Composition and Antimicrobial Activity of the Skin ... - ACS Publications

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Composition and Antimicrobial Activity of the Skin Peptidome of Russian Brown Frog Rana temporaria T. Yu. Samgina,1 E. A Vorontsov,1 V. A. Gorshkov,1 E. Hakalehto,2 O. Hanninen,3 R. A. Zubarev,4 and A. T. Lebedev1,* 1

Organic chemistry Department, Moscow State University, Moscow, Russia Department of Biosciences, University of Eastern Finland, P.O.B. 1627, FI-70211 Kuopio, Finland 3 Department of Physiology, University of Eastern Finland, P.O.B. 1627, FI-70211 Kuopio, Finland 4 Department of Medicinal Biochemistry and Biophysics, Division of Molecular Biometry, Karolinska Institutet, Stockholm, Sweden 2

ABSTRACT: A nano-HPLC-ESI-OrbiTrap study involving HCD and ETD spectra has been carried out to clarify the composition of the skin peptidome of brown Russian frogs Rana temporaria. This approach allowed determinantion of 76 individual peptides, increasing 3-fold the identified portion of the peptidome in comparison to that obtained earlier with FTICR MS. A search for the new bradykinin related peptides (BRPs) was carried out by reconstructing mass chromatograms based on the ion current of characteristic b- and y-ions. Several peptides were reported in the secretion of R. temporaria for the first time. The overall antibacterial activity of the skin secretion in general and of one individual peptide (Brevinin 1Tb) was determined using PMEU Spectrion (Portable Microbe Enrichment Unit) technology. The inhibitory effects of these peptides on Staphylococcus aureus and Salmonella enterica Serovar typhimutium were equal in scale to that reported for some antibiotics. KEYWORDS: peptides sequencing, mass spectrometry, HCD, ETD, Salmonella, Staphylococcus aureus, MRSA, antimicrobial frog peptides, antibiotics, PMEU, bacterial growth inhibition

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INTRODUCTION Host-defense antimicrobial peptides constitute the major portion of amphibian skin secretion, being a part of their innate immunity. They may be treated as a natural weapon against predators and pathogenic microorganisms.1−4 This feature was used by the native population of Russia and Finland a long time ago. For example, milk souring could be prevented if a frog was placed into the milk vessel. Also, it was believed that frogs were living only in wells with clean water. The skin secretion of Brown frog Rana temporaria has been studied earlier rather thoroughly.1−5 Various biologically active peptides of European species of R. temporaria are listed in review.6 They were discovered in the skin secretion, being predicted earlier by cDNA analysis. Some of the determined peptides became the first representatives of the new families, later discovered during the studies of other ranid frog species (temporins, melittinrelated peptides). Russian species of R. temporaria were studied by means of tandem mass spectrometry.7 It was demonstrated that the composition of the skin peptidome is slightly different depending on the territory of inhabitation.8 Thus, 21 peptides were identified in the skin peptidome of Zvenigorod (Moscow region, Russia) population of R. temporaria,7 while three of them were new ones: disulfide containing brevinin-1Tb, ornithokinin [Thr 6 ,Leu 8 ]bradykinin, and temporin-M © XXXX American Chemical Society

FLPLLGKVLSRVL−NH2. The work was done with FTICR mass spectrometer LTQ FT (Thermo) at 100 000 resolving power. Complementary collisionally induced dissociation (CID) and electron capture dissociation (ECD) spectra were recorded. This approach has demonstrated its efficiency for the elucidation of the skin secretion composition of various frogs.9−12 Among 21 peptides characteristic for R. temporaria, 4 belonged to disulfide containing brevinins-1 and -2. Besides that, bradykinin and two of its analogs (ornithokinin [Thr6,Leu8]bradykinin and [Thr6]bradykinin), four bradykinin-related peptides (BRPs), representing nonactive proteolytic copies of bradykinin with C-extension, a melittin-related peptide, and 9 short antimicrobial temporins in amide form were identified. The permanently improving properties of the new types of mass spectrometers, for example, higher acquisition rates accompanied by high mass accuracy, new methods of triggering fragmentation, allow more information about complex mixtures of natural compounds. The present work deals with the study of the peptidome of Kolomna (Moscow region, Russia) population of R. temporaria with nano-HPLC coupled to Received: September 21, 2012

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dx.doi.org/10.1021/pr300890m | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Article

Table 1. Peptides Identified in the Skin Secretion of R. temporaria from the Kolomna Population (Moscow Region, Russia) no. 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 61

sequences

exp. MH+ (Da)

VNPIILGVLPKFVCLITKKC−OH FITLLLRKFICSITKKC−OH LVPLFLSKLICFITKKC−OH GLLSGLKKVGKHVAKNVAVSLMDSLKCKISGDC−OH GLLSGLKKVGKHVAKNVAVSLMoxDSLKCKISGDC−OH GLLSGLKKVGKHVAKNVAVSLM2oxDSLKCKISGDC−OH GLLDGLKKVGKHVAKNVAVSLMDSLKCKISGDC−OH GLLDGLKKVGKHVAKNVAVSLMoxDSLKCKISGDC−OH GLLDGLKKVGKHVAKNVAVSLM2oxDSLKCKISGDC−OH KKFTLNLFHQLKCKIGGGC-OH FIGSALKVLAGVLPSVISWVKQ−NH2 FIGSALKVLAGVLPSVISWoxVKQ−NH2 FIGSALKVLAGVLPSVISW2oxVKQ−NH2 FLPLIGRVLSGIL−NH2 LLPIVGNLLKSLL−NH2 LLPILGNLLNGLL−NH2 LLPIVGNLLNSLL−NH2 VLPIIGNLLNSLL−NH2 FLPLIGKVLSGLL−NH2 FFPVIGRILNGIL−NH2 LSPNLLKSLL−NH2 LLPNLLKSLL−NH2 FVQWFSKFLGRIL−NH2 FVQWoxFSKFLGRIL−NH2 FVQW2oxFSKFLGRIL−NH2 FLPILGKVLSRVL−NH2 FLHypILGKVLSRVL−NH2 FLGALGNALSRVL−NH2 RPPGFSPFR−OH KVRPPGFSPFR−OH FSPFR−OH PGFSPFR−OH HVRPPGFSPFR−OH (416.20)RPPGFSPFR−OH (386.27)RPPGFSPFR−OH PGRPPGFSPFR−OH (400.28)RPPGFSPFR−OH (246.05)RPPGFSPFR−OH V(246.05)RPPGFSPFR−OH VVPPGFSPFR−OH NVRPPGFSPFR−OH AVRPPGFSPFR−OH DDRPPGFSPFR−OH DERPPGFSPFR−OH LDRPPGESPFR−OH (His+27.9932)VRPPGFSPFR−OH WRPPGFSPFR−OH HVRPPGFSPFRIA−OH HVRPPGFSPFRIAPAS−OH LVRPPGFSPFR−OH DVRPPGFSPFR−OH FVRPPGFSPFR−OH LLRPPGFSPFR−OH LRPPGFSPFR−OH (LS)PFR−OH FLRPPGFSPFR−OH GLLRPPGFSPFR−OH FLPRPPGFSPFR−OH GLLSGLKRPPGFSPFR−OH RPPGFTPFR−OH RPPGFSY−OH

2197.31 2026.18 1965.16 3397.86 3413.86 3429.85 3425.86 2294.57 3457.85 2134.15 2311.37 2327.36 2343.36 1396.88 1391.91 1361.87 1377.86 1377.86 1368.87 1457.88 1096.68 1122.74 1639.92 1655.92 1687.92 1453.94 1469.93 1329.78 1060.57 1287.74 653.34 807.42 1296.70 1476.77 1446.84 1214.65 1460.85 1306.65 1405.69 1102.60 1273.68 1230.68 1290.63 1304.64 1270.66 1324.69 1246.65 1480.82 1735.94 1272.72 1274.67 1306.71 1286.74 1173.66 619.36 1320.72 1343.76 1417.78 1728.98 1074.59 823.41

peptides Brevinin 1T Brevinin 1Ta Brevinin 1Tb Brevinin 2T [Metox]brevinin 2T [Met2ox]brevinin 2T Brevinin 2Te* [Metox]brevinin 2Te* [Met2ox]brevinin 2Te* Brevinin 2Tf * (MRP-1) [Trpox]MRP 1 [Trp2ox]MRP 1 Temporin A Temporin B Temporin C Temporin D Temporin E Temporin F Temporin G Temporin H Temporin K Temporin L [Trpox]temporin L [Trp2ox]temporin L Temporin M [Hyp3]temporin M* Temporin N* Bradykinin Bradykinin KR-11* Bradykinin 5−9* Bradykinin 3−9* Bradykinin HR-11* BRP 1* BRP 2* Bradykinin PR-11* BRP 3* BRP-4* BRP-5* [Val2]bradykinin VR-11* Bradykinin NR-11* Bradykinin AR-11* Bradykinin DR-11* Bradykinin DR-11−1* Bradykinin LR-11* BRP-6* Bradykinin WR-10* Bradykinin HI-13* Bradykinin HS-16* Bradykinin LR-11−1* Bradykinin DR-11−1* Bradykinin FR-11* Bradykinin LR-11−2* Bradykinin LR-10* [Leu5]bradykinin 5−9* Bradykinin FR-11* Bradykinin GR-12* Bradykinin FR-12* Bradykinin GR-16* [Thr6]bradykinin [Tyr7]bradykinin RY-7*

B



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Table 1. continued no.

peptides

62 63 64 65 66 67 68 69 70 71 72 73 74 75 76

Bradykinin RS-14 Bradykinin RT-15* Bradykinin RA-11 [desArg9,Thr6]bradykinin * [Thr6]bradykinin RA-11 Bradykinin RA-13* [Thr6]bradykinin RA-13* Bradykinin RI-10 Bradykinin RS-6* [Asp6]bradykinin RA-11* Bradykinin RP-12 [Thr6]bradykinin RI-10 Bradykinin RS-12* Bradykinin RD-13* [Thr6, Leu8]bradykinin

exp. MH+ (Da)

sequences RPPGFSPFRIAPAS−OH RPPGFSPFRIAPAST−OH RPPGFSPFRIA−OH RPPGFTPF−OH RPPGFTPFRIA−OH RPPGFSPFRIAVA−OH RPPGFTPFRIAVA−OH RPPGFSPFRI−OH RPPGFS−OH RPPGFDPFRIA−OH RPPGFSPFRIAP−OH RPPGFTPFRI−OH RPPGFSPFR(LY)S−OH RPPGFSPFRIAVD−OH RPPGFTPLR−OH

1499.81 1560.86 1244.69 918.49 1258.71 1414.80 1428.81 1173.66 660.34 1272.69 1341.75 1187.67 1423.75 1458.78 1040.59

Figure 1. LTQ Orbitrap HCD spectrum of brevinin 2Tf (MH44+). Spectrum represented after charge deconvolution.

resolving power, allowing us to distinguish between the most important isobaric pairs: Lys/Gln, Leu-Ile/Hyp, Phe/Metox, and Tyr/Met2ox. For several years we have carried out manual sequencing of the skin peptides of ranid frogs based on the complementary collisionally activated decomposition (CAD) and electron capture dissociation (ECD) spectra. This approach was proposed as quite reliable and efficient for tryptic peptides. However, it is useful for the nontryptic long peptides, including those containing disulfide bonds as well. The latter issue is very important for the ranid frog peptides.9−12 According to our results, manual sequencing possesses definite advantages, allowing structural analogies between the new and known peptides belonging to the same peptide families to be taken into account. This feature significantly helps with identifications of substitutions in the de novo sequencing peptides. Table 1 summarizes the sequences of the peptides, identified in the present study using the mentioned approach.

LTQ Orbitrap Velos instrument (Thermo). Manual interpretation of mass spectra was carried out to elucidate the sequence. Higher energy collisional dissociation (HCD) and electron transfer dissociation (ETD) mass spectra were used for this purpose. Antimicrobial activity of the crude skin secretion was tested with the PMEU Spectrion cultivator. An individual peptide brevinin 1Tb was tested similarly. Its potential biological activity was hypothesized earlier.13 The PMEU technology has been used before for evaluating both the antibiotic susceptibilities of various bacterial species14,15 as well as the inhibitory effect of defensin peptides on Escherichia coli strains.16

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RESULTS AND DISCUSSION

Identification of Peptides by Mass Spectrometry

The main advantage of Velos Orbitrap over FTICR in the present study involves its higher acquisition rate at adequate C



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Figure 2. LTQ OrbiTrap spectra of temporin N: a) HCD; b) ETD.

(23) were detected together with [Trpox] (12 and 24) and [Trp2ox] (13 and 25). Brevinin 2Te possesses one substitution in its sequence (Ser4→Asp4) in comparison with one of the major component of the peptide secretion of R. temporaria brevinin 2T. Brevinin 2T is a wide spectrum antibiotic,4 while biological activity of brevinin 2Te remains unknown. A proteolytic fragment of a new peptide entitled brevinin 2Tf (10) was identified in the present study. Figure 1 represents HCD spectrum of its quadruply protonated molecule of m/z 534.04.

Table 1 contains 76 peptide sequences. These peptides belong to four families: (1) disulfide containing brevinins-1 and brevinins-2 (1−10); (2) melittin-related peptides, MRPs (11− 13); (3) temporins (14−29); (4) bradykinin and bradykininrelated peptides (30−76). Peptides with Met and Trp in the backbone exist in an oxidized form together with the intact one. Thus, brevinin 2T and brevinin 2Te (4 and 7) were accompanied by methioninesulfoxide (5 and 8) and methioninesulfone (6 and 9). A similar situation was observed with tryptophane: both melittin related peptide (11) and temporin L D



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Figure 3. HCD spectra of BRPs detected by reconstructed mass chromatograms based on the ion current of the characteristic fragment ions: (a) m/z 904.47 and 419.24 (N-extended); (b) m/z 642.33 (C-extended).

The absence of ions below y7 and above b12 in the spectrum proves the presence of C-terminal S−S bond. The accurate mass of ion y7 is 635.264 Da. It coincides with that of y7 of brevinin 2Tl and brevinin 2 Td, with the known sequences, predicted by c-DNA cloning of European frog R. temporaria. However, it was not discovered so far in the skin secretions.3 The sequence clarified by the fragmentation (b-y series) and mass of y7 ion (characteristic of disulfide cycle) allowed defining the final sequence of this proteolytic fragment of brevinin 2Tf (11−29): KKFTLNIFHQLKCKIGGGC−OH.

This sequence contains two substitutions in comparison with that of predicted brevinin-2Tl: Leu18→Phe18 and Lys20→Glu20. Unfortunately a complete version of brevinin-2Tf containing Nterminus was not detected. Two new peptides belonging to temporins were identified in the studied samples: [Hyp3]temporin M (27), distinguished from temporin M by substitution of Pro3 for oxyproline, and temporin N (28). Figure 2 represents HCD and ETD spectra of temporin N: Complementary series of b-y ions, forming due to amide bond cleavages in HCD mode (Figure 2a), provide E



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Figure 4. (a) Growth of Staphylococcus aureus strain in the TYG medium without peptide additions; (b) growth of Staphylococcus aureus strain in the presence of 0.01 mg/mL of M3 sample; (c) growth of Staphylococcus aureus strain in the presence of 0.03 mg/mL of M3 sample; (d) growth of Salmonella enterica Serovar typhimurium strain in the TYG medium without peptide additions; (e) growth of Salmonella enterica Serovar typhimurium strain in the presence of 0.01 mg/mL of M3 sample; (f) growth of Salmonella enterica Serovar typhimurium strain in the presence of 0.03 mg/mL of M3 sample.

complete sequence of temporin N, with the exception of Phe1Leu2 and Arg11-Val12 amino acid residues. These missed sequence details were obtained by registering ions z12 of m/z 1166.77 and c11 of m/z 1117.77, formed by the rupture of the corresponding N−Cα bonds in ETD mode (Figure 2b). These complementary data allowed elucidating complete structure of temporin N: FLGALGNALSRVL−NH2. Temporin M (26) was originally identified in the analysis of R. temporaria skin secretion with LTQ ICR FT.7 Its biological activity remains unknown. As was mentioned already, [Hyp3]temporin M (27) distinguishes from temporin M by only one residue substitution. It is known that any substitution in the sequence

of a peptide may change its biological activity. However, substitution of Pro for Hyp (oxyPro) does not lead to the decrease of activity in the case of bradykinin.6 Bradykinins (29−76) constitute more than a half of the identified peptides (Table 1). Signal nonapeptide bradykinin itself plays an important role in the maintenance of numerous functions of amphibian skin.17 Its several analogs with substitutions in the backbone were detected in the skin secretion of R. temporaria as well. Besides, a variety of bradykinin-related peptides represented by N- or C-terminally extended inactive copies of these nonapeptides were identified. They are formed from prepropeptides as intermediate inactive F



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Staphylococcus aureus with the same peptide concentration (see Figure 4b). The onset of bacterial growth was detected only after about 18 h of cultivation, probably due to the number of cells slowly surviving the peptide addition. This fact allowed for the growth to be seen only after a certain amount of time. The final cfu after 24 h, as seen in Table 2, corresponded to approximately

peptides in the process of releasing active mature peptides under the influence of proteases. Thus, bradykinin itself is formed from its precursor kininogen due to kallikrein, an active C-arginin protease. Therefore, the observed array of intermediate proteolytic fragments gives a clue concerning the pathway of formation of bradykinin itself and its analogs in the amphibian organisms. Such a variety of BRPs in the skin secretion of a single amphibian species has never been reported before. Partially this result may be rationalized by the high data acquisition rate of LTQ Velos Orbitrap instrument. Another important issue realized in the present work deals with the directed search for these compounds (BRPs) using reconstructed mass chromatograms based on the ion current of the characteristic fragment ions. This approach is extremely efficient in the classic environmental mass spectrometry.18 In case of peptides this search may be realized using any intensive b, y, c, z ion. The authors19 reported the detection of N-extended copies of bradykinin using ion y3. In the present work, we were looking for BRPs with N-terminal extension using ions of m/z 419.24 or 904.47 and those with C-terminal extension using ions of m/z 642.33. Figure 3 demonstrates HCD spectra of two BRPs determined that way. A number of bradykinin analogs, for example, [Thr6]bradykinin and [Thr6,Leu8]bradykinin, were detected similarly. Bradykinins with partially resolved sequence were called according to the accepted nomenclature (34, 35, 37−39, 46, Table 1). We used the names BRP-1, BRP-2 and so on. Table 1 contains 49 BRPs detected this way. Peptides reported in the secretion of R. temporaria for the first time are marked with asterisks. Some of them were earlier identified in the secretion of other ranid frogs inhabiting the Moscow region.17 Among the other peptides listed in Table 1 are acidic spacers and numerous proteolytic N- and C-terminal fragments of brevinins-1 and -2, MRPs and temporins. While the acidic spacers are known to be biologically inactive, there is no reliable data on the activity of proteolytic fragments of brevinis and temporins.

Table 2. Effect of the Sample M3 on the Bacterial Growth in the PMEU Spectrion Cultivator as Detected by Plate Culturing after 24 h onto the TYG Medium peptide addition bacterial strain S.aureus S.aureus S.aureus S.typhimurium S.typhimurium S.typhimurium

M3 without peptide 0.01 mg/mL 0.03 mg/mL without peptide 0.01 mg/mL 0.03 mg/mL

cfu/mL (as colonies counted on a plate)

observable growth in the PMEU curves

dilution 10−2a

dilution 10−6a

dilution 10−7a

hours of inoculation

Nd

Full

286

7

Nd

Full

217

7

5

Nd

Nd

no growth

Nd

Full

165

7

Nd

full

45

10

Nd

103

Nd

20

a

The dilutions mean the exponential dilutions of the samples taken from PMEU cultures for inoculation onto the Petri plates. Consequently, 5 colony forming units (cfu) on the plating from dilution −2 means 500 cells or equivalent in a milliliter (the sample volume of a dilution applied onto one plate) of the broth, and 165 colonies from dilution −7 equals to 1 650 000 000 cfu per mL of the enrichment broth. These numbers illustrate the final growth yield of the PMEU culture with or without the peptide addition. Nd = not determined; Full = fully grown plate, usually more than 300 cfu’s, and uncountable.

100 million cells per milliliter. Regarding that value, the growth curve was rising up relatively slowly (Figure 4f). This could be an indication of some cell abnormalities, such as small size or flocculation. Therefore, in future studies, microscopic evaluation of the bacterial cells treated with frog peptides is warranted. When investigating the potential effects of the antibiotic additions on microbial strains, it is important to pay attention to the relative acceleration of metabolism in the PMEU by 2−4 times. Besides speeding up the growth of bacteria, the PMEU environment is fortifying the effect of antibiotics in many cases, such as aerobic bacilli23 and campylobacteria.24 On the other hand, the recovery of the isolated strains is clearly improved in the PMEU when comparing it with the traditional culture methods.25 As such, the PMEU approach offers a sensitive tool to monitor the influences of antimicrobial frog skin peptides on the growth of bacterial strains. Both the different staphylococcal species and the salmonellas had been previously studied using the enhanced enrichment with the PMEU.20,26 Antibiotic resistant variants such as MRSA (methicillin resistant Staphylococcus aureus) and other hazardous strains have been monitored with the PMEU method as well.15 The salmonellas artificially contaminating the tap water with one cell per milliliter were detected by PMEU enrichment in about 10 h.29 In the Salmonella cultivation with the sample

Biological Activity Studies

Biological activity of the frog peptides was tested with the PMEU method. This approach has earlier been used for cultivating and detecting very small concentrations of various bacteria. For example, septic contaminations by staphylococci, streptococci and enteric bacteria were verified in a few hours only on the basis of the volatiles emitted from the cultures in the PMEU Scentrion.20 The effects of any antimicrobial substances could be studied also on the basis of the transmitted light or IR radiation in the PMEU Spectrion.21 The effects of the peptide secretion (sample M3) on the growth curves of Staphylococcus aureus and Salmonella enterica Serovar typhimurium are presented in Figure 4. Note that although the bacterial growth almost reaches the level of the control (ca. Figure 4a,b), there is a “shoulder” in the growth after 10 h of detectable growth. This phenomenon could be related to some peptide molecules remaining active during the course of cultivation. In case of Figure 4c, the bacterial growth was completely inhibited, indicating that an adequate number of cells was not surviving for the required CO2 formation in the growth initiation.22 There is a clear cessation of growth with a detectable “shoulder” in the curve (Figure 4e) occurring after 10 h of upward trend. This observation is equal to the growth curve for G



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equally observable with both Gram-positive staphylococci and Gram-negative salmonellas. According to the present results, these peptides could be potentially useful for the prevention of both pathogenic and antibiotic resistant bacterial strains while their action may also explain the traditional experience of rural populations.

M3, the period of time required for the observable upward turn in the growth curves was 7 h after inoculation without peptide addition, and 10 and 18 h with the additions of 0.01 and 0.03 mg into one milliliter of the broth, respectively. However, the delayed rising up of the growth curves in Figure 4e,f could be explained by the diminished cell number in the lower peptide concentration or by rare surviving cells in the higher peptide concentrations. As a consequence, the growth reached the detection limit of the PMEU IR sensors later than in the reference cultivation (Figure 4d). In the case of staphylococci, the onset of growth seemed not to be delayed by the lower peptide concentration, but it was almost completely inhibited by the three times higher concentration. The corresponding bacterial densities after 24 h of cultivation are presented in Table 2. The secretions of individual frogs (samples M7, M8) and a mixture of the secretion of 10 individual frogs (sample M10) with the concentration of 0.015 mg/mL were able to silence the growth of both the staphylococci and the salmonellas in the PMEU environment (results not shown here). Their effect completely matched that of the sample M3, demonstrating the absence of differences in the secretions of individual frogs of the same population. This effect is comparable with the inhibition of E. coli with 0.025 mg/mL of the antibiotic netilmicin sulfate.16 Consequently, it could be stated that the influence of the frog skin peptides on the bacterial cultures equal to the effect of the antibiotic treatments. When the purified individual brevinin 1Tb was studied in PMEU, the final concentration of 0.015 mg/mL (7.6 × 10−9 M) completely prevented the growth of S. aureus, whereas ten times more diluted brevinin 1Tb sample exhibited no observable inhibition. On the basis of this relatively small distinction in concentration between fully influential peptide solution and the lack of any effect, it could be postulated that the effect of brevinin 1Tb is merely bacteriocidic than bacteriostatic on the cellular level. On the other hand, in both of the growth curves (Figure 4b,e) of staphylococci and salmonellas there is a “shoulder” after 10 h of active growth when exposed to lower peptide concentration. This could suggest that in PMEU the inhibitory effect could be somewhat prolonged when compared to the conditions in nature with some part of the peptides remaining active in the enrichment unit, thus influencing the growth of bacteria during the growth of new generations. It is also noteworthy that the proteolytic activities of the animal enzymes on the frog skin are directed also toward the bacterial cells and their surface proteins (besides the peptides of frog origin). Consequently, the peptide mixtures in vivo may also contain peptides of bacterial origin.28

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MATERIALS AND METHODS

Skin Secretions

Ten male species of R. temporaria were caught in the Kolomna district of Moscow region (Russia). Preparation of frog skin secretion was accomplished as described earlier.29 Secretion from the skin glands was provoked by mild electrical stimulation during 40 s using laboratory electrostimulator ESL-1 equipped with platinum electrodes. The duration of impulse was 3 ms with amplitude of 10 V at 50 Hz. The skin secretion was washed from the animals with Milli-Q water (about 25 mL) into the plastic container containing equal volume of methanol. The mixtures were centrifuged during 15 min at 3000 rpm and filtered through the PTF membrane filter (MillexHV, 0.45 μm, Millipore, USA). The solutions obtained were concentrated at 35 °C using rotary evaporator to a volume of 1 mL and lyophilized. The final samples were kept at −26 °C. HPLC Purification of the Skin Secretions

Separation of brevinin 1Tb from crude secretion was performed with HPLC system (Thermo Separations, USA) with a binary pump “Thermo System P2000” by injecting 20 μL of a peptide sample into a reverse-phase chromatographic analytical column C18 (5 μm, 100 Å, 150 × 4 mm, Dr. Maisch, Germany) equilibrated with 10% acetonitrile/aqueous 0.1% trifluoroacetic acid. The separation was achieved using a linear gradient from 10 to 70% (40 min) acetonitrile containing 0.1% trifluoroacetic acid. The flow rate was 0.8 mL/min. UV-detector was fixed to register the peptides’ signal at λ = 214 nm. The fractions with retention time 43.55−43.61 min containing brevinin 1Tb were collected and lyophilized as mentioned above. Mass Spectrometric Sequencing

All experiments were carried on with LTQ Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany) in electrospray ionization (ESI) mode, coupled with nanoHPLC system (Thermo Separation Products, NJ, USA) equipped with nanocolumn (15 cm length, 75 μm i.d., 375 μm o.d. homepacked with 3 μm C18 beads (Dr Masch, Germany) integrated with a sprayer, from Proxeon Biosystems, Odense, Denmark). Lyophilized samples were introduced into the column being dissolved in acetonitrile/water mixture (1:1) with addition of 0.1% formic acid. The following solvents were used: A − 99.9% HPLC grade water with 0.1% formic acid, B − 90% acetonitrile with 0.1% formic acid. Separation was achieved using a linear gradient from 4 to 80% (30 min) solvent B. Higher energy collisionally activated dissociation (HCD) and electron transfer dissociation (ETD) were consecutively applied to induce fragmentation of the most intense ions. The resolving power was 60 000 in MS mode and 7500 in MS/MS mode to improve duty cycle of the instrument. Interpretation of the spectra was performed manually.30

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CONCLUSIONS The technical properties of the Orbitraps Velos instrument allowed us to obtain detailed and rich information on the composition of skin peptidomes of ranid frogs. Thus, 76 natural nontryptic peptides were identified in the skin secretion of brown frogs R. temporaria from the Kolomna population (Moscow region, Russia). The sequences of brevinin 2Tf, temporin N and [Hyp3]temporin M were reported for the first time. Directed search with the use of characteristic fragment ions resulted in the discovery of 49 peptides belonging to bradykinin family. The majority of these peptides were not reported so far. In the testing with the rapid cultivator PMEU units, the biological activities of the frog skin peptides in concentrations above 0.01 mg per milliliter of growth medium against bacterial strains was well detectable. This effect was

Solvents and Chemicals

All the solvents used in the study were of HPLC grade and purchased from Panreac (Spain). H



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Antimicrobial Assay

Zvenigorod Population. J. Anal. Chem. 2011, 66 (14), 1353−1360. Original Russian text: Mass-spektrometria 2011, 8, 7−14. (8) Apponyi, M. A.; Pukala, T. L.; Brinkworth, C. S.; Maselli, V. M.; Bowei, J. H.; Tyler, M. J.; Booker, G. W.; Wallas, J. S.; Carver, J. A.; Separovic, F; doyle, J.; Liewellyn, L.E.. Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance. Peptides 2004, 25 (6), 1035−1054. (9) Samgina, T. Yu.; Artemenko, K. A.; Gorshkov, V. A.; Ogourtsov, S. V.; Zubarev, R. A.; Lebedev, A. T. De novo sequencing of peptides secreted by the skin glands of the Caucasian Green Frog Rana ridibunda. Rapid Commun. Mass Spectrom. 2008, 22, 3517−3525. (10) Samgina, T. Yu.; Artemenko, K. A.; Gorshkov, V. A.; Ogourtsov, S. V.; Zubarev, R. A.; Lebedev, A. T. Mass spectrometric study of peptides secreted by the skin glands of the brown frog Rana arvalis from the Moscow region. Rapid Commun. Mass Spectrom. 2009, 23, 1241−1248. (11) Samgina, T. Yu.; Gorshkov, V. A.; Vorontsov, Y. A.; Artemenko, K. A.; Ogourtsov, S. V.; Zubarev, R. A.; Lebedev, A. T. Investigation of skin secretory peptidome of Rana lessonae frog by mass spectrometry. J. Anal. Chem. 2011, 66 (13), 1298−1306. Original Russian text: Mass-spektrometriya 2010, 7, 261−270. (12) Samgina, T. Yu; Gorshkov, V. A.; Artemenko, K. A.; Vorontsov, E. A.; Klykov, O. V.; Ogourtsov, S. V.; Zubarev, R. A.; Lebedev, A. T. LC-MS/MS with 2D mass mapping of skin secretions’ peptides as a reliable tool for interspecies identification inside Rana esculenta complex. Peptides 2012, 34, 296−302. (13) Samgina, T. Yu.; Gorshkov, V. A.; Vorontsov, Ye. A.; Demkina, E. V.; Ogourtsov, S. V.; Shakhparonov, V. V.; El_Registan, G. I.; Lebedev, A. T. HPLC and MALDI Investigation of the Stress Influence on the Composition of Skin Secretion of the Common Frog Rana temporaria. J. Anal. Chem. 2011, 66 (14), 1361−1368. Original Russian text: Mass-spektrometriya 2011, 8 (2), 91−98. (14) Hakalehto, E.; Hell, M.; Bernhofer, C.; Pesola, J.; Heitto, A.; Humppi, T.; Paakkanen, H. Enhanced growth and gaseous emissions of pure and mixed small intestinal bacterial cultures in the PMEU (Portable Microbe Enrichment Unit) with gas sensor. Demonstration of the effects of bile and vancomycin on various strains. Pathophysiology 2010, 17, 45−53. (15) Hakalehto, E. Antibiotic resistance traits of facultative Enterobacter cloacae strain studied with the PMEU (Portable Microbe Enrichment Unit). In Science against microbial pathogens: communicating current research and technological advances; Formatex Microbiology Book Series No 3; Méndez-Vilas, A., Ed.; Formatex: Badajoz, Spain, 2011; pp 786−796. (16) Hakalehto, E. Simulation of enhanced growth and metabolism of intestinal Escherichia coli in the Portable Microbe Enrichment Unit (PMEU). In E. coli infections: causes, treatment and prevention; Rogers, M. C., Peterson, N. D., Eds.; Nova Publishers: New York, 2011; pp 159−175. (17) Samgina, T. Yu.; Gorshkov, V. A.; Vorontsov, Y. A.; Artemenko, K. A.; Zubarev, R. A. Lebedev. Mass Spectrometric Study bradykininrelated peptides (BRPs) from the skin secretions of Russian ranid frogs. Rapid Commun. Mass Spectrom. 2011, 25, 933−940. (18) Lebedev, A. T. Gas chromatography/mass spectrometry − a workhorse of the modern environmental analysis. In Comprehensive Environmental Mass Spectrometry; Lebedev, A. T., Ed.; ILMPublications: U.K., 2012; pp 22−40. (19) Chen, T.; Orr, D.; Bjourson, A.; McClean, S.; O'Rourke, M.; Hirts, D.; Rao, P.; Shaw, C. Novel Bradykinins and their precursor cDNAs from European yellow-bellied toad (Bombina variegata) skin. Eur. J. Biochem. 2002, 296, 4693−4700. (20) Hakalehto, E.; Pesola, J.; Heitto, A.; Bhanj Deo, B.; Rissanen, K.; Sankilampi, U.; Humppi, T.; Paakkanen, H. Fast detection of bacterial growth by using portable microbe enrichment unit (PMEU) and ChemPro100i® gas sensor. Pathophysiology 2009, 16, 57−62. (21) Hakalehto, E. Hygiene monitoring with the Portable Microbe Enrichment Unit (PMEU). 41st R3 − Nordic Symposium. Cleanroom technology, contamination control and cleaning; VTT (State Research

The samples of crude secretion of three individual frogs (male, M3, M7, and M8) and of a mixture of secretion of 10 individual frogs (males M10) belonging to the Kolomna population of R. temporaria, as well as of individual peptide brevinin 1Tb isolated from the crude secretion by HPLC were tested for their biological activity using bacterial laboratory strains of Gram-positive Staphylococcus aureus 178 (a patient strain from the Kuopio University Hospital neonatal ward) and Gramnegative Salmonella enterica Serovar typhimurium Sa 111/01 (originating from Evira Salmonella Center in Kuopio, Finland). These strains were grown as aerobic pure cultures at 37 °C in the PMEU Spectrion cultivator (Samplion Oy, Kuopio and Siilinjärvi, Finland).14,15 The initial concentrations of these bacteria were 37 000 cfu/mL and 15 000 cfu/mL, respectively. The peptides were added as stock solutions (1 mg/mL) into 10 mL of TYG (Tryptone, Yeast extract, Glucose) culture media prior to its inoculation with the bacteria. The final concentrations of these peptide additions were 0.01 mg and 0.03 mg per mL (sample M3), 0.015 mg per mL (Brevinin1Tb, or M7, M8, and M10 samples), or 0.015 mg per 10 mL (Brevinin 1Tb only). During cultivation, the cultures were aerated with sterilized air bubbles. The results were monitored by the growth curves of the PMEU devices and by plate culturing the PMEU enrichment samples onto TYG plates after cultivation.

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AUTHOR INFORMATION

Corresponding Author

*Phone: + 7 495 939 1407. E-mail: [email protected]. ru. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are thankful to Anneli Heitto, M.Sc. of Finnoflag Oy laboratory for the expert assistance in carrying out the PMEU cultivations. The authors thank Shakhparonov V. V. for keeping the frogs.

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REFERENCES

(1) Anastasi, A.; Erspamer, V.; Bertaccini, G. Occurence of bradykinin in the skin of Rana temporaria. Comp. Biochem. Physiol. 1965, 14, 43. (2) Simmaco, M.; Mignogna, G.; Canofeni, S.; Miele, R.; Mangoni, M.; Barra, D. Temporins, antimicrobial peptides from the European red frog Rana temporaria. Eur. J. Biochem. 1996, 242 (3), 788−792. (3) Simmaco, M.; Mignogna, G.; Barra, D. Antimicrobial peptides from amphibian skin: what do they tell us? Biopolymers 1998, 47 (6), 435−450. (4) Barra, D.; Simmaco, M.; Boman, H. Gene-encoded peptide antibiotics and innate immunity. Do ‘animalcules’ have defense budget? FEBS Lett. 1998, 430 (1), 130−134. (5) Conlon, J. M.; Aronsson, U. Multiple bradykinin-related peptides from the skin of the frog Rana temporaria. Peptides 1997, 18 (3), 361− 365. (6) Pukala, T. L.; Bowei, J. H.; Maselli, V. M.; Musgrave, I. F.; Tyler, M. J. Host-defence peptides from the glandular secretions of amphibians: structure and activity. Nat. Prod. Rep. 2006, 23 (3), 368−393. (7) Samgina, T. Yu.; Gorshkov, V. A.; Vorontsov, Y. A.; Artemenko, K. A.; Ogourtsov, S. V.; Zubarev, R. A.; Lebedev Mass Spectrometric Study of the Skin Peptidome of Brown frog Rana temporaria from I



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Centre of Finland) Publications: Espoo, Finland, 2010; Vol. 266, pp 164−176. (22) Hakalehto, E.; Hänninen, O. Gaseous CO2 signal initiates growth of butyric-acid-producing Clostridium butyricum in both pure culture and mixed cultures with Lactobacillus brevis. Can. J. Microbiol. 2012, 58, 928−931. (23) Mentu, J.; Heitto, L.; Keitel, H. V.; Hakalehto, E. Rapid microbiological control of paper machines with PMEU method. Paper Timber (Paperi ja Puu) 2009, 91, 54−57. (24) Pitkänen, T.; Bräcker, J.; Miettinen, I.; Heitto, A.; Pesola, J.; Hakalehto, E. Enhanced enrichment and detection of thermotolerant Campylobacter species from water using the Portable Microbe Enrichment Unit (PMEU) and realtime PCR. Can. J. Microbiol. 2009, 55, 849−858. (25) Pesola, J.; Vaarala, O.; Heitto, A.; Hakalehto, E. Enrichment in Portable Enrichment Unit in rapid characterization of infant intestinal enterobacterial microbiota. Microbial Ecol. Health Dis. 2009, 21, 203− 210. (26) Hakalehto, E.; Pesola, J.; Heitto, L.; Närvänen, A.; Heitto, A. Aerobic and anaerobic growth modes and expression of type 1 fimbriae in Salmonella. Pathophysiology 2007, 14, 61−69. (27) Hakalehto, E.; Heitto, A.; Heitto, L.; Humppi, T.; Rissanen, K.; Jäas̈ keläinen, A.; Paakkanen, H.; Hänninen, O. Fast monitoring of water distribution system with portable enrichment unit − Measurement of volatile compounds of coliforms and Salmonella sp. in tap water. J. Toxicol. Environ. Health Sci. 2011, 3, 223−233. (28) Hakalehto, E.; Santa, H.; Vepsäläinen, J.; Laatikainen, R.; Finne, J. Identification of a common structural motif in the disordered Nterminal region of bacterial flagellins. Evidence for a new class of fibrilforming peptides. Eur. J. Biochem. 1997, 250, 19−29. (29) Tyler, M.; Stone, D.; Bowie, J. A novel method for the release and collection of dermal, glandular secretions from the skin of frogs. J. Pharmacol. Toxicol. Methods 1992, 28 (4), 199−200. (30) Kinter, M., Sherman, N. Protein Sequencing and Identification Using Tandem Mass Spectrometry. Wiley-Interscience Series on Mass Spectrometry; Desiderio, D. M., Nibbering, N. M. M., Eds.; John Wiley & Sons Inc.: New York, 2000; p 320.

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