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Antimicrobial Agents and Chemotherapy, November 2007, p. 4148-4156, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00635-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Antimicrobial Effect of Halocidin-Derived Peptide in a Mouse Model of Listeria Infection{triangledown}

Woong Sik Jang,1,{dagger} Sang-Chul Lee,2,{dagger} Young Shin Lee,1 Yong Pyo Shin,1 Kyoung Hwa Shin,1 Boo Hee Sung,2 Byung S. Kim,2 Soo Han Lee,3 and In Hee Lee1*

Department of Biotechnology, Hoseo University, Asan City, Chungnam, South Korea,1 Immunomodulation Research Center, University of Ulsan, Ulsan, South Korea,2 Department of Clinical Pharmacology and Therapeutics, Asan Medical Center, Seoul, South Korea3

Received 14 May 2007/ Returned for modification 25 July 2007/ Accepted 29 August 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Halocidin is an antimicrobial peptide found in the tunicate. A series of experiments were previously conducted in an attempt to develop a novel antibiotic derived from halocidin, as the peptide was determined to evidence profound antimicrobial activity against a variety of antibiotic-resistant microbes, with significantly less toxicity to human blood cells. In this study, we assessed the validity of one of the halocidin congeners, called Khal, as a new antibiotic for the treatment of systemic bacterial infections. Our in vitro antimicrobial tests showed that the MICs of Khal against several gram-positive bacteria were below 16 µg/ml in the presence of salt. We also determined that Khal retained sufficient target selectivity to discern microbial and human blood cells and was therefore capable of efficiently killing invading pathogens. Furthermore, Khal caused no aggregation problems upon incubation with human serum and also proved to be resistant to proteolysis by enzymes occurring in human serum. In the following experiments conducted with a mouse model of Listeria monocytogenes infection, we demonstrated that a single intravenous inoculation with Khal resulted in significant therapeutic effects on the survival of mice. In addition, our bacterial-enumeration analysis showed that after Listeria infection, livers and spleens from Khal-treated mice generated a great deal fewer recoverable CFU. Finally, the antibiotic effects of Khal were evaluated under confocal microscopy after we immunostained the liver sections with anti-Khal antibody. It was concluded that Khal bound specifically to the surfaces of bacteria colonized in the mouse liver and killed the bacteria rapidly.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Over the last 2 decades, antimicrobial peptides (AMPs) have come to be considered a promising candidate for novel antibiotics used for the control of rapidly emerging bacteria with resistance against the currently utilized antibiotic drugs (1, 5, 22). Many AMPs have been shown to evidence a broad antimicrobial spectrum and appear to be capable of killing microbes quite quickly, via a mode of action distinctly different from that exploited by conventional antibiotics (10, 24). Thus far, more than 880 AMPs have been isolated from a wide variety of organisms, including vertebrates, invertebrates, and plants (20, 23). On the basis of the determined structure of these host-derived AMPs, a number of analogues have been synthesized de novo and examined for their antimicrobial activities against microbial human pathogens (6). Halocidin is an AMP found in the hemocytes of the tunicate Halocynthia aurantium (15). AMP is a heterodimeric peptide consisting of two monomers with 18 and 15 amino acid residues, which are referred to as 18Hc and 15Hc, respectively. As natural halocidin evidences a significant degree of antimicrobial activity against clinically isolated bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa, several structural congeners of halocidin have been synthesized in efforts to design a lead compound with far more potent antimicrobial activity and less toxicity than the original peptide. The antimicrobial activities of these congeners were then compared under a variety of conditions, such as under elevated salt concentration conditions or in the presence of divalent cations (13). As a result, di-K19Hc, a homodimeric version of a peptide (named K19Hc) designed via the addition of a lysine (K) residue to the N terminus of 18Hc, was determined to be the best candidate.

In order to develop AMP-based therapies for the treatment of infections, several obstacles remain, although the agents discovered thus far have exhibited strong in vitro antibacterial activity against a range of bacteria under physiological concentrations of salts and also were safe for use in human cells. Once the peptides were systemically introduced, they were determined to bind frequently to serum proteins and be aggregated, consequently abrogating their activity (17). In addition, AMPs may prove susceptible to proteolytic degradation via endogenous proteases, thereby exhibiting too short a half-life for the exertion of their in vivo activities (10). Accordingly, the stability of peptides in the serum and the successful delivery of the peptides via circulation to the target sites may afford them in vivo antibacterial efficacy, which would clearly increase the possibility of their therapeutic use in treatments for systemic infection.

The present study was conducted to determine the antibiotic effects of halocidin-derived peptides in a mouse model of bacterial infection. At the inception of this work, we attempted to determine whether our first candidate, di-K19Hc, would retain its antibacterial activity in the presence of human serum. We found that the activity of di-K19Hc was abrogated completely in the presence of 50% human serum. Additionally, in the following experiment we noted that di-K19Hc bound to a serum protein upon incubation in human serum, thus causing an aggregation problem. Due to these unexpected results, we withdrew di-K19Hc from our candidates for a halocidin-based antibiotic. An alternate congener was then selected on the basis of its in vitro antibacterial potency. The peptide, referred to as Khal, is a heterodimer connected by an interdisulfide linkage between K19Hc and 15Hc. Prior to our evaluation of its therapeutic efficacy in a mouse model of infection, Khal was assessed with regard to its antimicrobial activity and stability in human serum. The peptide was confirmed not only to maintain its antimicrobial activity but also to be stable in human serum, which permitted us to employ it in the following experiments.

In this article, we demonstrate that Khal evidenced significant therapeutic effects against systemic Listeria monocytogenes infection. Furthermore, herein we present data demonstrating the antibiotic efficacy of Khal; these data were obtained from laser scanning confocal microscopic observations.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Peptides. The primary structures of halocidin and its two analogues (di-K19Hc and Khal) are provided in Table 1. According to the amino acid sequences, each monomer (18Hc, K19Hc, or 15Hc) was synthesized with an automated solid-phase peptide synthesizer (Pioneer Applied Biosystems, Foster, CA) at the Korea Basic Science Institute and then purified using a C18 reverse-phase, high-pressure liquid chromatography (RP-HPLC) column (Vydac 218TP54; The Separation Group, Hesperia, CA). The purification of HPLC was conducted with a variety of linear gradients of acetonitrile containing 0.1% trifluoroacetic acid. For the first 5 min after loading the sample, the column was washed with 5% acetonitrile at a flow rate of 0.5 ml/min. Next, the acetonitrile concentration was increased in a linear fashion by 1%/min for 60 min. To prepare the dimeric peptides, we followed the procedures described in a study by Jang et al. (14). While only K19Hc was used for the production of di-K19Hc, equal amounts of K19Hc and 15Hc were employed for the preparation of Khal. Di-K19Hc and Khal were repurified to >95% homogeneity via C18 RP-HPLC (Fig. 1). The identity of each dimer was verified via measurements of molecular mass via matrix-assisted laser desorption-time of flight analysis. Synthetic halocidin was generated via the mixture of 18Hc and 15Hc, via the same procedure used to generate Khal (data not shown). In one of our experiments, we used WLBU2 as a control peptide. WLBU2 (4) is an antimicrobial peptide designed from lentivirus lytic peptide 1 (LLP1), which was derived from the C terminus of the human immunodeficiency virus type 1 transmembrane protein (6). From the amino acid sequence provided in reference 4, the peptide was synthesized at the Korea Basic Science Institute and repurified, as was the case for Khal.


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  		TABLE 1. Amino acid sequences and molecular masses of antimicrobial peptides used in this study

 

Figure 1
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  		FIG. 1. RP-HPLC profile for the purification of Khal and di-K19Hc. Khal and di-K19Hc were eluted at 41% and 47% acetonitrile, respectively. Additionally, the homodimer of 15Hc (di-15Hc) was also eluted from the column at 34% acetonitrile.

 
Bacteria and animals. Two clinically isolated antibiotic-resistant bacteria, MRSA and vancomycin-resistant Enterococcus faecium (VRE), were acquired from the Culture Collection of Antibiotic-Resistant Microbes (CCARM) at Seoul Women's University in Korea. Listeria monocytogenes ATCC 19111 was employed for both the in vitro and in vivo experiments. For the animal experiments, wild-type male BALB/c mice were purchased from Hyo Chang BioScience (Tae-Ku, Korea). All mice were maintained under specific-pathogen-free conditions in the animal facility of the Immunomodulation Research Center, University of Ulsan, and were used at 8 to 10 weeks of age.

Antibacterial assays. The antibacterial activities of the peptides were tested via a broth microdilution assay, with slight modifications from the procedure recommended by the Clinical and Laboratory Standards Institute (2). Bacteria were grown overnight to stationary phase in Mueller-Hinton broth (MHB) (50 ml in a 250-ml Pyrex flask) at 200 rpm and at 37°C. The cultures were diluted in fresh MHB to a final concentration of 2 x 105 CFU/ml. To determine the antibacterial activity of the peptides in the presence of salt, we used MHB supplemented with 150 mM NaCl as a culture medium. Stock solutions of each peptide were prepared in 0.01% acetic acid at 640 µg/ml in polypropylene microtubes. The peptide solution was then twofold diluted serially in 0.01% acetic acid to 10 µg/ml. After 100-µl aliquots of the bacterial suspension were dispensed into each well of a 96-well polypropylene microtiter plate (Costar 3790; Corning), 11 µl of peptide solution was added. The antibacterial activity of the peptides was assessed by visible turbidity in each well of the plates after 18 h of incubation at 37°C. The MICs were expressed as the minimum concentration of Khal required to visibly inhibit growth.

In addition, the antibacterial activity of each peptide in the presence of human serum was evaluated via modifications of a previously described radial diffusion assay (25). Prior to testing, human blood was collected from three healthy volunteers and incubated for over 3 h at 4°C. After 10 min of centrifugation at 300 x g, normal human serum (NHS) was obtained from the supernatant and heat treated for 30 min at 56°C in order to inactivate an inherent antimicrobial activity resultant from the complement activation. The NHS was stored at –20°C until use. Peptide solutions were prepared in phosphate-buffered saline (PBS) containing 50% (vol/vol) NHS at predetermined concentrations, ranging from 25 to 200 µg peptides/ml. After 10 min of incubation at 37°C, 5-µl peptide samples were introduced into the wells (3 mm in diameter), which had been punched in the underlay agar, in which washed mid-logarithmic MRSA isolates (4 x 106 CFU/ml) were trapped. The underlay gel consisted of 9 mM sodium phosphate, 1 mM sodium citrate buffer, 1% (wt/vol) agarose (Sigma; catalog no. A-6013), and 0.3 mg of tryptic soy broth (TSB; Difco, Sparks, MD)/ml. The gel solution was adjusted to pH 7.4 prior to sterilization by autoclaving. After 3 h of incubation at 37°C, a 10-ml overlay gel of 1% agarose and 6% (wt/vol) TSB was poured over the underlay gel. After the plates were incubated overnight at 37°C, the clear zone diameters were measured to the nearest 0.1 mm. To further assess the effects of human serum on the antibacterial activity of the peptides, 2 mg of each peptide was incubated in 2 ml of PBS containing 50% (vol/vol) NHS or no NHS for 10 min at 37°C. Each sample was then centrifuged for 3 h at 295,000 x g at 4°C. The supernatant was extracted from the tube, and 5 µl was used for the radial diffusion assay. Also, the supernatant and the precipitate resuspended in PBS were subjected to tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis in order to detect the presence of Khal in each fraction. A Mini-Protean 3 cell apparatus (Bio-Rad) was used to conduct SDS-PAGE. In the experiment in which we evaluated the specific binding of Khal to the cell wall components of L. monocytogenes, the peptide was first incubated at a final concentration of 200 µg/ml for 10 min at 37°C in 10 mM sodium phosphate buffer (pH 7.4), which contained peptidoglycan (PGN), lipoteichoic acid (LTA), pustulan (Pus), or mannan (Man) in differing amounts. Then a 5-µl sample of each mixture was tested for its anti-Listeria activity via radial diffusion assay.

Cell count and hemolytic assay. Washed mid-logarithmic-phase L. monocytogenes was resuspended in 190 µl of PBS containing 108 human blood cells and 10 µl containing 8 µg Khal or each control peptide, melittin (Sigma), or WLBU2. The final concentrations of the assay mixture were 1 x 105 CFU/ml for bacteria, 5 x 108 cells/ml for the blood cells, and 40 µg/ml for the peptides. The mixtures were then incubated at 37°C for predetermined times, and 10-µl aliquots were extracted from each sample and plated on tryptic soy agar (TSA) directly or after dilution. The resultant colonies were counted following overnight incubation. On the other hand, human blood cells in the remaining mixture were counted at different time points using a hemocytometer (Marienfeld, Germany) under light microscopy. For the hemolytic assay against human red blood cells (RBC), 20 µl of Khal or each control peptide at different concentrations was mixed with 180 µl of a 2.5% (vol/vol) suspension of RBC in PBS. After 30 min of incubation at 37°C, 600 µl of PBS was added to each of the mixtures. After 3 min of centrifugation at 10,000 x g, the supernatant was removed and the optical density at 540 nm was assessed. For colony count assay in the absence of RBC, peptides were mixed with mid-logarithmic-phase L. monocytogenes in a 10 mM sodium phosphate buffer (pH 7.4) containing 0.3 mg/ml of TSB powder. Each mixture, typically 40 µg/ml of peptide in a final volume of 200 µl (2 x 107 CFU/ml for bacteria), was incubated at 37°C, and 10-µl aliquots were removed at intervals and directly, or after dilution, plated on TSA. The resulting colonies were counted after overnight incubation. All experiments were conducted in triplicate on different days. Representative results were utilized to generate data, as each experiment produced similar results.

Duration of Khal in human serum. Sixty microliters of stock Khal solution (1 mg/ml) was added to 1.2 ml of 25% NHS in PBS and incubated in a shaking incubator at 37°C. After incubation for the predetermined times (0, 2, 4, and 8 h), each 200-µl aliquot was extracted and mixed with 20 µl of 1% trifluoroacetic acid to stop any possible enzyme reactions. The resultant mixtures were then maintained at 4°C for 10 min and centrifuged for an additional 10 min at 15,000 rpm. Twenty microliters of the supernatant was loaded onto a C18 RP-HPLC column (Vydac 218TP54; The Separation Group, Hesperia, CA) and separated under conditions identical to those utilized in the peptide purification protocol. Fractions eluted from the column at a retention time equivalent to that for Khal were collected, and the peptide was identified via measurements of its molecular mass by matrix-assisted laser desorption-time of flight analysis. Also, the relative amount of Khal eluted from the HPLC column was calculated via measurements of the area of the peak in the chromatogram by using specialized software (UniPoint 3.3 for Windows 2000) in a Gilson HPLC system, transferred into the percentage of peptide remaining after incubation in NHS, and then compared to measurements from the sample injected without incubation. All of the experiments were conducted in triplicate, and a representative result was used for the data expressed herein.

Mouse infection model and Khal treatment. In the animal study, we used a mouse model of Listeria infection. Each mouse was infected via tail vein injection with bacteria and treated with a single dose (4 mg/kg of body weight) which had been determined previously to ensure 100% mouse survival after four cycles of intravenous (i.v.) administration into noninfected mice in a control experiment in which the safety of Khal was assessed. L. monocytogenes was grown in brain heart infusion broth (BHIB; Difco Laboratory, Detroit, MI) for 18 h at 37°C, and aliquots were frozen at –80°C until use. Prior to the infection experiment, the viability of the frozen aliquot was consistently confirmed via plating onto BHIB agar (Difco). Thirty mice were infected with 2 x 105 L. monocytogenes isolates per mouse via tail vein injections. Then each of 10 mice received a dose of 4 mg/kg of Khal in 100 µl PBS via i.v. injection, and another 10 mice were treated with an identical dosage of Khal 2 h after infection. As a vehicle control, 100 µl of PBS was administered i.v. to each of 10 Listeria-infected mice. All experiments with mice were approved by the Animal Care Committee of the University of Ulsan.

Quantitative organ culture. Mice were infected with L. monocytogenes at a rate of 1 x 105 cells/mouse, and each mouse was immediately inoculated i.v. with 100 µl of PBS containing 100 µg of Khal or no peptide for a control. At different time points, mice were sacrificed via CO2 euthanasia. Livers and spleens were removed and washed in sterile RPMI 1640 medium containing 10% fetal bovine serum. The organs were homogenized separately into PBS containing 0.1% bovine serum albumin by using a tissue grinder. Aliquots of each organ suspension were diluted serially in PBS and plated onto TSA containing 5% sheep blood. Colonies were counted after 18 h of incubation at 37°C.

Bacterium labeling and histology. For the in situ detection of bacteria in the mouse livers after infection, we utilized L. monocytogenes labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). The Listeria stock was resuspended in 1 ml of sterile PBS and washed twice via centrifugation at 10,000 x g for 15 min at 4°C. The bacterial suspension was then incubated with 5 (and 6)-CFSE (Molecular Probes, Eugene, OR) to a final concentration of 5 µM for 20 min at 37°C in darkness. After incubation, the cells were washed three times in PBS to remove excess dye and resuspended in 1 ml of PBS to a concentration of 1 x 109 CFU/ml. To ensure that the viability of the bacteria was not affected by the CFSE labeling, aliquots of bacterial suspensions before and after staining were diluted serially and plated on sheep blood TSA. Also, in accordance with the standard method for bacterial analysis (11), flow cytometry was conducted on the bacteria to determine whether our Listeria strain had been stained efficiently with CFSE. CFSE fluorescence was detected using a FACSCalibur system (Becton-Dickinson Bioscience, San Jose, CA). As a result, it was shown that 99.9% of L. monocytogenes cells were labeled successfully with CFSE via the aforementioned procedures (data not shown).

Mice were infected via tail vein injection with CFSE-labeled L. monocytogenes (4 x 108 cells/mouse), and each of the mice was inoculated immediately with 100 µl of PBS containing 200 µg Khal of peptide or no peptide for control via tail vein injection. According to the above-described procedure, the mice were sacrificed 10 min after peptide inoculation and the livers were removed. Then frozen 6-µm-thick liver sections were prepared from each peptide-treated or control mouse. The slides were stained with rabbit anti-Khal polyclonal antibodies (1:200 dilution) and then stained with Cy3-conjugated goat anti-rabbit immunoglobulin G antibodies (Jackson ImmunoResearch, West Grove, PA) in order to detect the primary antibodies. In this experiment, we employed anti-18Hc antiserum that had been previously generated via the injection of 18Hc into a rabbit (12) as an anti-Khal antibody, as the antiserum was verified to cross-react with Khal as well as with di-K19Hc (14). The slides were observed under a laser scanning confocal microscope (model FV500; Olympus, London, United Kingdom) with an argon-krypton laser by using double-fluorescence channels (488 nm/543 nm).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In vitro antibacterial activity of peptides. From the broth microdilution assay conducted in accordance with CLSI (formerly NCCLS) recommendations, the MICs of three peptides (halocidin, di-K19Hc, and Khal) for MRSA, L. monocytogenes, and VRE were determined in the presence of 150 mM NaCl and the absence of salt (Table 2). As reported previously (13), di-K19Hc was verified to be quite active against all bacteria tested under both conditions. Khal was also determined to retain an antibacterial activity that was significantly stronger than that of halocidin, although it evidenced MICs that were approximately twofold those of di-K19Hc, particularly in the presence of salt.


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  		TABLE 2. MICs for antimicrobial activities of halocidin, di-K19Hc, and Khal

 
Antibacterial activity of peptides in the presence of human serum. After the incubation of the peptides in 50% human serum, twofold serially diluted samples of each peptide were subjected to ultrasensitive radial diffusion assays. As a result, we determined that di-K19Hc evidenced no antibacterial activity even at the highest concentration (200 µg/ml) tested (Fig. 2A). By way of contrast, Khal largely maintained its activity after incubation in NHS, although the clearing zone of sample in 50% NHS was a little smaller than that of sample in PBS, which may be attributable to the difference between the diffusion rates of two samples on an agar plate. Therefore, we concluded that the antibacterial activity of Khal was unaffected by the presence of human serum. To further investigate the effects of serum on the activities of the three peptides, each peptide sample in 50% human serum was ultracentrifuged after 1 h of incubation. We then attempted to determine whether activity remained in the supernatants of each of the samples. Whereas the supernatants of the halocidin and Khal samples evidenced activity almost equivalent to that of the control samples (in PBS), the supernatant of the di-K19Hc sample evidenced no such antibacterial activity (Fig. 2B). In addition, the supernatant and the precipitate remaining after centrifugation of the di-K19Hc/serum mixture were subjected to tricine SDS-PAGE analyses. As shown in Fig. 2C, di-K19Hc was detected in the precipitate but not in the supernatant, thereby suggesting that di-K19Hc was bound to serum proteins and precipitated. Altogether, we concluded that the incubation of di-K19Hc in human serum caused an aggregation problem, as has been frequently noted in cases of other hitherto-evaluated peptides.


Figure 2
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  		FIG. 2. Antibacterial activity of peptides in the presence of 50% NHS. (A) After each peptide was serially diluted with PBS containing 50% NHS and incubated, their antibacterial activities against MRSA were assessed via radial diffusion assays. For the control, each peptide solution (200 µg/ml) in PBS with no NHS was subjected to an identical radial diffusion assay (upper left photo). Clearing zone diameters were expressed in units (1 mm = 10 units), and the mean values obtained from triplicate experiments were utilized to construct a graph. con., concentration. (B) Anti-MRSA activity of the supernatants of each sample following the ultracentrifugation of the peptide in 50% NHS or PBS. (C) Tricine SDS-PAGE analysis for the precipitate and supernatant after ultracentrifugation of the di-K19Hc-50% NHS mixture. The gel (16.5%) was stained with Coomassie blue. Lane M, standard molecular mass markers; lane 1, di-K19Hc; lane 2, supernatant; lane 3, precipitate. The arrow designates di-K19Hc detected in the precipitate.

 
Target selectivity of Khal on bacteria and human blood cells and hemolytic activity. The target selectivity of Khal was determined by counting of bacterial and human blood cells following the incubation of the peptide in an assay tube containing both cells (Fig. 3A). While the human blood cells were little affected by 40 µg/ml of Khal, a concentration higher than the MIC of Khal for L. monocytogenes, the number of viable bacterial cells was significantly reduced by 3 log10 after 30 min of incubation. As control peptides for this assay, we used melittin and WLBU2, which are a bee venom toxin and an LLP1-derived peptide, respectively. Melittin evidenced no target selectivity toward microbial and human cells. WLBU2, however, evidenced stronger anti-Listeria activity but less target selectivity than was observed with Khal. In addition, our hemolysis assay showed that 25, 50, and 100 µg/ml of Khal disrupted 2.3%, 4.5% and 8.5% of human RBC, respectively (Fig. 3B).


Figure 3
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  		FIG. 3. Cell counting assay for L. monocytogenes and human blood cells after incubation with Khal, WLBU2, or melittin (40 µg/ml) in a tube containing a mixture of two cells. (A) Blood cells were counted at 10, 20, and 30 min after incubation. To determine the anti-Listeria activity of the peptides, we plated each 10-µl aliquot on TSA at 10, 20, and 30 min postincubation and counted viable Listeria colonies after overnight incubation. Closed and open symbols indicate the human RBC per milliliter and bacterial CFU per milliliter, respectively. The controls (no added peptides) are represented by inverted triangles. (B) The percentage of hemolysis was calculated in accordance with the following equation: % hemolysis = (A540 of sample – A540 of peptide-free control sample)/(A540 of 100% control sample – A540 of peptide-free control) x 100 (right graph). For the peptide-free control sample, peptide-free PBS was added to a 2.5% RBC solution. Triton X-100 (1%, vol/vol) was used for the 100% hemolysis control. (C) Bacterial colony count assay for the anti-Listeria activity of peptides in the absence of blood cells.

 
Stability of Khal in human serum. Apart from the aggregation problem caused by binding with serum proteins, peptides may be susceptible to proteolytic degradation by certain proteases that exist in human serum. To determine the stability of Khal toward endogenous proteases, we measured the amounts of remaining peptide by using an RP-HPLC system at predetermined time points after incubation with human serum. The area of the Khal-containing peak in the HPLC chromatogram for each of the samples was calculated by using specialized software, and the values were converted into the remaining percentage of Khal compared to that of the control samples. Also, the Khal-containing fraction was confirmed via matrix-assisted laser desorption analysis, which generated only a single mass value that corresponded to Khal (data not shown). More than 70% of Khal peptide was detected in the peptide-serum mixture 4 h after incubation, and 39% of the peptide was determined to remain in the sample after 8 h of incubation (data not shown). Therefore, we concluded that the half-life of Khal in 25% human serum might be at least 6 h. However, when an identical experiment was conducted with di-K19Hc, we were unable to generate data regarding the remaining percentage of di-K19Hc because the retention time of di-K19Hc in the HPLC column consistently overlapped with that of a certain protein in the human serum (data not shown).

Antibiotic effect of Khal on systemic Listeria infection in a mouse model. Kaplan-Meier survival curves for the control and Khal-treated mice infected with L. monocytogenes are shown in Fig. 4B. First, we attempted to determine whether Khal evidences lethal toxicity against mice upon systemic inoculation. Khal was injected i.v. into each of the noninfected mice (n = 10) at a dose of 4 mg/kg; this injection was repeated four times at 1-day intervals. None of these mice perished during our experimental period, indicating that the administration of Khal had no lethal effect on the mice for at least 2 weeks (Fig. 4A). For systemic bacterial infection, L. monocytogenes was injected i.v. into mice (n = 10). The ability of Khal to protect mice against lethal infection was then tested via the i.v. administration of Khal into mice immediately or 2 h after infection. While none of the mice had survived by 4 days after Listeria infection in the vehicle control group, a single Khal inoculation administered immediately after bacterial challenge resulted in 100 and 90% survival during the first 6 days and over the entire period, respectively. In the case of Khal treatment delayed 2 h, it was determined that only 50% of the mice survived for 15 days, although none of the mice died until 4 days after infection.


Figure 4
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  		FIG. 4. Therapeutic effect of Khal on mice infected with L. monocytogenes. (A) Khal peptide was repeatedly injected into noninfected mice at a dose of 100 µg/mouse. Arrows indicate the injection points. No dead mice were noted for 2 weeks. (B) Listeria-infected mice were treated via single i.v. inoculations with Khal immediately or 2 h after infection. The numbers of surviving mice were determined at daily intervals for 15 days.

 
Antibacterial effects of Khal on the livers and spleens of Listeria-infected mice. We also assessed the antibiotic effects of Khal on systemic Listeria infection via bacterial-enumeration analyses of the liver and spleen. As it was previously shown that L. monocytogenes extensively colonizes the livers and spleens of mice at 2 to 4 days postinoculation (3, 18), we selected the liver and spleen as target organs for evaluations of the antibacterial efficacy of Khal administered intravenously. Mice were treated with vehicle or Khal immediately after L. monocytogenes infection and further reared for an additional 1 or 3 days. The two organs were then removed from mice at different time points, and recoverable colonies of L. monocytogenes in each organ were counted. As shown in Fig. 5, both organs of the Khal-treated mice were determined to evidence significantly reduced CFU compared to the vehicle control samples. Notably, at 3 days postinfection, liver samples from mice treated with Khal evidenced few viable L. monocytogenes colonies. To further assess the in vivo antibacterial activity of Khal, the livers were removed from each of the mice treated with Khal or vehicle after infection with CFSE-labeled L. monocytogenes and observed under confocal microscopy, via immunostaining with anti-Khal antibody (Fig. 6). The confocal sections of the mouse livers were visualized separately under two different fluorescence channels, which indicated many light green (Fig. 6A and D) and red (Fig. 6E) spots, representing bacteria and Khal peptides, respectively. Then the two images were merged so we could observe the antibiotic efficacy of Khal against bacteria which had invaded the liver. As shown in the merged image of the vehicle control sample (Fig. 6C), all bacterial colonies detected in Fig. 6A also appeared as intact spots in a green color, thereby indicating that our anti-Khal antibody did not nonspecifically bind to L. monocytogenes invading into the mouse liver. In contrast, many red spots were noted in the confocal sections of the livers obtained from the Khal-treated mice after Listeria infection (Fig. 6E), indicating that the i.v. inoculated Khal had been successfully transferred to the liver via circulation. Furthermore, from the merged images, we determined that many of the Listeria colonies shown in green overlapped the Khal peptides shown in red and thereby appeared as yellow-orange (Fig. 6F). In addition, to confirm the specific binding of Khal to the cell wall components of gram-positive bacteria, we attempted to ascertain whether the antibacterial activity of Khal might be inhibited by LTA or PGN. As shown in Fig. 7, we determined that the anti-Listeria activity of Khal clearly declined as the concentration of solubilized LTA or PGN increased. By way of contrast, two control carbohydrates (pustulan and mannan) used in this experiment were not determined to affect the antibacterial activity of Khal. Altogether, we concluded that Khal administered intravenously immediately after Listeria infection exerted its bactericidal activity in the mouse livers via specific binding to the surface components of L. monocytogenes.


Figure 5
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  		FIG. 5. Mice were i.v. infected with L. monocytogenes at 2 x 105 CFU/mouse and treated immediately with 100 µg of Khal or PBS as a vehicle control. The numbers of recoverable colonies in the liver and spleen were then determined on days 1 and 3 after infection. Data are from three independent experiments and are expressed as means ± standard deviations (P < 0.01).

 

Figure 6
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  		FIG. 6. Confocal microscopic observation of livers removed from Listeria-infected mice treated with Khal or PBS as a vehicle via i.v. injection. Confocal sections of the livers removed from two mice were immunostained with anti-Khal antibody. (A) Fluorescence image for detection of CFSE-labeled bacteria in livers from vehicle-treated mice. (B) The same confocal section was observed under different fluorescence channels in order to monitor the nonspecific binding of anti-Khal antibody to the liver. (C) The two images (A and B) were merged. (D) Fluorescence image for the detection of CFSE-labeled bacteria in the liver from a Khal-treated mouse. (E) The same confocal section was observed to detect the secondary antibody in a different fluorescence channel. Many red spots indicating Khal peptides were found in the liver section. (F) When the two images were merged, the majority of the green spots (indicating bacteria) were determined to have overlapped with the red spots (indicating the Khal peptides).

 

Figure 7
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  		FIG. 7. Specific binding of Khal to PGN and LTA. Binding/radial diffusion assay was conducted via the mixing of various amounts of PGN, LTA, Pus, or Man with Khal. The anti-Listeria activity of Khal was attenuated as the concentration (con.) of PGN or LTA in the mixture increased. By way of contrast, neither Pus nor Man affected the anti-Listeria activity of Khal.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Over the last two decades, a number of studies have reported that cationic AMPs exhibit several advantages over conventional antibiotics with regard to the ability of the peptides to kill pathogenic microbes (19, 26, 27). Accordingly, AMPs have become a focus of considerable interest as a lead compound for the development of novel antibiotics. Nonetheless, no AMP-based antibiotic has become available for the treatment of systemic infections, as the clinical applications of these agents have been hampered significantly by four major barriers: (i) toxicity to the host due to less selectivity for target cells, (ii) the loss of antimicrobial activity under physiological conditions, (iii) an aggregation problem caused by binding to proteins occurring in human serum or tissue fluid, and (iv) susceptibility to proteolytic degradation by endogenous proteases (10, 16, 17, 28). Therefore, prior to clinical tests conducted with certain AMP-derived peptides, it is important to determine whether the selected peptide evidences favorable properties that are capable of overcoming these four obstacles.

Recently, we have been conducting a series of experiments in an effort to develop a novel peptide antibiotic derived from halocidin, a cationic {alpha}-helical AMP existing within the hemocytes of tunicates (15). We focused our works on the selection of a suitable candidate for clinical testing. Previously, we selected one of the halocidin congeners (di-K19Hc) as a lead candidate, as it was shown to exhibit profound antimicrobial activities against a wide range of antibiotic-resistant bacteria under a variety of conditions (13). At the inception of the present work, di-K19Hc was tested with regard to whether it might bind serum proteins, a phenomenon which has been noted frequently with many other peptides (8, 17). As shown in Fig. 2, di-K19Hc caused an aggregation problem upon incubation with human serum; this problem compelled us to discontinue any further work with di-K19Hc. For the following experiments, we selected an alternate candidate, Khal, which is the principal subject of this study. Our in vitro experiments showed that Khal evidenced significant target selectivity for the discernment of bacteria and human blood cells and also rapidly killed bacteria, as was observed in our previous study concerning the antifungal activity of di-K19Hc (14). Furthermore, unlike di-K19Hc, this peptide was determined to retain its activity as well as stability in the presence of human serum. These favorable characteristics of Khal prompted us to exploit the in vivo potential of this peptide against lethal bacterial challenge in mice.

L. monocytogenes is a facultative intracellular bacterium, with a life cycle that enables it to survive and multiply in an intracellular environment (21). It has been reported that Listeria injected i.v. into mice arrives at the liver within 10 min after inoculation, although the bulk of the bacteria is rapidly cleared from the bloodstream (7). Then, Listeria in the liver invades host cells, such as macrophages and hepatocytes, via certain pathways, including an interaction of bacterial surface proteins with receptors on the host cell (9). It is likely that Listeria remains for some time at the early phase of infection in the extracellular space of the liver under the condition of adherence or nonadherence to the cell surface. In accordance with this theory, we now assume that the in vivo anti-Listeria efficacy of Khal may be attributable to its bactericidal action against L. monocytogenes, which occurs in the blood or extracellular fluid prior to its entry into host cells. This is considered to be one of the reasons why immediate inoculation with Khal after bacterial injection evidenced a stronger therapeutic effect than the effect when treatment was delayed for 2 h. Considering the minimal cytolytic activity of Khal toward the blood cells, the kinetics of its antibacterial action (Fig. 3), and the mode of antimicrobial action of the Khal-like peptide (di-K19Hc) (14), it is by no means clear that Khal is also capable of killing intracellular resident Listeria after its penetration into the host cell without membrane damage.

In the present study, we made a particular effort to determine the antibiotic efficacy of our peptide in the livers of bacterially infected mice via immunostaining with anti-Khal antibody, a test which has been performed only rarely with the hitherto-known AMPs. As shown in Fig. 6, Khal peptides arriving at the liver from the tail vein were generally bound to the surfaces of bacteria. This specific binding of Khal to the bacterial surface was made apparent by the results of a radial diffusion assay conducted in the presence of several polysaccharides, which revealed that Khal bound specifically to LTA and PGN on the cell walls of gram-positive bacteria (Fig. 7). Collectively, these results strongly bolstered our conclusion that Khal has sufficient target selectivity to locate certain invading gram-positive bacteria and kill them efficiently with little toxicity toward mammalian hosts. Although the therapeutic window presented in this study is narrow, our results still provide encouraging evidence that Khal might be utilized as an agent for the treatment of bacterial infections.


   ACKNOWLEDGMENTS
 
This work was supported by a grant from the World-Class 2030 Project of Hoseo University. B. H. Sung and S.-C. Lee were supported by the second project of BK21, the Ministry of Education and Human Resources Development.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biotechnology, Hoseo University, 165 Sechuli, Baebangmyun, Asan City, Chungnam 336-795, South Korea. Phone: 82-41-540-5626. Fax: 82-41-548-6231. E-mail: leeih{at}office.hoseo.ac.kr Back

{triangledown} Published ahead of print on 10 September 2007. Back

{dagger} W.S. Jang and S.-C. Lee contributed equally to this study. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Antimicrobial Agents and Chemotherapy, November 2007, p. 4148-4156, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00635-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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