Antibacterial Peptide Nucleic Acid–Antimicrobial Peptide (PNA–AMP

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Antibacterial peptide nucleic acid - antimicrobial peptide (PNAAMP) conjugates: Antisense targeting of fatty acid biosynthesis Anna Mette Hansen, Gitte Bonke, Camilla Josephine Larsen, Niloofar Yavari, Peter E Nielsen, and Henrik Franzyk Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00013 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 5, 2016

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Antibacterial peptide nucleic acid − antimicrobial peptide (PNAAMP) conjugates: Antisense targeting of fatty acid biosynthesis Anna Mette Hansen,† Gitte Bonke,† Camilla Josephine Larsen,† Niloofar Yavari‡, Peter E. Nielsen,†‡ and Henrik Franzyk†* †

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark ‡

Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark Supporting Information ABSTRACT: Antisense peptide nucleic acid (PNA) oligomers constitute a novel class of potential antibiotics that inhibit bacterial growth via specific knock-down of essential gene expression. However, discovery of efficient, non-toxic delivery vehicles for such PNA oligomers has remained a challenge. In the present study we show that antimicrobial peptides (AMPs) with an intracellular mode of action can be efficient vehicles for bacterial delivery of an antibacterial PNA targeting the essential acpP gene. The results demonstrate that buforin2-A (BF2-A), drosocin, oncocin 10, Pep-1-K, KLW-9,13-a, (P59→W59)-Tat48-60, BF-2A-RXR and drosocin-RXR are capable of transporting PNA effectively into E. coli (MICs of 1-4 µM). Importantly, presence of the innermembrane peptide transporter SbmA was not required for antibacterial activity of PNA-AMP conjugates containing Pep-1-K, KLW-9,13-a, or drosocin-RXR (MICs of 2-4 µM).

Continued emergence of multidrug-resistant (MDR) bacterial strains of human pathogens are now directly translating into major healthcare problems as even last-resort antibiotics are losing efficacy worldwide. Furthermore, almost untreatable infections with Gram-negative panresistant strains are 1,2 being reported, while antibiotic research by the pharma3 ceutical industry has declined. Consequently, discovery of antibacterial agents with novel targets and/or novel mode of action (MoA) is crucial. Genetic antibiotics in the form of antisense PNA (peptide nucleic acid) oligomers targeting essential bacterial genes were introduced 15 years ago as such 4,5 an approach. A large number of subsequent studies using both PNA and PMO (phosphorodiamidate morpholino) oligomers conjugated to delivery peptides have validated this 6-13 concept. In particular, expression of the acpP gene, essential for fatty acid biosynthesis, has been targeted by using 4,5,10-13 both PNA and PMO peptide conjugates. Surprisingly, it was found that quite short oligomers (10-12 nucleobases) are 5,12 optimal for antibacterial effects, and this has been ascribed to restrictions imposed by bacterial uptake. Furthermore, an antisense MoA has been validated for an extended series of PMO and PNA conjugates by using both control mis-match

5,10,13

PNA and as well as mutated acpP RNA targets. However, the approach is still challenged by relatively low potency of the PNA-peptide conjugates, and general toxicity of the delivery peptides used. Only a limited number of delivery peptides, centered around the originally employed (KFF)3K peptide and eukaryotic cell-penetrating peptides (CPPs) based on repetitive arginine/6-aminohexanoic acid motifs, 5-15 have so far been studied for PNA and PMO conjugation. Finally, recent studies have shown that bacterial resistance to these types of conjugates arise quite readily by mutations and deletions in the non-essential sbmA gene coding for an inner membrane ABC transporter that is crucial to the antibacterial activity of most PNA- and PMO-peptide conju13,16 gates. Hence, alternative carriers that both promote SbmA-independent bacterial delivery of PNA and confer improved pharmacokinetic properties without significant systemic toxicity are highly warranted. We hypothesized that antimicrobial peptides (AMPs) which constitute an essential 17,18 part of the innate immune system of higher organisms, might also act as bacterial delivery vehicles. Generally, the MoA of AMPs involves a selective disruption or permeabilization of bacterial membranes facilitated by a combination 19 of cationic and hydrophobic residues, however, several AMPs without pronounced cytotoxicity appear to kill bacteria via intracellular targets facilitated by non-lytic internali20,21 zation processes. Consequently, we decided to explore the applicability of such AMPs as delivery vehicles for PNA oligomers targeting an essential bacterial gene. A literature survey of AMPs reported to have an intracellular target was performed in order to identify an array of such peptides comprising different subtypes with characteristic amino acid compositions. Initially, twelve AMPs (1-12; Table 1) were selected by the key criteria that they: (i) appear to rely on an intracellular MoA, (ii) consist of less than 25 residues, and (iii) do not contain any Cys within the sequence as a terminal Cys was to be utilized for conjugation to PNA. In addition, NLS-Gb3 (13), an antimicrobial hybrid combining a nuclear localization signal (NLS) peptide and a globotriaosyl (Gb3) mimic was consid22 ered. Finally, the CPP variant (P59→W59)-Tat48-60 (14) was included in the study, as another similar analog and

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Table 1. Sequences and molecular properties of the peptides tested as vehicles for bacterial delivery of PNA. No.

AMP

Amino acid sequence

Molecular Mass (Da)

Net charge

Ref.

1

Buforin2-A (BF2-A)

RAGLQFPVGRVHRLLRK-NH2

2

Drosocin

GKPRPYSPRPTSHPRPIRV-NH2

2001.23

+7

26

2198.26

+7

22

3

Apidaecin-1a

GNNRPVYIPQPRPPHPRI-NH2

2108.18

+5

23

4

Pyrrhocoricin

VDKGSYLPRPTPPRPIYNRN-NH2

2340.28

+4

24

5

Oncocin 10

VDKPPYLPRPRPPRRIYNR-NH2

2390.39

+6

25

6

Bac1-15

RRIRPRPPRLPRPRP-NH2

1806.09

+9

30

7

IsCT-p

ILKKIWKpIKKLF-NH2

1653.12

+6

31

8

Pep-1-K

KKTWWKTWWTKWSQPKKKRKV-NH2

2844.65

+10

32

9

TPk

VRRFkWWWkFLRR-NH2

1964.16

+7

33

10

SA-3

IKWAGKWWKLFK-NH2

1589.95

+5

34

11

K6L2W3

KLWKKWKKWLK-NH2

1571.01

+7

35

12

KLW-L9, 13-a

KWKKLLKKaLKLaKKLLK-NH2

2178.55

+10

36

13

NLS-Gb3

WHWTWLRIRKKLR-NH2

1877.42

+7

17

14

(P59→W59)-Tat48-60

GRKKRRQRRRPWQ-NH2

1919.23

+8

19

15

BF2-A-RXR

RAGLQFPVGRVHRLLRXR-NH2

2142.32

+7

-

16

Drosocin-RXR

GKPRPYSPRPTSHPRPIRXR-NH2

2367.38

+8

-

a = NAla; k = NLys; p = D-Pro; X = Ahx = 6-aminohexanoic acid; -NH2 indicates a C-terminal amide 23,24

Tat48-60 itself exhibit weak antibacterial activity.

All peptides were obtained via microwave-assisted Fmocbased solid-phase peptide synthesis. Besides the native AMPs analogues incorporating a Cys residue at either the N- or Cterminus were prepared in order to enable subsequent conjugation via the bifunctional succinimidyl-4-(Nmaleimidomethyl)-cyclohexane-1-carboxylate (SMCC) linker. Thus, the resulting Cys-containing peptides were conjugated 5,13 to the previously validated anti-acpP PNA oligomer HTCTCATACTC-NH2 to afford the type of conjugates depicted in Figure 1 (see SI).

Figure 1. Structure of PNA-AMP conjugates with an SMCC linker. The PNA oligomer is depicted in blue, the SMCC linker in black, while the AMP displaying an N-terminal Cys is shown in red. R: side chain of amino acids, Base: a nucleobase: adenine (A), guanine (G), thymine (T) or cytosine (C). To test whether the selected AMPs were capable of internalizing an antisense PNA oligomer, the wild-type E. coli strain MG1655 was incubated with AMP/CPP 1-14 and with the corresponding anti-acpP PNA conjugate (1a-14a) for 15 hours while continuously measuring the turbidity of the bacterial culture. The minimal inhibitory concentration (MIC value) was determined from the resulting growth curve as the lowest concentration of the compound preventing growth (see SI for representative data sets). Comparison of the MIC for the AMP and the corresponding anti-acpP PNA conjugate was used to assess whether the observed antimicrobial activi-

ty of the conjugate was due to silencing of the targeted gene or caused by the carrier peptide moiety (results are listed in Table 2). The subclass of Pro-rich peptides, for which a non-lytic mechanism has been established, is represented by drosocin 25 26 27 (2), apidaecin (3), and pyrrhocoricin (4). For AMPs 3 and 4 only modest antibacterial activity was found, and somewhat surprisingly their corresponding PNA conjugates displayed quite similar MIC values, indicating that the peptides are not capable of internalizing the PNA oligomer with a functional cytosolic delivery. Oncocin 10 (5) is a synthetic Pro-rich AMP that also appears to act via a non-lytic mecha28 nism, albeit its exact intracellular target remains unknown. Our results are in line with this, as both oncocin 10 (5) and its antisense PNA conjugate (5a) were quite potent with similar MIC values (3 µM and 0.9 µM, respectively). By contrast, the MICs for compounds 2 and 2a (>33 µM vs. 0.9 µM) differed more than 30-fold, with conjugate 2a being most potent, inferring that the antisense PNA oligomer was indeed 29,30 internalized. The same trend was seen for BF2-A (1) and its PNA conjugate 1a in agreement with an intracellular tar31 get reached without membrane disruption. Translocation of PNA oligomers across the bacterial membrane with subsequent growth inhibition via an antisense mechanism appear likely from our results, thus corroborating an intracellular target for buforin2-A (1). To rule out that the increased antibacterial activity of conjugates 1a and 2a simply arise from potentiation of AMPs 1 and 2 by linkage to the PNA, a mismatch anti-acpP PNA oligomer was conjugated to each of these. The resulting conjugates (1c and 2c) were tested similarly, and their activity proved to be at least 10- to 30-fold lower than that of the matched conjugates, indicating that for conjugates 1a and 2a an antisense mechanism via the anti-acpP PNA moiety is indeed most likely.

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A shortened version (6) of Bac7 was examined as carrier due to its antimicrobial activity and cell-penetrating properties in 32,33 both bacterial and mammalian cell systems. Expectedly, 32,33 Bac1-15 (6) displayed significant antimicrobial activity as did the corresponding PNA conjugate 6a. However, both compounds displayed identical MIC values (3 µM) inferring that the activity of the conjugate arises from the peptide part. Peptide 13 had moderate activity (MIC of 10 µM) in contrast to the inactive CPP 14 (MIC > 10 µM), while the corresponding conjugates 13a and 14a had enhanced potency (MIC of 3 µM). AMPs 7-14 have a high content of cationic amino acids (i.e. Lys/Arg) combined with hydrophobic Trp residues that 22,34-39 confer efficient initial membrane association. For these AMPs the increase in antibacterial activity, induced by conjugation to the anti-acpP PNA, was 3- to 10-fold or less resulting in MIC values of 2-4 µM, indicating moderate intracellular delivery efficiency of the anti-acpP PNA oligomer. Interestingly, synthetic AMP analogue 12 originates from an αhelical model peptide KLW (i.e. KWKKLLKKLLKLLKKLLKNH2), in which two Leu residues are replaced by two NAla 39 residues (i.e. peptoid units corresponding to Ala). This disrupts the α-helix towards a less folded structure in the presence of membrane models, and its MoA changes from membrane disruption to a non-lytic mechanism as fluorescently labeled 12 localized internally in E. coli as visualized by 39 confocal laser-scanning microscopy. To investigate the influence of the direction of peptide-PNA conjugation, i.e. whether a free N- or C-terminal part of the delivery vehicles is critical for the internalization and hence for their antisense antibacterial activity, AMPs 1, 2, 5, and 8 were also linked via a C-terminal Cys to the PNA oligomer resulting in conjugates 1b-2b, 5b, and 8b, for which the antibacterial activity was evaluated. The results showed only minor differences between N- and C-terminal conjugation of the AMP to the PNA oligomer, indicating that linkage of PNA to a specific terminus may be not crucial for enhanced antibacterial activity of the conjugate. Because sbmA is required for antibacterial activity in E. coli of most of the PNA- and PMO-peptide conjugates examined 13,40,42,43 so far, and is only present in some (mostly Gram41-42 such dependency could severely narnegative) bacteria, row the spectrum of PNA-peptide antibiotics. Furthermore, mutations in the sbmA gene could readily lead to clinical development of resistance towards such new antibiotics. Finally, since SbmA has also been reported to be involved in the inner membrane transport of structurally diverse substrates, e.g. Pro-rich peptides Bac7 and PR-39, microcins B17 16,17,41,43 and Mcc25, , it was pertinent to determine whether active sbmA is required for the activity of the present PNApeptides. a

Table 2. MICs (in µM) for peptides and PNA conjugates No.

MIC toward E. coli MG1655

1

33

MG1655 ∆sbmA 33

1a

0.9

>10

1b

3

16

Peptide properties Effective PNA carrier +

SbmA dependency +

1c

>10

-

2

>33

>33

2a

0.9

>10

2b

≤0.9

-

2c

>16

-

3

>33

>33

3a

>16

>16

4

10

>33

4a

>16

>16

5

3

>10

5a

0.9

>10

5b

3

>16

5c

>16

-

6

3

3

6a

3

3

7

10

>10

7a

3

>10

8

3

>10

8a

2

2

8b

2

2

8c

>16

>16

8d

4-8

4-8

9

3

>10

9a

2

>10

10

3

3

10a

3

10

11

3

>10

11a

3

>10

12

10

10

12a

4

4

12c

16

16

13

10

>10

13a

3

>10

14

>10

>10

14a

3

>10

15

>10

10

15a

≤0.9

10

15c

>16

>16

16

>10

>10

16a

3

3

16c

>16

>16

+

+

-

+

-

+

+

+

-

-

-

+

+

-

-

+

+

-

-

-

(+)

-

+

+

+

+

+

+

+

-

a

Anti-acpP-PNA: H-TCTCATACTC-NH2; conjugation via the N- or C-terminus (denoted by a and b, respectively) of the AMP. Mismatch-anti-acpP-PNA: H-TCACATTCTC-NH2; conjugation via the N- or C-terminus (denoted by c and d) of the AMP. MG1655 and MG1655∆sbmA denote a wild-type E. coli strain and the corresponding SbmA knock-out strain,

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respectively. Effective PNA carrier: increased activity of the conjugate as compared to the peptide alone. SbmA dependency: activity is significantly diminished in MG1655∆sbmA strain (typically to the level of peptide alone). As seen from Table 2 most of the peptides (i.e. 1-5, 7, 9, 11, 13 and 14) as well as the corresponding PNA conjugates exhibited reduced or similarly low activity toward E. coli MG1655 ΔsbmA as found against the wild-type strain. AMPs 2-6 belong to the Pro-rich AMPs reported to be trans40,42,44,45 ported by SbmA, however, the N-terminal Bac7 frag47 ment 6 and its PNA conjugate both exhibited similar activity toward MG1655 and MG1655 ΔsbmA indicating that SbmA may not be required for translocation of this AMP and/or that the activity of the conjugate arises from the peptide part. Interestingly, peptides 1, 7, 9, 11, 13 and 14 containing several Lys/Arg and Trp residues (but no Pro) and the corresponding PNA conjugates exhibited insignificant activity against MG1655ΔsbmA, and thus like Pro-rich AMPs 2-5 appear to depend on the presence of SbmA for efficient translocation. The slightly lower MIC of the PNA-AMP conjugate 12a as compared to the mis-match conjugate 12c as well as to peptide 12, indicates that this synthetic AMP containing two NAla residues is an effective carrier of the PNA independently of the presence of SbmA. Second-generation AMPs were designed for the well-known AMPs BF2-A and drosocin (1 and 2) for which the most prominent increase in activity of the corresponding PNA conjugates was observed. This involved replacement of the two C-terminal residues with an RXR motif, i.e. RK→RXR and RV→RXR in AMPs 1 and 2, respectively. The rationale for this alteration was the finding that delivery vehicles based on the RXR repeating unit confers general cell penetration of 8,9,15 both eukaryotic and prokaryotic cells. The peptides displaying the RXR motif (i.e. 15 and 16) both had significantly higher MICs as compared to the corresponding PNA conjugates 15a and 16a, indicating that bacterial delivery of PNA was retained. Notably, the activity of conjugate 16a was SbmA-independent, whereas that of 1a/15a was not. Thus the present results for conjugate 16a corroborate that the RXR motif in some instances may support an SbmA-independent uptake mechanism, as previously reported for the (RXR)4X13 βAla peptide. The PR motif occurs in the sequences of many AMPs with intracellular targets (e.g. 2-6), and its presence has been suggested to be a prerequisite for AMPs to undergo translo40,41 cation by SbmA, however, a few exceptions like BF2-A (1) have been reported. Interestingly, our investigation imply that both Arg/Lys- and Trp-rich AMPs and CPPs should be added to the list of diverse substrates for SbmA. Hence, SbmA displays a surprisingly unrestrained specificity. For interpretation of the data in Table 2, it is assumed that peptides constitute functional antisense PNA carriers when both these prerequisites are met: (i) the PNA-AMP conjugate exhibits higher activity than the parent peptide, and (ii) the mismatched conjugate has significantly lower activity than the antisense (i.e. the matched) conjugate. Additionally, bacterial uptake is considered SbmA-independent when the activity against the ΔsbmA strain is similar to that found for the wild type. In conclusion, peptides BF2-A (1), drosocin (2),

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oncocin 10 (5), Pep-1-K, (8), KLW-9,13-a (12), (P59→W59)Tat48-60 (14), BF-2A-RXR (15) and drosocin-RXR (16) are all capable of transporting PNA into E. coli. Of these, PNA conjugates with peptides 8, 12, and 16 exhibit SbmA-independent activity, and of these 16a in particular constitutes an interesting antibacterial lead compound.

ASSOCIATED CONTENT Supporting Information Experimental procedures, HPLC chromatograms, and HRMS data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; Tel: (+45)35336255

Funding Sources The research was supported by Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen.

ACKNOWLEDGMENT The authors thank Dr. Kenneth T. Kongstad for the HRMS results; while technicians Birgitte Simonsen and technician Jolanta Ludvigsen are acknowledged for excellent technical assistance in HPLC and synthesis/purification of the PNA conjugates, respectively.

REFERENCES (1) Theuretzbacher, U. (2013) Global antibacterial resistance: The never-ending story, J. Glob. Antimicrob. Resist. 1, 63-69. (2) Livermore, D. M. (2012) Current epidemiology and growing resistance of Gram-negative pathogens, Korean J. Intern. Med. 27, 128-142. (3) Jabes, D. (2011) The antibiotic R & D pipeline: an update. Curr. Opin. Microbiol. 14, 564-569. (4) Good, L., and Nielsen, P. E. (1998) Antisense inhibition of gene expression in bacteria by PNA targeted to mRNA. Nat. Biotechnol. 16, 355-358. (5) Good, L., Awasthi, S. K., Dryselius, R., Larsson, O., and Nielsen, P. E. (2001) Bactericidal antisense effects of peptide-PNA conjugates, Nat. Biotechnol. 19, 360-364. (6) Mondhe, M., Chessher, A., Goh, S., Good, L., and Stach, J. E. M. (2014) Species-Selective Killing of Bacteria by Antimicrobial PeptidePNAs, PLoS One 9, e89082. (7) Nekhotiaeva, N., Awasthi, S. K., Nielsen, P. E., and Good, L. (2004) Inhibition of Staphylococcus aureus Gene Expression and Growth Using Antisense Peptide Nucleic Acids. Mol. Ther. 10, 652659. (8) Mellbye, B. L., Puckett, S. E., Tilley, L. D., Iversen, P. L., and Geller, B. L. (2009) Variations in Amino Acid Composition of Antisense Peptide-Phosphorodiamidate Morpholino Oligomer Affect Potency against Escherichia coli In Vitro and In Vivo. Antimicrob Agents Chemother. 53, 525-530. (9) Tilley, L. D., Hine, O. S., Kellogg, J. A., Hassinger, J. N., Weller, D. D., Iversen, P. L., and Geller, B. L. (2006) Gene-Specific Effects of Antisense Phosphorodiamidate Morpholino Oligomer-Peptide Conjugates on Escherichia coli and Salmonella enterica Serovar

4

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Typhimurium in Pure Culture and in Tissue Culture. Antimicrob. Agents Chemother. 50, 2789-2796

antibacterial peptides from a hemipteran insect, the sap-sucking bug Pyrrhocoris apterus. Biochem. J. 300, 567-575.

(10) Mellbye, B. L., Weller, D. D., Hassinger, J. N., Reeves, M. D., Lovejoy, C. E., Iversen, P. L., and Geller, B. L. (2010) Cationic phosphorodiamidate morpholino oligomers efficiently prevent growth of Escherichia coli in vitro and in vivo. J. Antimicrob Chemother. 65, 98106.

(28) Knappe, D., Kabankov, N., and Hoffmann, R. (2011) Bactericidal oncocin derivatives with superior serum stabilities, Int. J. Antimicrob. Agents 37, 166-170.

(11) Mellbye, B.L., Puckett, S. E., Tilley, L. D., Iversen, P. L., and Geller, B. L. (2009) Variations in amino acid composition of antisense peptide-phosphorodiamidate morpholino oligomer affect potency against Escherichia coli in vitro and in vivo. Antimicrob Agents Chemother. 53, 525-530. (12) Deere, J., Iversen, P., Geller, B. L. (2005) Antisense phosphorodiamidate morpholino oligomer length and target position effects on gene-specific inhibition in Escherichia coli. Antimicrob Agents Chemother. 49, 249-255. (13) Ghosal, A., Vitali, A., Stach, J. E. M., and Nielsen, P. E. (2012) Role of SbmA in the Uptake of Peptide Nucleic Acid (PNA)-Peptide Conjugates in E. coli, ACS Chem. Biol. 8, 360-367. (14) Nekhotiaeva, N., Elmquist, A., Rajarao, G. K., Hällbrink, M., Langel, Ü., and Good, L. (2004) Cell entry and antimicrobial properties of eukaryotic cell-penetrating peptides. FASEB J. 18, 394-396. (15) Abes, R., Moulton, H. M., Clair, P., Yang, S.-T., Abes, S., Melikov, K., Prevot, P., Youngblood, D. S., Iversen, P. L., Chernomordik, L. V. et al. (2008) Delivery of steric block morpholino oligomers by (R-XR)4 peptides: structure–activity studies. Nucl. Acids Res. 36, 63436354. (16) Pränting, M., Negrea, A., Rhen, M., and Andersson, D. I. (2008) Mechanism and Fitness Costs of PR-39 Resistance in Salmonella enterica Serovar Typhimurium LT2. Antimicrob. Agents Chemother. 52, 2734-2741. (17) Zasloff, M. (2002) Antimicrobial peptides of multicellular organisms. Nature 415, 389-395. (18) Brown, K. L., and Hancock, R. E. W. (2006) Cationic host defense (antimicrobial) peptides. Curr. Opin. Immunol. 18, 24-30. (19) Toke, O. (2005) Antimicrobial peptides: New candidates in the fight against bacterial infections. Pept. Sci. 80, 717-735. (20) Nicolas, P. (2009) Multifunctional host defense peptides: intracellular-targeting antimicrobial peptides, FEBS J. 276, 6483-6496. (21) Scocchi, M., Tossi, A., and Gennaro, R. (2011) Proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action. Cell. Mol. Life Sci. 68, 2317-2330. (22) Yamada, Y., Miura, Y., Sakaki, A., Yoshida, T., and Kobayashi, K. (2006) Design of multifunctional peptides expressing both antimicrobial activity and shiga toxin neutralization activity. Bioorg. Med. Chem. 14, 77-82. (23) Bahnsen, J. S., Franzyk, H., Sandberg-Schaal, A., and Nielsen, H. M. (2013) Antimicrobial and cell-penetrating properties of penetratin analogs: Effect of sequence and secondary structure. Biochim. Biophys. Acta – Biomembr. 1828, 223-232. (24) Zhu, W. L., and Shin, S. Y. (2009) Effects of dimerization of the cell-penetrating peptide Tat analog on antimicrobial activity and mechanism of bactericidal action, J. Pept. Sci. 15, 345-352. (25) Bikker, F. J., Kaman-van Zanten, W. E., de Vries-van de Ruit, A.M. B. C., Voskamp-Visser, I., van Hooft, P. A. V., Mars-Groenendijk, R. H., de Visser, P. C., and Noort, D. (2006) Evaluation of the Antibacterial Spectrum of Drosocin Analogues. Chem. Biol. Drug Des. 68, 148-153. (26) Casteels, P., Ampe, C., Jacobs, F., Vaeck, M., and Tempst, P. (1989) Apidaecins: antibacterial peptides from honeybees, Embo J. 8, 2387–2391. (27) Cociancich, S., Dupont, A., Hegy, G., Lanot, R., Holder, F., Hetru, C., Hoffmann, J. A., and Bulet, P. (1994) Novel inducible

(29) Park, C. B., Kim, M. S., and Kim, S. C. (1996) A Novel Antimicrobial Peptide from Bufu bufo gargarizans. Biochem. Biophys. Res. Commun. 218, 408-413. (30) Park, C. B., Yi, K.-S., Matsuzaki, K., Kim, M. S., and Kim, S. C. (2000) Structure–activity analysis of buforin II, a histone H2Aderived antimicrobial peptide: The proline hinge is responsible for the cell-penetrating ability of buforin II. Proc. Natl. Acad. Sci. U.S.A. 97, 8245-8250. (31) Hao, G., Shi, Y.-H., Tang, Y.-L., and Le, G.-W. (2013) The intracellular mechanism of action on Escherichia coli of BF2-A/C, two analogues of the antimicrobial peptide Buforin 2. J. Microbiol. 51, 200-206. (32) Skerlavaj, B., Romeo, D., and Gennaro, R. (1990) Rapid membrane permeabilization and inhibition of vital functions of gramnegative bacteria by bactenecins. Infect. Immun. 58, 3724-3730. (33) Sadler, K., Eom, K. D., Yang, J.-L., Dimitrova, Y., and Tam, J. P. (2002) Translocating Proline-Rich Peptides from the Antimicrobial Peptide Bactenecin 7. Biochemistry 41, 14150-14157. (34) Lim, S. S., Kim, Y., Park, Y., Kim, J. I., Park, I.-S., Hahm, K.-S., and Shin, S. Y. (2005) The role of the central L- or D-Pro residue on structure and mode of action of a cell-selective α-helical IsCTderived antimicrobial peptide. Biochem. Biophys. Res. Commun. 334, 1329-1335. (35) Zhu, W. L., Lan, H., Park, I.-S., Kim, J. I., Jin, H. Z., Hahm, K.-S., and Shin, S. Y. (2006) Design and mechanism of action of a novel bacteria-selective antimicrobial peptide from the cell-penetrating peptide Pep-1. Biochem. Biophys. Res. Commun. 349, 769-774. (36) Zhu, W. L., Lan, H., Park, Y., Yang, S.-T., Kim, J. I., Park, I.-S., You, H. J., Lee, J. S., Park, Y. S., Kim, Y. et al. (2006) Effects of Pro→Peptoid Residue Substitution on Cell Selectivity and Mechanism of Antibacterial Action of Tritrpticin-Amide Antimicrobial Peptide. Biochemistry 45, 13007-13017. (37) Joshi, S., Bisht, G. S., Rawat, D. S., Kumar, A., Kumar, R., Maiti, S., and Pasha, S. (2010) Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775. Biochim. Biophys. Acta – Biomembr. 1798, 1864-1875. (38) Park, K. H., Nan, Y. H., Park, Y., Kim, J. I., Park, I.-S., Hahm, K.S., and Shin, S. Y. (2009) Cell specificity, anti-inflammatory activity, and plausible bactericidal mechanism of designed Trp-rich model antimicrobial peptides. Biochim. Biophys. Acta – Biomembr. 1788, 1193-1203. (39) Song, Y. M., Park, Y., Lim, S. S., Yang, S.-T., Woo, E.-R., Park, I.S., Lee, J. S., Kim, J. I., Hahm, K.-S., Kim, Y. et al. (2005) Cell Selectivity and Mechanism of Action of Antimicrobial Model Peptides Containing Peptoid Residues. Biochemistry 44, 12094-12106. (40) Runti, G., Lopez Ruiz, M. d. C., Stoilova, T., Hussain, R., Jennions, M., Choudhury, H. G., Benincasa, M., Gennaro, R., Beis, K., and Scocchi, M. (2013) Functional Characterization of SbmA, a Bacterial Inner Membrane Transporter Required for Importing the Antimicrobial Peptide Bac7(1-35). J. Bacteriol. 195, 5343-5351. (41) Corbalan, N., Runti, G., Adler, C., Covaceuszach, S., Ford, R. C., Lamba, D., Beis, K., Scocchi, M., and Vincent, P. A. (2013) Functional and Structural Study of the Dimeric Inner Membrane Protein SbmA. J. Bacteriol. 195, 5352-5361. (42) Mattiuzzo, M., Bandiera, A., Gennaro, R., Benincasa, M., Pacor, S., Antcheva, N., and Scocchi, M. (2007) Role of the Escherichia coli SbmA in the antimicrobial activity of proline-rich peptides. Mol. Microbiol. 66, 151-163.

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(43) Puckett, S. E., Reese, K. A., Mitev, G. M., Mullen, V., Johnson, R. C., Pomraning, K. R., Mellbye, B. L., Tilley, L. D., Iversen, P. L., Freitag, M. et al. (2012) Bacterial resistance to antisense peptide phosphorodiamidate morpholino oligomers. Antimicrob. Agents Chemother. 56, 6147-6153. (44) Narayanan, S., Modak, J. K., Ryan, C. S., Garcia-Bustos, J., Davies, J. K., and Roujeinikova, A. (2014) Mechanism of Escherishia coli resistance to pyrrhocoricin. Antimicrob. Agents Chemother. 58, 27542762. (45) Guida, F., Benincasa, M., Zahariev, S., Scocchi, M., Berti, F., Gennaro, R., and Tossi, A. (2015) Effect of size and N-terminal residue characteristics on bacterial cell penetration and antibacterial activity of the proline-rich peptide Bac7. J. Med. Chem. 58, 1195-1204.

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