peptides 27 (2006) 2585–2591
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journal homepage: www.elsevier.com/locate/peptides
Synthetic peptides derived from human antimicrobial peptide ubiquicidin accumulate at sites of infections and eradicate (multi-drug resistant) Staphylococcus aureus in mice Carlo P.J.M. Brouwer a, Sylvia J.P. Bogaards a, Marty Wulferink a, Markwin P. Velders a, Mick M. Welling b,* a
AM-Pharma, Bunnik, The Netherlands Department of Radiology, Section of Nuclear Medicine, Leiden University Medical Center (LUMC), C4-R Room 77, P.O. Box 9600, NL-2300 RC Leiden, The Netherlands
b
article info
abstract
Article history:
The presence and antimicrobial activity of antimicrobial peptides (AMPs) has been widely
Received 28 March 2006
recognized as an evolutionary preserved part of the innate immune system. Based on
Received in revised form
evidence in animal models and humans, AMPs are now positioned as novel anti-infective
22 May 2006
agents. The current study aimed to evaluate the potential antimicrobial activity of ubiqui-
Accepted 22 May 2006
cidin and small synthetic fragments thereof towards methicillin resistant Staphylococcus
Published on line 11 July 2006
aureus (MRSA), as a high priority target for novel antibiotics. In vitro killing of MRSA by synthetic peptides derived from the a-helix or b-sheet domains of the human cationic
Keywords:
peptide ubiquicidin (UBI 1–59), allowed selection of AMPs for possible treatment of MRSA
Antimicrobial peptide
infections. The strongest antibacterial activity was observed for the entire peptide UBI 1–59
Synthetic fragments
and for synthetic fragments comprising amino acids 31–38. The availability, chemical
MRSA
synthesis opportunities, and size of these small peptides, combined with their strong
In vitro killing
antimicrobial activity towards MRSA make these compounds promising candidates for
Experimental infections
antimicrobial therapy and detection of infections in man. # 2006 Elsevier Inc. All rights reserved.
Microbicidal activity Technetium Detection of infections
1.
Introduction
The emerging resistance of micro-organisms to currently available antibiotics has raised great concerns regarding the battle against infections in man. This is underlined by the resolution of the World Health Assembly (WHA) on improving the containment on antimicrobial resistance. Based on proof of concept experiments in animal models for infection,
treatment of bacterial infections with cationic antimicrobial peptides (AMPs) has been proposed [8]. However, disadvantages of the application of natural AMPs as antibiotics need to be addressed before implementation. The elaborate purification procedures to obtain large quantities of pure peptide and their possible immunological and toxic side effects [16], may have limited the systemic use of AMPs this far. Consequently, mainly topical applications for AMPs are being pursued [5,10]
* Corresponding author. Tel.: +31 71 5261880; fax: +31 71 5266751. E-mail address:
[email protected] (M.M. Welling). 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.05.022
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2 0 1 4 0 1 1 0 5 11 2 3 7 6 4 9 0.39 0.78 0.67 0.28 0.83 0.62 0.75 0.79 1–18 18–35 36–41 42–59 18–29 29–41 31–38 22–35
Amino acids from both the a-helix (in italics) and b-sheet are underlined.
0.88 2.5 2.57 0.43 2.42 2.47 2.31 2.8 1890 2169 867 1904 1444 1693 1108 1733
12.0 11.8 10.9 10.3 10.4 12.2 11.7 12.0
7 20 0.39 1.36 6648
12.2
Cationic amino acids Linear charge density pI-value (pH) Lipophilicity (kcal/mol) Mol. weight (D)
UBI UBI UBI UBI UBI UBI UBI UBI
Ubiquicidin (UBI 1–59 KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPNANS, Mw: 6648 D) was isolated from mouse RAW macrophages as described [11]. Characteristics of synthetic peptides derived from human cationic antimicrobial peptide ubiquicidin and the natural peptide UBI 1–59 were analyzed using PepTool 2.0 software (BioTools Inc., Edmonton, Canada). Fragments were selected by calculation of Garnier-Robson and Chou-Fasman test as depicted in Table 1. Peptides UBI 1–18, UBI 18–35, UBI 36–41, UBI 42–59, UBI 18–29 and UBI 29–41 were synthesized as described [7] by the Department of Immunohematology and
KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPNANS KVHGSLARAGKVRGQTPK KVAKQEKKKKKTGRAKRR MQYNRR FVNVVPTFGKKKGPNANS KVAKQEKKKKKT TGRAKRRMQYNRR RAKRRMQY QEKKKKKTGRAKRR
Peptides
UBI 1–59
2.1.
Amino acids
Materials and methods
Peptide
2.
Table 1 – Characteristics of synthetic peptides derived from human cationic antimicrobial peptide ubiquicidin and the natural peptide UBI 1–59
whereas systemic applications are limited, despite the results of systemic use for various synthetic AMPs. This paper focuses on the use of synthetic fragments of the antimicrobial peptide ubiquicidin (UBI) for detection and treatment of bacterial infections. This 6.6 kD linear peptide is found at low concentrations as a first line of defense inside human airway epithelial cells, activated macrophages [11], and, in human colon mucosa [15]. However, in contrast to most other AMPs, ubiquicidin is only present intracellularly and only released after severe damage during acute infection. It has been shown to be microbicidal towards a broad spectrum of pathogens. To select fragments that contain strong microbial binding and/or antimicrobial properties [4]. The structure of UBI has been investigated through computer analysis and the amino acid sequence and various cationic and lipophilic derivates in the a-helix and b-sheet domains are well characterized. Recently, the binding of one specific (radiolabeled) synthetic fragment from UBI to various bacteria and fungi was established and enabled accumulation and visualization of experimental infections [18,19] and the monitoring of antimicrobial therapy [14]. The observation that the synthetic fragments of UBI target to infection sites [12] opens perspectives for antimicrobial treatment with UBI derived peptides in patients [1,13]. UBI 1–59 is of human origin and is not expected to be immunogenic for man and resistance induction is expected to be significantly lower than for classical antibiotics. Selection of UBI fragments through determination of clusters consisting of cationic and/or lipophilic domains, has been based on calculations according to Garnier et al. [9] and Chou and Fasman [6]. Selection of MRSA as a target for the antimicrobial peptides from UBI is driven by the significant health risks that are associated with these bacteria. MRSA is mentioned as the first priority target on the list by IDSA (March 2006) of ‘‘Bad Bugs that need drugs’’. The reason being that: ‘‘MRSA infections constitute the majority of health care-associated infections, increasing lengths of hospital stay, severity of illness, deaths, and costs’’. While these infections used to be limited primarily to hospitals, the increase in number of MRSA carriers in communities is striking, especially where groups of people are in close quarters, including military facilities, sports teams, and prisons. (http://www.idsociety.org/). Based on the results presented here, a set of new UBI derived peptides for systemic antimicrobial use are proposed.
Aromatic amino acids
peptides 27 (2006) 2585–2591
peptides 27 (2006) 2585–2591
Bloodtransfusion, LUMC, The Netherlands. Stock solutions of the various peptides in 0.01% (v/v) of acetic acid (HAc, pH 4) were stored at 20 8C in Eppendorf vials.
2.2.
Micro-organisms
The Staphylococcus aureus strain 2141 (MRSA, resistant to methicillin MIC > 256 mg/L, cloxacillin MIC > 256 mg/L, penicillin MIC > 0.25 mg/L, gentamycin MIC > 8 mg/L, displaying limited sensitivity MIC < 2 mg/L to teicoplanin and rifampicin and to vancomycin MIC < 1) is a clinical isolate (Department of Infectious Diseases, LUMC, Leiden, The Netherlands). Virulent bacteria were maintained in mice through in vivo passage. Briefly, about 0.5–2.0 107 colony-forming units (CFU) in 0.1 mL of the micro-organisms were injected into a tail vein of mice and 24 h thereafter the mice were sacrificed. The spleen was aseptically removed, homogenized, and appropriate dilutions of the homogenate were plated onto diagnostic sensitivity test agar (DST, Oxoid) or tryptic soy agar (TSA, Oxoid). After 24 h of incubation at 37 8C a single colony was transferred into 25 mL of the appropriate broth and incubated for 24 h at 37 8C and 1 mL aliquots of these suspensions containing about 0.5–5.0 108 virulent microorganisms per mL were stored at 20 8C.
2.3.
In vitro killing assay
To assess the microbicidal activity of the various peptides towards MRSA, bacteria were washed twice in 14 mM sodium phosphate buffer pH 7.4 (NaPB). Next, they were diluted to about 1 106/mL in NaPB supplemented with 1% (wt/vol) of tryptic soybean broth (TSB, Difco). Micro-organisms were exposed to various amounts of peptides for 2 h at 37 8C and thereafter, the number of viable micro-organisms was assessed microbiologically using DST plates. All negative cultures were assigned the value of less than 4000 CFU/mL, being the detection limit. Data are expressed as inhibition concentration (IC) to eradicate 50% (IC 50), 90% (IC 90), or 99% (IC 99) of the number of viable microorganisms as compared to control incubations.
2.4.
Animals
Specific pathogen-free, male Swiss mice (Broekman Institute, Someren, The Netherlands) weighing between 22 and 42 g were used in this study. The animals were housed in the animal housing facilities of the LUMC for at least 1 week before the onset of the experiments. Food and water were given ad libitum. All animal studies were performed in compliance with the Dutch laws related to the conduct of animal experiments and approved by the local Committee for Animal Experiments.
2.5.
MRSA infections in mice
Mice were anaesthetized with a single intraperitoneal injection of 0.1 mL of saline containing 1 mg fluanisone and 0.03 mg fentanyl citrate (Hypnorm, Janssen Pharmaceutics, Tilburg, The Netherlands). Next, approximately 0.5–2.0 107 CFU MRSA in 0.1 mL of saline were injected into the right thigh muscle. After 18 h the mice were anaesthetized as described above, i.v. injected with various amounts of the UBI peptides,
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24 h thereafter, the animals were sacrificed through an i.p. injection of 12 mg of sodium pentobarbiturate (Nembutal, Sanofi BV, Division Algin, Maassluis, The Netherlands). The infected thigh muscles were removed, homogenized, and plated onto DST agar plates as described above. The number of bacteria was determined microbiologically.
2.6. Targeting MRSA-infections in mice with technetium99 m labeled UBI peptides All peptides were directly labeled with technetium-99 m in order to visualize their infection-targeting characteristics. Briefly, 10 mL of a peptide stock solution was added to 4 mL of an aseptic mixture of 950 mg/L SnCl22H2O and 2 g/L sodium pyrophosphate10H2O in saline in a 1.5 mL Eppendorf vial. Immediately thereafter, 2 mL of a solution containing 10 mg/ mL of KBH4 in 0.1 M NaOH (LUMC) were pipetted into this mixture. After addition of 0.1 mL of 99mTc-sodium pertechnetate solution (99mTc, approximately 200–500 MBq/mL, Technekow, Mallinckrodt Medical BV, Petten, the Netherlands) the mixture was gently stirred at room temperature for 1 h [17]. This preparation is further referred to as 99mTc-UBI peptide. The mixture has a final pH of 8.6. Next, 2–5 MBq of radiolabeled peptide diluted in 0.1 mL of saline was injected into a tail vein of an anaesthetized mouse bearing a thigh muscle infection with MRSA as described above. Until 2 h after the tracer was injected accumulation of radioactivity in infected tissues and various organs was determined using scintigraphy. For this purpose, mice were placed in supine position on a low-energy general-purpose parallel-hole collimator that was connected to a dedicated computer (Toshiba GCA 7100/UI, Tokyo, Japan). Dynamic whole body images of the animals were collected in a 128 128 matrix at three times enlargement. The energy peak was set at 140 keV with a window of 20%. Anatomically fitted regions of interest (ROI) were drawn over various tissues and the entire mouse and radioactive counts in the ROI were expressed as a percentage of the injected dose (%ID) in the mouse. On the scintigrams regions of interest were drawn over the heart area and after correction for total body activity of the mice between 0 and 120 min after injection of the peptides the pharmacological half-life of the tracers was calculated.
2.7.
Statistical analysis
To understand the relation between in vitro killing activity and various parameters such as pI-value, lipophilicity and linear charge the two-tailed Pearson’s correlation coefficient (and the corresponding P-value) was calculated. Decrease in the number of viable micro-organisms in mice after treatment with various amounts of UBI peptides at various intervals was evaluated with Student’s t-test. The level of significance was set at a P-value of 0.05.
3.
Results
3.1.
Peptides
Based on the outcome of computer software analysis (PepTool 2.0, BioTools Inc., Edmonton, Canada) of the UBI 1–59 peptide
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Fig. 1 – Lipophilicity characteristics of ubiquicidin and depiction of potential antimicrobial domains according to GarnierRobson and Chou-Fasman. Domains containing a a-helix are indicated in blue and domains containing a b-sheet are indicated in yellow. Lipophilicity is expressed in kcal/mol. Lipophilic and cationic residues are printed in red and blue, respectively.
using the algorithms of Garnier-Robson [9] and Chou-Fasman [6] we synthesized a UBI peptide library (Fig. 1): UBI 1–18, UBI 18– 35, UBI 36–41, and UBI 42–59 (Garnier-Robson) and UBI 18–29 and UBI 29–41 (Chou-Fasman). The amino acid sequence and other characteristics, such as calculated lipophilicity, pI-value, and linear charge of the various individual synthetic peptides as well as the location of a-helix and b-sheet according to GarnierRobson and Chou-Fasman algorithms are summarized in Table 1. Purity of the synthetic peptides usually exceeded 95%, as determined by reverse phase high performance liquid chromatography (HPLC) using water/methanol as eluent [7]. Contaminating traces of lipopolysaccharides (LPS) in the various peptide preparations were lower than 20 pg/mL assessed by Limulus assay (Chromogenix, Mo¨lndal, Sweden).
3.2.
From the peptides listed in Table 1, the treatment mediated decrease of viable MRSA numbers in vitro expressed as IC 50, IC 90 and IC 99 was determined (Table 2). Highest killing of MRSA was observed with the natural peptide UBI 1–59 followed by synthetic peptides UBI 31–38, UBI 29–41, UBI 18–35, and UBI 1–18.
Table 2 – Microbicidal activity of UBI peptides to MRSA strain 2141 in vitro
UBI UBI UBI UBI UBI UBI UBI UBI UBI a
1–59 1–18 18–35 36–41 42–59 18–29 29–41 31–38 22–35
Antimicrobial efficacy in vivo
Next, the decrease in viable MRSA isolated from the thigh muscle of infected mice after systemic treatment with a single dose of UBI derived peptides was determined and the results expressed as IC 50, IC 90 or IC 99 (Table 3). In vivo, the highest antimicrobial efficacy was observed for the natural peptide UBI 1–59 and synthetic peptide UBI 31–38. Moderate anti-MRSA activity was observed for UBI 1–18, UBI 18–35, UBI 36–41, UBI 29–41, and UBI 22–35.
3.4. Targeting MRSA-infections in mice with technetium99 m labeled UBI peptides As depicted on the scintigrams (Fig. 2), within 1 hour after injections all peptides visualized the MRSA infected thigh muscles. Most striking are the differences in biodistribution as
Antimicrobial efficacy in vitro
Peptide
3.3.
IC 50a (mM)
IC 90a (mM)
IC 99a (mM)
0.60 24.5 23.5 >200 >200 82.5 20.0 16.0 38.0
0.75 31.2 30.65 >200 >200 84.8 25.5 19.5 49.0
1.35 76.0 42.0 >200 >200 96.5 40.5 29.0 74.0
IC 50, 90 and 99 were determined after 2 h of incubation and were calculated from the results of at least 3 independent experiments.
Table 3 – Microbicidal activity of UBI peptides to MRSA in thigh muscles of mice Peptide UBI UBI UBI UBI UBI UBI UBI UBI UBI
1–59 1–18 18–35 36–41 42–59 18–29 29–41 31–38 22–35
IC 50 (mmol/mouse)
IC 90 (mmol/mouse)
IC 99 (mmol/mouse)
0.42 8.2 2.9 6.6 8.0 68.0 3.1 0.10 5.8
0.67 13.4 5.1 12.9 28.0 80.0 5.5 1.3 24.8
0.73 24.3 41.2 24.0 >100 >100 17.0 2.4 115
Values are expressed as mmol/mouse required to determine IC 50, IC 90, and IC 99 of viable micro-organisms/g tissue measured in mice compared with the number of viable bacteria in mice without antimicrobial treatment. Bacterial numbers were determined 24 h after administration of the peptide, i.e. 18 h after the onset of infection (1 107 CFU/mouse). The number of animals used is at least 3 per concentration (for at least three concentrations).
peptides 27 (2006) 2585–2591
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the peptides with the fastest clearance (pharmacological halflife between 17 and 27 min) UBI 36–41, UBI 42–59, and UBI 31–38 showing highest accumulation in the urinary bladder and kidneys (Table 4). UBI 18–29 shows highest (24%) accumulation of radioactivity in the liver. No significant relations could be calculated between the biodistribution data (accumulation in the infected thigh muscle or clearance) and the in vivo killing activity of the various AMPs.
3.5. Correlations between UBI peptide characteristics and biological assays No correlation could be established between the biodistribution data and the differences in biological behavior (i.e. antimicrobial activity) of the peptides. Also, those AMPs with the highest accumulation in infected tissues are not necessarily the peptide that kills bacteria most efficiently. Next, physiological parameters of the peptides were studied to explain the efficacy differences. Pearson’s (two-tailed) correlations and P-values were calculated to assess a relation between various UBI peptide characteristics such as lipophilicity, pI value, and linear charge density as prediction for in vitro or in vivo killing of MRSA. We found a good correlation between the pI value and the in vitro (R = 0.813, P = 0.041) and in vivo killing (R = 0.645, P = 0.04) of MRSA at IC 90 and IC 99 values. After linear regression analysis the pI value predicted significantly reliable (P = 0.003) the killing of MRSA both in vitro and in infected mice. Finally, we calculated relations between our data concerning the in vitro and in vivo killing of MRSA for IC 90 and IC 99 values and after linear regression analysis the in vitro killing of MRSA predicted significantly reliable (P < 0.001) the killing of MRSA in infected mice.
4.
Discussion
The main conclusion of this study is that testing the antibacterial activity of natural AMP ubiquicidin (UBI 1–59) and synthetic derivates thereof in an in vitro bacteria killing assay, enabled the selection of UBI peptides suitable for antimicrobial treatment of thigh muscle infections with multidrug resistant Staphylococcus aureus in mice. This conclusion is based on a significant correlation between the in vitro killing of AMPs and their antibacterial activity in mice as determined by the viable bacterial colony count after single treatment with the various UBI derived AMPs. Secondly, a relation between the pI value of the peptide and its antimicrobial activity could be established for the peptides used in this study. However, no significant relation between the number of cationic and/or lipophilic residues was observed, which indicates that the order of amino acids and the peptide conformation play an important role [3]. This is confirmed by experiments with a scrambled version of UBI 29– 41 that showed strongly reduced infection targeting capacity
Fig. 2 – Typical scintigrams of 99mTc-labeled UBI peptides 1 h after injection into mice infected 18 h before with 0.5–
2.0 107 viable MRSA. The infected thigh muscles are indicated with an arrow. Contrast in each image is adjusted to show optimal accumulation of radioactivity in both hind legs.
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Table 4 – In vivo biodistribution characteristics of 99mTc-labeled peptides derived from the natural human cationic peptide ubiquicidin (UBI 1–59) at 1 h post-injection Peptide UBI UBI UBI UBI UBI UBI UBI UBI UBI
1–59 1–18 18–35 36–41 42–59 18–29 29–41 31–38 22–35
Pharmacological half-life (min)
T/NT ratios
Urinary bladder (%ID)
Kidneys (%ID)
Liver (%ID)
49 126 49 17 21 38 142 27 36
1.49 0.31 2.06 0.43 2.26 0.31 1.44 0.42 2.20 0.16 3.06 0.74 2.04 0.12 2.10 0.36 1.71 0.24
13.2 4.8 33.0 2.0 19.5 2.1 66.1 2.5 42.3 4.2 12.8 2.0 32.0 5.2 56.2 5.6 23.6 8.4
23.0 2.6 22.1 2.6 25.9 1.6 10.6 1.2 18.4 3.1 21.8 8.3 22.3 2.5 16.9 4.0 28.0 13.2
4.3 2.6 9.2 2.9 6.3 0.8 7.4 3.1 14.1 3.6 24.6 10.9 13.5 2.2 4.4 1.8 9.5 4.9
Data are expressed as the mean (S.D.) of at least three observations. T/NT: Target (infected thigh muscle) to non-target (non-infected thigh muscle) ratio. %ID: Percentage of the injected dose (corrected for decay).
as compared to the normal UBI 29–41 peptide sequence [17] indicating the importance of the order of amino acids. Remarkably, in contrast to the fragment containing the bsheet (UBI 42–59), synthetic fragments comprising an a-helix displayed the highest, dose-dependent antibacterial activity towards MRSA. Poor activity of the UBI fragment comprising the b-sheet may be attributed to low linear charge density and lipophilicity. Additional experiments have shown that the strong antimicrobial activity of the peptides containing the a-helix was also seen against Staphylococcus aureus, Klebsiella pneumoniae, Escherichiae coli, and Candida albicans (unpublished results) indicating that this domain plays an important role in the antimicrobial activity of UBI towards a variety of pathogens. An explanation for this phenomenon [18] is, that domain 31–38 is responsible for binding to microorganisms. The previous observation that the number of cationic residues alone cannot fully explain the antimicrobial activity of antimicrobial peptides [2,3], was confirmed in this study, as for UBI 18–29, despite the fact that it contain seven cationic amino acids, displayed poor in vitro and in vivo antimicrobial activity. In contrast, UBI 1–18, UBI 42–59, UBI 29–41, and UBI 31– 38 that contain less cationic residues than UBI 18–29 demonstrated higher antimicrobial activity. This phenomenon could be explained by differences in biodistribution and in accumulation of the peptides in the infected tissues. One important question related to antimicrobial peptides (AMPs) is whether in vitro killing assays allow predicting in vivo efficacy. For some AMPs, especially those with strong immunomodulatory activity, such correlation cannot be established. Furthermore, it is encouraging that large amounts of peptide injected into mice are well tolerated and that recently, one of the UBI peptides (UBI 29–41) has been successfully tested in patients for the detection of infections [1,13]. Although, the strongest antibacterial activity is observed for natural peptide UBI 1–59, the high costs to synthesize this peptide under GMP production and, alternatively, the elaborating purification procedures to obtain natural ubiquicidin 1–59, limits its use for treatment and detection. In this respect, a selected synthetic peptide, comprising the potent microbicidal domain 31–38, offers a promising alternative. Its availability, ease of synthesis and production under GMP, as well as its strong antimicrobial activity towards multi-drug resistant micro-organisms make this compound a promising
candidate for antimicrobial therapy and detection of infections in man.
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