Identification of Api88 Binding Partners in ... - ACS Publications

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Identification of Api88 Binding Partners in Escherichia coli Using a Photoaffinity-Cross-Link Strategy and Label-Free Quantification Daniela Volke,†,‡ Andor Krizsan,†,‡ Nicole Berthold,†,‡ Daniel Knappe,†,‡ and Ralf Hoffmann*,†,‡ †

Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and ‡Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, Leipzig, Germany S Supporting Information *

ABSTRACT: Gene-encoded antimicrobial peptides (AMPs) kill bacteria very efficiently by either lytic mechanisms or inhibition of specific bacterial targets. Proline-rich AMPs (PrAMPs), for example, produced in insects and mammals rely on the second mechanism. They bind to the 70 kDa bacterial heat shock protein DnaK and the 60 kDa chaperonin GroEL and interfere with protein folding, but this does not explain their strong bactericidal effects. Thus, we looked for further binding partners of apidaecin 1b, originally identified in honey bees, and two rationally optimized analogues (Api88 and Api137). Because affinity chromatography using Api88 as an immobilized ligand enriched only a few proteins at low levels besides DnaK, we synthesized Api88 analogues substituting Tyr7 with p-benzoylphenylalanine (Bpa), which can cross-link the peptide to binding partners after UV irradiation. Escherichia coli was incubated with biotinylated Api88 Tyr7Bpa or the corresponding all-D-peptide, irradiated, and lysed. The protein extract was enriched by streptavidin, separated by SDS-PAGE, digested with trypsin, and analyzed by nanoRP-UPLCESI-QqTOF-MS/MS. Among the 41 proteins identified, 34 were detected only in the L-Api88 Tyr7Bpa sample, including five 70S ribosomal proteins, DNA-directed RNA polymerase, and pyruvate dehydrogenase, indicating that PrAMPs might interfere with protein translation and energy metabolism. KEYWORDS: 70 kDa bacterial heat shock protein DnaK, proline-rich antimicrobial peptide (PrAMP), apidaecin, mass spectrometry, protein targets, ribosome

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INTRODUCTION Infectious diseases have threatened humans, animals, and plants during the whole of evolution, triggering a battle between innate and adaptive immune systems of hosts versus the virulence of pathogens adapted by high mutagenesis rates and horizontal gene transfer to provide novel resistance mechanisms. This evolutionary race between pathogens and hosts has dramatically accelerated since humans started to treat infectious diseases with antibiotics ∼70 years ago. The broad use of antibiotics radically reduced the number of serious infections and death tolls but simultaneously selected bacteria less susceptible or resistant to antibiotic drugs. On average, resistant pathogens appear in clinics ∼2−5 years after an antibiotic is applied on a larger scale.1 One of the first clinically relevant examples was methicillin-resistant Staphylococcus aureus (MRSA), which reminded humans about the constant health threat of bacterial infections. Nowadays, it is expected that current treatment options will most likely fail after a few years. This consideration in the face of epidemics known from previous centuries has triggered the search for new antimicrobial substances that can kill bacteria by novel mechanisms to overcome current resistances. Antimicrobial peptides (AMPs), discovered in vertebrates and invertebrates as part of their innate immune systems, have © XXXX American Chemical Society

often been suggested as therapeutic options for treating systemic infections.2 Despite many promises, these geneencoded linear or cyclic peptides have either not been investigated in animal models or failed in preclinical studies for different reasons, such as small therapeutic windows (especially AMPs exhibiting membranolytic mechanisms) or unfavorable pharmacokinetics, such as fast proteolytic degradation or (renal) clearance rates. Proline-rich AMPs (PrAMPs), which were identified in insects and mammals around 25 years ago, appear to be more promising. Insect-derived PrAMPs, such as apidaecin, drosocin, and pyrrhocoricin isolated from the hemolymph of honeybees (Apis mellifera), fruit flies (Drosophila melanogaster), and the European sap sucking-bug (Pyrrhocoris apterus), respectively, are ∼20 amino acid residues long and mostly active against Gram-negative bacteria.3−6 PrAMPs isolated from granulocytes in mammalian species are typically ∼40−60 residues long, such as bactenicin Bac7, PR-39, ChBac5, and OaBac5α isolated from cattle (Bos taurus), pig (Sus domesticus), sheep (Ovis aries), and goats (Capra aegagrus hircus).7−9 Because of their low cytotoxicity and slow degradation by serum proteases, native, structurally modified Received: April 1, 2015

A

DOI: 10.1021/acs.jproteome.5b00283 J. Proteome Res. XXXX, XXX, XXX−XXX

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Journal of Proteome Research Table 1. Peptide Codes and Sequences of Peptides Used in the Current Studya no. 1 2 3 4 5 6 7 8 9 10 11 12

code L-apidaecin

1b Cf-L-Api1b Tyr7Bpa L-Api88 Cf-L-Api88 Cf-L-Api88 Tyr7Bpa Bi-L-Api88 Bi-L-Api88 Tyr7Bpa Bi-D-Api88 Tyr7Bpa L-Api88-Ahx-Ahx-Cys D-Api88-Ahx-Ahx-Cys L-Api137 Cf-L-Api137 Tyr7Bpa

sequenceb

MIC [μg/mL]

Kdc [μmol/L]

GNNRPVYIPQPRPPHPRL-NH2 Cf-GNNRPVBIPQPRPPHPRL-NH2 gu-ONNRPVYIPRPRPPHPRL-NH2 gu-O(Cf-SG)NNRPVYIPRPRPPHPRL-NH2 gu-O(Cf-SG)NNRPVBIPRPRPPHPRL-NH2 gu-O(Bi-SG)NNRPVYIPRPRPPHPRL-NH2 gu-O(Bi-SG)NNRPVBIPRPRPPHPRL-NH2 gu-o(Bi-SG)nnrpvbiprprpphprl-NH2 gu-ONNRPVYIPRPRPPHPRL-ffC-NH2 gu-onnrpvyiprprpphprl-ffC-NH2 gu-ONNRPVYIPRPRPPHPRL-OH gu-O(Cf-SG)NNRPVBIPRPRPPHPRL-OH

1 128 2 16 8 2 2 n.d. n.d. n.d. 1 32

n.d. 2.9 ± 1.6 n.d. 5.0 ± 1.2 1.3 ± 0.4 n.d. n.d. n.d. n.d. n.d. n.d. 2.9 ± 0.6

a

Minimal inhibitory concentrations against E. coli BL21 AI were determined in 33% TSB Medium. Dissociations constants (Kd) were determined using fluorescence polarization. bB, CF, f, and gu denote L-p-benzoyl-phenylalanine (Bpa), 5(6)-carboxyfluorescein, 6-aminohexanoic acid (Ahx), and N,N,N′,N′-tetramethylguanidino, respectively. Small letters indicate D amino acids. cKd values were determined by fluorescence polarization and are therefore only reported for Cf-labeled peptides.

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MATERIAL AND METHODS Materials were obtained from the following manufacturers: Applichem (Darmstadt, Germany), nonfat dried milk powder and tris(hydroxymethyl)aminomethane (Tris base); Bio-Rad Laboratories GmbH (Munich, Germany), Precision Plus Protein Dual Xtra protein standard; Biosolve BV (Valkenswaard, Netherlands), dimethylformamide (DMF, peptide synthesis grade) and acetonitrile (HPLC-S gradient grade); Biozym Scientific GmbH (Hessisch Oldendorf, Germany), streptavidin-APC conjugate and Advansta Washing buffer; Carl Roth GmbH (Karlsruhe, Germany), dithiothreitol (DTT, ≥99%) and dichloromethane (DCM); Gibco (Paisley, UK), phosphate buffered saline (PBS, pH 7.4); Life Technologies GmbH, SulfoLink Coupling Resin; Merck KGAa (Darmstadt, Germany), diethyl ether (puriss); MultiSynTech GmbH, Rink amide 4-methylbenzhydrylamine (MBHA) resin and 2-(1Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronoium hexafluorophosphate (HBTU); Merck Schuchardt OHG (Hohenbrunn, Germany), D- and L-Fmoc-p-benzoylphenyl-alanine (FmocBpa); Orpegen Pharma GmbH (Heidelberg, Germany) or MultiSynTech GmbH (Witten, Germany) or Iris Biotech (Marktredwitz, Germany), all 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids, Fmoc-6-aminohexanoic acid (Fmoc-Ahx)-OH, and Fmoc-(Nδ-4-methyltrityl)ornithine-OH (Fmoc-Orn(Mtt)-OH); Phenomenex Inc. (Torrance, CA, USA), Jupiter C18-columns (internal diameter (ID), 21.2 mm; length, 250 mm; particle size, 15 μm; pore size, 30 nm; ID, 10 mm; length, 250 mm; particle size, 5 μm; pore size, 30 nm; and ID, 2 mm; length, 150 mm; particle size, 5 μm; pore size, 30 nm); Serva Electrophoresis GmbH (Heidelberg, Germany), acrylamide/bis(acrylamide) (30% T, 2.67% C), TEMED, ammonium persulfate (99%), glycine (>98.5%), protease inhibitor mix M, Tween 20 (pure), and trypsin (sequencing grade, MS approved); Sigma-Aldrich GmbH (Taufkirchen, Germany), biotin, 1,2-ethandithiole (≥98%), m-cresole (99%), thioanisole (≥99%), N,N′-diisopropylcarbodiimide (DIC, >98% by GC), N,N-diisopropylethylamine (DIPEA), 5(6)carboxyfluorescein (Cf, for fluorescence), 1-hydroxy-benzotriazole (HOBt, >98%), trifluoroacetic acid (TFA, UV-grade for HPLC), TFA (purum) for peptide synthesis; N-methylmorpholine (NMM, >95% GC), triisopropylsilane (TIS), tryptic soy broth (TSB), and sodium chloride (NaCl, ≥99.5%).

natural or artificial PrAMPs (“designer peptides”) with superior antibacterial activities and high serum stabilities and antibacterial efficacy have been studied in different murine infection models by several research groups.10−15 Api88, for example, is a chemically optimized analogue of apidaecin 1b with improved antibacterial activities. This 18-residue peptide is highly efficient in systemic Escherichia coli and Klebsiella pneumoniae septicemia infection mouse models.13,14 In contrast to most other AMPs, PrAMPs do not kill bacteria by membrane-active mechanisms but are internalized by bacteria via the ATP-binding cassette transporter (ABC transporter) SbmA16,17 and bind to intracellular targets. First, it was shown that pyrrhocoricin binds specifically to the 70 kDa bacterial heat shock protein DnaK and most likely nonspecifically to 60 kDa chaperonin GroEL.18 Further studies revealed that PrAMPs bind to the substrate-binding domain of DnaK in two different binding modes (forward and reverse) with micromolar dissociation constants (Kd).19−21 A recent study comparing protein profiles in Escherichia coli using an iTRAQ approach showed that chaperonin GroEL and its cofactor GroES are both downregulated in the presence of apidaecin 1b.22 Overexpression of GroEL and GroES reduced the susceptibility of E. coli even more than overexpression of DnaK, DnaJ, and GrpE. The same group also identified the cell division protease ftsH, which participates in membrane lipid homeostasis and might interfere with the biosynthesis of lipopolysaccharides (LPS) and phospholipids.23 It remains undetermined, however, if these proteins are indeed lethal targets or only highly expressed binding partners of PrAMPs, as indicated in the latter two studies as potential resistance mechanisms against apidaecin 1b. Thus, we applied two approaches to identify bacterial binding partners of Api88. Affinity chromatography with immobilized Api88 enriched only a few proteins at low quantities, besides DnaK, as dominant proteins. The cross-linking strategy relied on Api88 substituted with photoreactive p-benzoyl-phenylalanine (Bpa) at Tyr7 and a biotin for specific enrichment.24,25 E. coli was incubated with this biotinylated L-Api88 Tyr7Bpa, the corresponding D-Api88 Tyr7Bpa, or without peptide as controls and irradiated with UV light. After enrichment, 41 proteins were identified by nanoRP-UPLC-ESI-QqTOF-MS/ MS in the L-Api88 Tyr7Bpa samples to have higher quantities than in both controls. B


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Journal of Proteome Research

7.5) containing protease inhibitor mix M (1%, v/v) and sonicated on ice. The lysate was centrifuged (12,000g, 5 min, RT), and the protein concentration of the supernatant (cell lysate) was determined using a Bradford assay, adjusted with Tris-HCl (0.1 mol/L, pH 7.5) to a protein concentration of 5 g/L, and stored at −20 °C. The cell lysate (5 μL) was mixed with Tris-HCl (0.1 mol/L, pH 7.5; 1.25 μL) and either water (6.25 μL) or an aqueous solution of L-Api88 (3.64 mmol/L; 6.25 μL) for 10 min (RT), and Cf-L-Api88 Tyr7Bpa (2.5 μL, 2.89 μmol/L) dissolved in Tris-HCl (0.05 mol/L, pH 7.5) was added. The samples (15 μL) were incubated (10 min, RT), irradiated (350 nm) with a dosage of 4.5 J/cm2 (controlled by a dosimeter; ∼10 min), mixed with Laemmli buffer (10 μL), and heated (5 min, 95 °C). The sample was separated by SDS-PAGE immediately, as described below.

Water was produced in house using a Purelab Ultra water purification system (resistance >18.2 MΩ*cm; total organic content 95% probability) were considered as confident. Minimal inhibitory concentrations (MICs) were determined in a serial dilution assay using 33% TSB medium.13,26 Dissociation constants (Kd) were obtained for Cf-labeled peptides using fluorescence polarization.32

Figure 1. SDS-PAGE of an E. coli MC4100 lysate before (1) and after enrichment by affinity chromatography using L-/D-Api88-Ahx-AhxCys-SulfoLink resin (lanes 2 and 3). MW indicates the molecular weight standard containing proteins with the masses shown on the left.

Despite different attempts to optimize the washing and elution conditions of the affinity chromatography, to load larger protein quantities, or to use the lysate of a DnaK-null mutant of E. coli, it was not possible to detect further significant differences in the band patterns (data not shown). More problematic, the differences in the band patterns were difficult to reproduce. Thus, we changed the strategy and synthesized peptide analogues of L-Api88 substituting Tyr7 with the photoreactive amino acid Bpa, which can cross-link the peptide to binding partners after UV irradiation,24,25 as well as the corresponding D-Api88 derivative as a negative control. To detect specifically cross-linked proteins, we coupled 5(6)-carboxyfluorescein via the δ-amino group of Orn1 to L-Api88 and L-Api88 Tyr7Bpa either directly or via a dipeptide linker (Gly-Ser), respectively (Table 1). As Cf-L-Api88 and Cf-L-Api88 Tyr7Bpa bound with similar dissociation constants of 5.0 ± 1.2 and 1.3 ± 0.4 μmol/ L, respectively, to DnaK, we assumed that the substitution does not disturb Api88-protein interactions significantly. MIC-values of 16 μg/mL (Cf-L-Api88) and 8 μg/mL (Cf-L-Api88 Tyr7Bpa) against E. coli BL21AI, which were 16- and 8-fold worse than that determined for L-Api88 indicated moderate antimicrobial activities, indicating that neither carboxyfluorescein nor substitution of Tyr7 abolished the antimicrobial activity. Thus, both Cf-labeled derivatives should kill bacteria by the same mechanisms as Api88. The cell lysate obtained from E. coli BL21AI after incubation and irradiation with Cf-L-Api88 Tyr7Bpa (in vitro cross-link) displayed a complex protein pattern (more precisely, Trpcontaining proteins) with many bands of different intensities in SDS-PAGE, as expected for such a complex protein mixture (Figure 2A). The protein pattern was not affected by the addition of L-Api88 in 63-fold excess. In contrast, the band pattern of the Cf-L-Api88 Tyr7Bpa sample obtained by recording the fluorescence of 5(6)-carboxyfluorescein looked completely different, displaying only a few intense bands beside many weak bands (Figure 2B, Api88). This clearly indicates that Cf-L-Api88 Tyr7Bpa did not just cross-link the dominant bacterial proteins but appeared to act more specifically on distinct binding partners present in the cell lysate. When the E. coli lysate was competitively incubated with Cf-L-Api88

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RESULTS The L- and D-Api88-based affinity chromatography enriched only a few proteins from the E. coli cell lysates based on the band pattern in SDS-PAGE (Figure 1). Most band intensities were very similar between both samples, but a few bands in the L-Api88 samples were more intense. After in-gel digestion, the corresponding proteins were identified as chaperon protein DnaK, transketolase 1, cold shock DEAD-box protein A, and 30S ribosomal protein S1 with the last protein also being detected in the corresponding gel piece of the D-Api88 sample. D


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experiment also considers the bacterial uptake of the peptide to reach certain cellular components and thus reflects more closely the mode of action than the in vitro experiments on cell lysates. An E. coli culture (mid-logarithmic growth phase) was split into three equal aliquots and incubated with Bi-L-, Bi-DApi88 Tyr7Bpa, or without a PrAMP. The corresponding Western blots probed with allophycocyanin-labeled streptavidin (streptavidin-APC) displayed in all three samples an intense band at an apparent molecular weight of 20 kDa (Figure 3A),

Figure 2. SDS-PAGE of an E. coli BL21AI lysate incubated in vitro with Cf-Api88 Tyr7Bpa without the addition of competing Api88 or with 63-fold molar excess of Api88 (+). Tryptophan-containing proteins were detected by the fluorescence of oxidized tryptophan residues (panel A). Cross-linked proteins were detected in the gel by the fluorescence of 5(6)-carboxyfluorescein (panel B).

Tyr7Bpa in the presence of a 63-fold molar excess of Api88, the fluorescence intensities of the previously intense bands decreased significantly (Figure 2B, lane Api88+). The weak bands of the image were typically not affected, indicating that they represent a nonspecific background (Figure 2B). This clearly demonstrates the specific interaction of L-Api88 Tyr7Bpa with several E. coli proteins. When the experiments were repeated with the structurally related peptides Cf-Lapidaecin 1b Tyr7Bpa and Cf-L-Api137 Tyr7Bpa instead of CfL-Api88 Tyr7Bpa, the band patterns obtained in the absence or presence of apidaecin 1b and Api137 as competitors (Figure S1, Supporting Information), respectively, displayed very similar band patterns. This additionally confirms that the peptides cross-linked mostly to specific binding partners. Unfortunately, we were not able to identify the cross-linked proteins because nanoRP-UPLC-ESI-QqTOF-MS/MS of the in-gel digests yielded too many proteins. It was also not possible to identify peptides cross-linked to Api88. Twodimensional gel electrophoresis resolved the protein pattern much better, but then the Cf-based intensities were relatively low even when loading the highest possible protein quantities (data not shown). Additionally, it was not possible to match the spots of the Cf-based image to the protein image. Most likely, only a small part of the binding protein was cross-linked to Api88 resulting in two distinct spots (i.e., the protein and the cross-linked protein) because of major shifts in the isoelectric point and the molecular weight related to the cationic 2.3 kDa Api88 sequence. As Api88, Api137, and apidaecin 1b showed identical behaviors in three different E. coli strains (Figures S1 and S2, Supporting Information), we continued all following experiments with only E. coli BL21AI and Api88. As the cross-link strategy worked well in vitro, we tested it also on E. coli BL21AI cell cultures in mid-logarithmic growth phase. Therefore, 5(6)-carboxyfluorescein was substituted with biotin for enriching the cross-linked proteins specifically with streptavidin, assuming that this will not affect the cross-linking experiment significantly. The cell cultures were incubated with Bi-L- or Bi-D-Api88 Tyr7Bpa using the same conditions typically used to determine MICs as a measure of the antimicrobial activity. Interestingly, L-Api88, Bi-L-Api88, and Bi-L-Api88 Tyr7Bpa were equally active (MIC = 2 μg/mL) and more active than the corresponding Cf derivatives. This

Figure 3. Western blot (A) and SDS-PAGE (B) of E. coli BL21AI lysates (50 μg) without prior enrichment and after enrichment on streptavidin beads, respectively. The cells were incubated without (control), with biotinylated D-Api88 Tyr7Bpa (D-Api88), or with LApi88 Tyr7Bpa (L-Api88) (in vivo cross-link). Lane 1 contains the protein standard (MW).

which most likely corresponds to the biotin carboxyl carrier protein of acetyl-CoA carboxylase known to be biotinylated in E. coli. This protein provided a good internal standard indicating that the sample preparation was very reproducible, including the Western blots. The control (no peptide added) showed only two more faint bands at approximately 30 and 45 kDa (Figure 3A). The respective protein preparations were enriched on streptavidin beads using native buffer conditions, eluted with sample buffer at 95 °C to accomplish maximal protein recovery, and separated by SDS-PAGE (Figure 3). The lane corresponding to the E. coli culture grown in the absence of PrAMPs displayed only a few very faint bands. The lanes of both other samples displayed very similar band patterns, including several intense bands that were not detected before (Figure 3B). Thus, the protein composition appeared very similar, at least for the detected proteins. In contrast, the LApi88 lane showed many intense bands that were typically more intense than the corresponding bands of the D-Api88 lane (Figure 3A). As the differences between the L-/D-Api88 samples appeared only in the anti-biotin blots, whereas the protein patterns in the gels were very similar, it seemed to be impossible to identify the binding proteins by simply cutting out the corresponding bands and identifying all proteins present, especially when considering their very low quantities relative to the dominant proteins. Furthermore, as the mobility differences between both protein versions depend on the protein and are thus unpredictable, it appears impossible to cut out a small gel piece for further analysis that contains both the cross-linked and the corresponding native proteins. Therefore, each of the three lanes (irradiated samples) was cut in 24 equal E


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Table 2. Proteins Identified by NanoRP-UPLC-ESI-QqTOF-MS/MS in the in Vivo Crosslink Samples in Alphabetical Ordera number of peptides (protein matched intensity sum; protein score) protein

string ID

30S ribosomal protein S1 30S ribosomal protein S10 30S ribosomal protein S4 50S ribosomal protein L1 50S ribosomal protein L3 5-methyltetrahydrofolate-homocysteine methyltransferase 60 kDa chaperonin (groEL protein) adenylosuccinate synthetase alanyl-tRNA synthetase aldehyde-alcohol dehydrogenase ATP synthase alpha chain bifunctional purine biosynthesis protein purH carbamoyl-phosphate synthase large chain cell division protein mukB chaperone protein dnaK ClpB protein dihydrolipoamide acetyltransferase dihydrolipoamide dehydrogenase DNA protection during starvation protein DNA/pantothenate metabolism flavoprotein DNA-directed RNA polymerase beta’ chain DNA-directed RNA polymerase beta chain elongation factor G elongation factor Tu (EF-Tu) formate acetyltransferase 1 glutamate synthase [NADPH] large chain precursor glutamine synthetase hypothetical protein yfbG leucyl-tRNA synthetase Mg(2+) transport ATPase_ P-type 1 outer membrane lipoprotein slyB precursor outer membrane protein F precursor phosphate acetyltransferase phosphoribosylformylglycinamidine synthase polyribonucleotide nucleotidyltransferase prolyl-tRNA synthetase pyruvate dehydrogenase E1 component pyruvate kinase I S-adenosylmethionine:2-demethylmenaquinone methyltransferase transcription termination factor rho trigger factor (TF)

rpsA rpsJ rpsD rplA rplC metH groL purA alaS adhE atpA purH carB mukB dnaK clpB aceF lpdA dps dfp rpoC rpoB fusA tufA pflB gltB gln A arnA leuS mgtA slyB ompF pta purL pnp proS aceE pykF rraA rho tig

control 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 3 1 4 0 0 4 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0

(2837; 65)

(21492; 311) (4474; 80) (29241; 269)

(44500; 904)

(259389; 1674)

D-Api88

0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 2 1 1 0 0 3 0 0 2 0 0 0 3 0 0 0 0 0 0 0 0 0 0

(8201; 261)

(15063; 344) (1345; 92) (1837; 82)

(22676; 424)

(15607; 171)

(124781; 1345)

L-Api88

4 5 3 3 3 3 14 3 4 6 3 4 15 3 11 4 15 3 1 3 35 22 6 8 4 10 7 3 5 4 3 5 6 10 5 4 8 9 3 6 13

(81899; 1958) (106889; 3704) (21313; 680) (55602; 1326) (21695; 798) (9664; 103) (1215222; 3844) (25818; 300) (20020; 175) (61216; 517) (13809; 198) (57064; 465) (485704; 1084) (18562; 110) (691975; 2259) (39133; 202) (856897; 2331) (69235; 452) (2915; 272) (54576; 513) (1896085; 3290) (1350034; 1710) (58213; 441) (532660; 2949) (42997; 246) (176602; 455) (587583; 2038) (36010; 196) (38884; 176) (27985; 254) (758633; 5588) (94096; 1172) (72050; 459) (182248; 413) (41898; 408) (22726; 190) (198761; 641) (852278; 2278) (14265; 349) (37434; 532) (633202; 2204)

band 5 18 14 13 13 3 6, 8 4 4 7 7 3 2 5 4 5 7 17, 8, 3 3 5 8, 5 3 7 6 4 4 19, 10 5 3 5 6 4 7 16 8 7

7

18 9

9

20

a

E. coli was incubated with L-Api88 Tyr7Bpa, D-Api88 Tyr7Bpa, or without either peptide (control), photo-crosslinked, and lysed. The crosslinked proteins present in the cell lysates were enriched with streptavidin and separated by SDS-PAGE. The lanes were cut into 24 pieces and digested with trypsin (Figure S3, Supporting Information). Given are the peptide sequences identified in the different bands of each sample, the corresponding m/z, charge state, and the peptide score obtained by PLGS. The proteins identified by these peptides are shown at the end of the peptide list (light green background) including their string ID and the pathway(s) involved. For further details about the identified peptides, see Table S1 in the Supporting Information.

pieces (Figure S3, Supporting Information) assuming that gel pieces at the same horizontal positions of the three lanes should contain the same proteins if present. This was at least indicated by the identical gel and blot band patterns obtained for all three sample lanes (Figure 3). Proteins present in the 72 gel pieces were digested with trypsin in parallel and analyzed by nanoRPUPLC-ESI-QqTOF-MS/MS using positive ion MSE. Because of the long analysis time of roughly 3 days, the three protein digests resulting from the same horizontal gel pieces of the three sample lanes were analyzed consecutively in the order of the expected protein contents (i.e., control, D-Api88, and then

L-Api88

sample) to minimize differences in the analytical conditions (e.g., retention time, temperature, and spray). The analysis of the gel was pursued from top to bottom (i.e., from high to low mass range). In total, we could identify six proteins in all three samples and an additional 35 proteins in the L-Api88 sample considering proteins identified by at least three peptides with confident scores (seven or more matching signals of the MS/MS) (Table 2). The number of identified proteins increased in the same order as the band intensities increased in the Western blot (Figure 3). The signal intensities of all peptides matching the F


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among these 41 proteins were calculated with high confidence score (0.7) using the MCL algorithm (parameter value of 3).34 Two large and four small clusters (2−4 proteins) were formed. Three separate proteins interacted with these clusters, whereas no interactions were displayed for six proteins. The largest cluster contained 16 proteins forming the 70S ribosome or closely related to protein translation, such as 30S (rpsA, rpsD, and rpsJ) and 50S ribosomal proteins (rplA and rplC) and transcription termination factor (rho). The second and third largest cluster contained six proteins involved in pyruvate metabolism (Figure 4, red) and four proteins of purine metabolism (Figure 4, orange). Ala-, Leu-, and Pro-tRNA synthetase (alaS, leuS, and proS) formed a separate cluster.

six proteins detected in all three samples calculated with the PLGS software (protein matched peptide intensity sum) indicated that four proteins appeared at equal quantities and three of the L-Api88 at significantly higher quantities (Table 2). Although the biotin carboxyl carrier protein of acetyl-CoA carboxylase presumably detected in the streptavidin blots (see above) was not identified here, two peptides corresponding to the protein were detected with confident scores and, importantly, very similar signal intensities in all three samples (10% error range). This confirmed again that the applied strategy was valid and allowed the relative quantification of proteins among the three sample lanes. Upon first view, the number of identified proteins appeared unexpectedly high, but it should be considered that isolation and enrichment with streptavidin using nondenaturing conditions do not enrich only the biotinylated cross-linked proteins, but also all proteins forming a complex with the crosslinked protein. This is most obvious from the four 30S ribosomal proteins (i.e., proteins S1, S4, and S10) and two 50S ribosomal proteins (i.e., L1 and L3) identified (Table 2). These proteins are components of the bacterial 70S ribosome and might be detected even if only one of the five proteins was cross-linked. The same is true for other proteins that bind to the ribosome, such as elongation factors G, and Tu as well as transcription termination factor rho and trigger factor. Thus, the list of binding proteins was further evaluated by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, version 9.1) analysis (Figure 4)33 [www.string-db.org; accessed

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DISCUSSION The increasing health threats triggered by resistant bacteria and the global spread of formerly regional bacterial pathogens have revived the interest in antibiotics and have especially triggered the search for new antimicrobials and valid bacterial targets.35 The interest in PrAMPs during the last two decades has focused much on their nonlytic mode of action and the development of analogues with superior efficacies.35 DnaK was the first suggested target of PrAMPs isolated, including a detailed characterization of the molecular interactions.18,20,21,27 However, similar susceptibilities of E. coli strain BW25113 and the corresponding DnaK null mutant JW0013 against different PrAMPs36 indicated that DnaK might not be the only target protein. The term “target” typically refers to a cellular component hit by a substance that triggers or inhibits certain biological processes, such as reduced bacterial growth (bacteriostatic) or cell death (bactericidal) in response to antibiotics. As this study did not intend to test biochemical and functional aspects in the cellular context, we use the terms binding partner or binding protein. It should be noted that most likely not all proteins are real binding partners, as the nondenaturing conditions of streptavidin enrichment will also yield protein complexes present in the cells or formed during protein isolation. The presented data clearly indicate that Api88 interacts with the 70S ribosome, which we were able to confirm in a parallel study showing that different PrAMPs bind strongly to the ribosome with dissociation constants often in the low nanomolar range.36 Oncocin analogues Onc72 and Onc112 even inhibited protein translation in vitro very efficiently, whereas apidaecins appeared to be less efficient, but further research is necessary to test these effects in vivo. Interestingly, an independent study on Bac7 showed shortly afterwards that this shortened bovine PrAMP also binds to the 70S ribosome in E. coli to inhibit protein translation but not transcription.37 Affinity chromatography using Api88 as ligand confirmed DnaK as a binding partner and indicated three additional proteins, which we did not trust at this stage. However, 30S ribosomal protein S1 was later confirmed by the cross-linking strategy and thus might represent a strong interaction partner of Api88 in vitro and in vivo, pointing to the major ribosome binding site of Api88. Several aspects can limit the L-Api88based affinity chromatography, although similar strategies are commonly and successfully applied to other ligands.27 Sterical hindrances between the stationary phase and a binding partner can prevent the enrichment of proteins and especially protein complexes, particularly if the binding sites are deep inside a protein complex. In contrast, the cross-link strategy relies on

Figure 4. Confidence view of the interactome of 41 proteins specifically enriched by L-Api88 Tyr7Bpa or at higher contents than in the controls. The calculations used the STRING database of E. coli K12 strain MG1655, a high confidence score (0.7), and an MCL clustering of 3 (accessed on June 4th, 2015). Some knots were manually shifted for better illustration.

on March 20, 2015]. The STRING database provides information on both known and predicted protein−protein interactions as well as cellular functions and pathways of query proteins allowing for judgement of interactions among cellular proteins. This interactome was studied for 41 proteins detected specifically or at higher contents in the L-Api88 Tyr7Bpa sample (Figure 4) using the STRING database of E. coli K12 strain MG1655 (accessed on June 4th, 2015). The interactions G


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split the proteins first into protein complexes and then look for potential targets or strong binding partners in this subgroup of proteins. STRING analysis allows for retrieving confirmed or predicted protein−protein interactions and thus to subgroup proteins into protein complexes, which also includes temporary complexes with short half-life times. Interestingly, DnaK did not cluster with any of the other proteins, indicating that it should be isolated as a single protein, which is in agreement with its enrichment by affinity chromatography using Api88 as a ligand. Though the exact binding site of Api88 to the bacterial ribosome is not known, it strongly binds to the 70S ribosome, indicating together with inhibition of in vitro translation that the ribosome is an important target. The identified ribosomal proteins indicate that Api88 might bind somewhere between the 50S and 30S subunits close to the 3′-end of the 23S rRNA. Furthermore, the 30S ribosomal subunit protein S4 (rpsD) binds to both beta (rpoB) and beta’ subunits (rpoC) of DNAdirected RNA polymerase, which might therefore be enriched in a complex, although it is tempting to speculate that an interaction with either RNA polymerase could inhibit DNA transcription. Further studies on individual proteins or smaller parts of the ribosome, such as the 30S and 50S subunits, or Xray crystallography on the Api88-ribosome complex, are needed to reveal the interaction sites specifically. The different clusters, however, indicate that Api88 and most likely other PrAMPs kill bacteria by a multimode mechanism interfering with proteins in different parts of the cell.

free compounds that should be able to reach all binding sites, as suggested here by only slightly increased MIC values, which depend on target binding but also on bacterial uptake. The structural similarities between Tyr and Bpa should only slightly affect the binding constants but not the binding mode. This prerequisite was confirmed with Cf-labeled derivatives, which showed only a few intense bands, indicative of strong interactions between Api88 derivatives and a few bacterial proteins in the cell lysate. The observation that the Cf-related fluorescence intensity did not correlate to the protein pattern further confirmed the specificity. When 5(6)-carboxyfluorescein was replaced by biotin, only a few labeled proteins were efficiently enriched. The band patterns seen in the Western blots and the Cf-fluorescence images were similar though they differed much in the relative band intensities. The minor changes might be related to the different imaging techniques and, more importantly, the differences between cell lysates and cell culture incubations. Assuming that streptavidin-enriched proteins cross-linked to Bi-L-Api88 Tyr7Bpa reflect the Api88-protein interactions closely at the time point of irradiation, it is surprising to see how much the SDS-PAGE of this sample differed from the sample enriched initially by affinity chromatography using Api88 as ligand. Importantly, DnaK was present at much lower amounts despite Cf-L-Api88 Tyr7Bpa and Cf-L-Api88 binding equally well to DnaK in vitro. The lower enrichment of DnaK probably indicates that Api88 cannot efficiently approach DnaK in the cell, although it is present at high quantities in E. coli. This might be related to the chaperone cycle where DnaJ binds the substrate first, forms a complex with DnaK, transfers the substrate to DnaK, and then is released again to catch another substrate.38 Supposing that biotin and Bpa substitution do not seriously affect protein binding, Bi-L-Api88 Tyr7Bpa will closely resemble the mechanism of Api88. Thus, two aspects will influence the amount of proteins enriched: the binding strength between Api88 and the protein as well as the content of this protein in E. coli. Analytical techniques can easily identify strongly binding abundant proteins but may miss less abundant targets. Even more challenging, weak unspecific interactions with highly abundant proteins will result in an “unspecific” background that makes it difficult to identify the real targets. Conversely, Api88 might strongly bind to some bacterial proteins without affecting their functions, which therefore would not be considered protein targets. These proteins will capture Api88, reduce the intracellular concentration of free Api88, and thereby reduce target inhibition. If present, such proteins might even represent a kind of bacterial defense mechanism. The experimental setup using a relatively short UV irradiation period to cross-link the proteins after preincubating an E. coli culture should capture all targets and strong binding partners, but unbound Bi-L-Api88 Tyr7Bpa will additionally produce an unspecific background. The free molecules will cross-link to “any” protein nearby (i.e., mostly dominant proteins having the highest probability to react). The identification of DnaK and GroEL, which have both been reported as target proteins, and the ribosome, which was confirmed by in vitro translation and binding studies, confirmed that the list of Api88 binding partners probably contains further target proteins (Table 2). The major challenge will be to identify valid targets. Assuming that Bpa in Api88 Tyr7Bpa will cross-link to only one protein or maybe a few proteins of a protein complex (e.g., 70S ribosome), it would be interesting to

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CONCLUSION The cross-linking strategy using L-Api88 Tyr7Bpa derivatives to identify binding proteins in E. coli and D-Api88 Tyr7Bpa to exclude nonspecific interactions was highly efficient, as the previously suggested target proteins DnaK and GroEL were confirmed here as interaction partners in vivo. Additionally, five binding partners of a likely target, the bacterial 70S ribosome, were identified. The remaining 36 proteins, of which 30 were separated into six clusters in a STRING analysis, most likely contain further protein targets of PrAMPs that will help to describe the multimodal mode of action assumed for most antimicrobial peptides. Thereby, AMPs disturb different bacterial functions, reducing the risk of resistance mechanisms.

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ASSOCIATED CONTENT

* Supporting Information S

(S1 and S2) Data for all strains and AMPs used for the in vitro cross-link in the absence or presence of the corresponding AMP. (S3) Illustration of the gel cutting used for in-gel digestion. Supplementary Table S1 contains detailed data of MS analyses.The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jproteome.5b00283.

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AUTHOR INFORMATION

Corresponding Author

*Tel. +49 (0)341 9731330. Fax. +49 (0)341 9731339. E-mail: hoff[email protected]. Notes

The authors declare the following competing financial interest(s): Prof. Dr. Ralf Hoffmann is a cofounder of AMP Therapeutics GmbH (Leipzig, Germany) and member of the scientific advisory boards. H


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ACKNOWLEDGMENTS We thank Tina Goldbach for technical assistance in peptide synthesis and purification. Financial support by the Federal Ministry of Education and Research (BMBF; Project no. 01GU1104A), the European Fund for Regional Structure Development (EFRE, European Union and Free State of Saxony; Project no. 100105139 and 100127675), the Deutsche Forschungsgemeinschaft, and the Free State of Saxony (Project no. INST 268/289-1) is gratefully acknowledged.

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ABBREVIATIONS ABC, ATP-binding cassette; Ahx, 6-aminohexanoic acid; AMPs, antimicrobial peptide; Bpa, p-benzoyl-phenylalanine; APC, allophycocyanin; Bi, biotin; Cf, 5(6)-carboxyfluorescein; DMF, dimethylformamide; DTT, dithiothreitol; Fmoc, 9fluorenylmethoxycarbonyl-; HOBt, 1-hydroxy-benzotriazole; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronoium hexafluorophosphate; Kd, dissociation constant; MHBA, 4methylbenzhydrylamine; MIC, minimal inhibitory concentration; MRSA, methicillin-resistant Staphylococcus aureus; PrAMP, proline-rich antimicrobial peptide; RP, reversedphase; TFA, trifluoroacetic acid; Tris base, tris(hydroxymethyl)aminomethane; TSB, tryptic soy broth; STRING, Search Tool for the Retrieval of Interacting Genes/ Proteins; UPLC, ultrahigh performance liquid chromatography

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