B., Jacks, T.J., Peter, J., and Walsh, T.J. (2000) D-

cecropin B: proteolytic resistance, lethality for pathogenic fungi and binding properties. Med Mycol. 38, 301-308. DOI NOT FOUND (47)

Strömstedt, A.A., Pasupuleti, M., Schmidtchen, A., and Malmsten, M. (2009) Evaluation of

strategies for improving proteolytic resistance of antimicrobial peptides by using variants of EFK17, an

internal

segment

of

LL-37.

Antimicrob

Agents

Chemother.

53,

593-602.

DOI:

10.1128/AAC.00477-08 (48)

Grassi, L., Maisetta, G., Esin, S., and Batoni, G. (2017) Combination Strategies to Enhance

the Efficacy of Antimicrobial Peptides against Bacterial Biofilms. Front Microbiol. 8, 2409. DOI:10.3389/fmicb.2017.02409 (49)

Yu, L., Tan, M., Ho, B., Ding, J. L., and Wohland, T. (2006) Determination of critical

micelle concentrations and aggregation numbers by fluorescence correlation spectroscopy: aggregation of a lipopolysaccharide. Anal Chim Acta 556, 216-225. DOI: 10.1016/j.aca.2005.09.008 (50)

Zhang, Y. (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics

9, 40. DOI: 10.1186/1471-2105-9-40 (51)

Mark, P., and Nilsson, L. (2001) Structure and dynamics of the TIP3P, SPC, and SPC/E

water models at 298 K. J. Phys. Chem. A 105, 9954-9960. DOI: 10.1021/jp003020w



Page 30 of 43

Table 1. E53 CecB and Q53 CecB antimicrobial activities against different microorganisms. Microorganism

E53 CecB MIC (µM)

Q53 CecB MBC (µM)

pH 5 pH 7 pH 8 pH 10

MIC (µM)

MBC (µM)

pH 5 pH 7 pH 8 pH 10

E. coli ATCC 25922

0.2

0.2

n.d.

2.5

0.15

0.15 0.15 n.d.

2.5

S. aureus ATCC BAA-44

> 20

> 20 > 20 n.d.

n.d.

> 20

> 20 > 20 n.d.

n.d.

0.2

P. aeruginosa ATCC 27853

4

4

4

4

20

2.2

2.2

2.2

2.2

11

P. aeruginosa ATCC 25668

4

4

4

4

20

2.2

2.2

2.2

2.2

11

S. epidermidis ATCC 700565

10

10

10

n.d.

25

8

8

8

n.d.

20

> 20 > 20 n.d.

n.d.

> 20

> 20 > 20 n.d.

n.d.

C. albicans ATCC 1023

> 20

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


Page 31 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Thermodynamic parameters of E53 CecB and Q53 CecB peptides in the presence of LPS* Peptide

n

E53 CecB 0.60 Q53 CecB 0.74

KD (M)

ΔH (kJ mol-1)

13.47 7.35

-81.34 ± 29.19 -51.81 ± 16.86

ΔS (J.mol-1 K-1) ΔG (kJ mol-1) -179.6 -75.31

-27.82 -29.37

*Buffer: 10 mM phosphate buffer with 150 mM NaCl (pH 7.4) at 298K



Page 32 of 43

Figure Legends Figure 1: Comparative activity of E53 CecB and Q53 CecB peptides against P. aeruginosa live cells, LUVs and LPS mimicking bacterial membrane. (A) E53 CecB and Q53 CecB peptide sequences. Arrow indicates the polymorphic site. (B) Time-course of bactericidal activity for CecB isoforms on P. aeruginosa [Percentage of dead cells (mean ± SEM), two independent experiments], via flow-cytometry analysis. 4 µM Q53 CecB (red striped bars) showed a significantly higher bactericidal activity compared to 4 µM CecB E53 (blue bars) (Two-way ANOVA: F 2, 18 = 6.107, p < 0.001). Red bars: 2 µM Q53 CecB. (C) Effect of the two CecB isoforms on a bacterial model membrane via CF dye leakage assay, using 3:1 POPE/POPG model membrane. (D) Membrane destabilization property of E53 CecB (blue line) and Q53 peptides (red line) on P. aeruginosa LPS bicelles via DPH fluorescence assay. The black line represents the fluorescence of the negative control (DPH-supplemented bicelles only). (E) Kinetics of P. aeruginosa membrane permeabilization, measuring the uptake of PI fluorescent dye after addition of CecB peptides. E53 CecB (blue line) and Q53 peptides (red line). Black line represents the negative control. AU: Arbitrary Unit.

Figure 2. TEM micrograph of control (A), Q53 CecB (B, C), and E53 CecB (D, E) treated P. aeruginosa cells. In bacteria incubated with 10 M E53 CecB and with 4 or 10 M Q53 CecB isoform, a damage to the cell membrane and the release of cytoplasm content are visible. Bars: 1 m (A), 200 nm (BE). Q53: Q53 CecB isoform; E53: E53 CecB isoform.

Figure 3. CD spectra elucidating thesecondary structure of E53 CecB (A) and Q53 CecB (B) isoforms when bound to live P. aeruginosa cells. In aqueous solution (black line; A,B) both the peptides adopted random coil conformation, confirmed by the presence of a strong negative minima at ~200 nm. However, immediately after addition of P. aeruginosa cell a drastic peak shift (green line; A,B) was obtained and incubation of sample for 2h, one peak was observed at ~208 and another at ~225 nm in both CD spectra (purple line; A,B).

Figure 4. STD NMR studies of the interaction between E53 CecB and Q53 CecB peptides and P. aeruginosa cells. 1D NMR analysis of E53 CecB (A) and Q53 CecB (B) isoforms interaction with P. 32 ACS Paragon Plus Environment

Page 33 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


aeruginosa cells; 1D proton NMR spectra of peptide alone (i), and upon titration with increasing concentrations of bacteria (ii, iii). Dashed lines show the emergence of a second peak in the W28 indole region (NH), indicating the presence of another conformation. For E53 CecB (C) and -Q53 (D) isoforms, reference 1H NMR spectrum, and STD NMR spectrum of the peptide (1 mM) in the presence of P. aeruginosa cells (in 100 % D2O, pH 6.5; stock:108 cells/ml), is showing non-exchangeable proton resonances. Light blue stripes indicate the aromatic protons of F31. E53 CecB (E; blue) and Q53 CecB (F; red) peptide models, in which spheres of three different colours were used to reflect the vicinity of protons to the binding of P. aeruginosa live cells (white: 70–100%; yellow: 50-69%; green: < 49%). A larger saturation transfer (70–100%) indicates that the ligand protons are in close proximity to the LPS micelles, whereas a weak STD total values (

Enhanced Silkworm Cecropin B Antimicrobial Activity against

Recommend Documents