CSIRO Livestock Industries, Private Bag 24, Geelong, Victoria 3220, Australia
Received 27 November 2002; returned 23 January 2003; revised 20 February 2003; accepted 25 February 2003
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Abstract |
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Methods: Groups of mice were challenged intravenously with Listeria monocytogenes and treated with purified P126 at varying times before and after challenge to determine whether administration of this peptide reduced numbers of colonizing L. monocytogenes and the symptoms of listeriosis.
Results: The bacteriocin P126 retained antilisterial activity after injection into the mouse. During the early time-points of listerial infection, the purified P126 was found to significantly reduce the listerial load in the liver and spleen and, further, that this reduction translated to reduced clinical signs of disease.
Conclusions: This is the first report of a Class IIA bacteriocin displaying in vivo antimicrobial activity. Such a result provides preliminary evidence that this class of molecules may be useful in controlling systemic bacterial infections.
Keywords: bacteriocin, in vivo activity, piscicolin 126
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Introduction |
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The most widely studied group of bacteriocins are the lantibiotics. Research on the lantibiotic nisin has shown this molecule to be active against Staphylococcus aureus and vancomycin-resistant enterococci when tested in vitro.6 Nisin has been used to prevent colonization of chicken skins by Salmonella sp.,7 whereas other reports have shown the effectiveness of nisin8 and lacticin 31479 (a two-component bacteriocin) at controlling surface-related infections such as mastitis. A related lantibiotic, mersacidin, cured systemic staphylococcal infections10 but was ineffective at controlling listerial infections,11 due to a proposed inability to cross the eukaryotic cell membrane.
Related work has also been carried out using cationic antimicrobial peptides such as buforin II, cecropin P1 and magainin II. In preliminary work, a number of these peptides were shown to produce in vitro activity against Pseudomonas aeruginosa and Escherichia coli.12,13 Expanding on this work, other reports show magainin II (an antimicrobial peptide originating from the African clawed frog) alone and in combination with a ß-lactam antibiotic has improved the survival rate of rats in a Gram-negative septic shock model.12,14 It was also noted in these reports that none of the animals displayed any adverse effects after administration of this peptide. Despite the promise of this work there has been little published information relating to the activity and safety of the Class IIA bacteriocins once injected into an animal. On this topic a single report shows pediocin AcH, derived from a lactic acid bacterium, to be non-immunogenic and non-toxic when injected into mice and rabbits.15 However, no report was made on the antimicrobial activity of this molecule following injection into the animal.
As far as we are aware no report has addressed the issue of antibacterial activity of a systemically injected Class IIA bacteriocin. In the present study, the previously described Class IIA bacteriocin, piscicolin 126 (P126), was expressed from Escherichia coli and purified.16 P126, originating from the bacterium Carnobacterium piscicola, is a 4.4 kDa peptide comprising 44 amino acids.16 Experiments were carried out to determine whether P126 retained antimicrobial activity within mice as evidenced by control of bacterial population development and disease progression following challenge of mice with Listeria monocytogenes, an experimental model of bacterial infection.
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Materials and methods |
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The sequence of the pisA gene coding for P126 has the GenBank Accession no. of AF275938. E. coli LE39217,18 carrying the plasmid pPOW2-mpisAHN,16 expressing P126 constitutively, was grown overnight in 2 L of LB-broth with 100 µg/mL of ampicillin. P126 is fused to a secretion signal in this construct that facilitates transfer of P126 into the culture medium. The supernatant, containing secreted P126, was harvested by removal of the cells following centrifugation (5000g, 25 min, 4°C). P126 was harvested by precipitation with 51.6% w/v (NH4)2SO2 at 4°C with stirring. The precipitated protein was pelleted by centrifugation (12 000g, 30 min, 4°C) and resuspended in 160 mL of 20 mM sodium phosphate buffer at pH 5.8. The solution was cleared by centrifugation (12 000g, 30 min, 4°C), and the cleared supernatant was applied to a Q-Sepharose Fast Flow column (1.0 x 2.5 cm; Pharmacia) previously equilibrated with 20 mM sodium phosphate buffer, at a flow rate of 2 mL/min. Following washing with the 20 mM sodium phosphate buffer, fractions were eluted with steps of 500 mM NaCl and 1 M NaCl in sodium phosphate buffer pH 5.8. Fractions were assayed for activity against L. monocytogenes by radial diffusion assays and active fractions pooled. The pooled active fractions are referred to as purified P126 throughout this work. The concentration of protein contained in these fractions was determined using the Bio-Rad commercially adapted method of Bradford.19
A P126-free control preparation designated MocBac was prepared in an identical fashion to P126 with the exception that the bacterial strain E. coli LE392 did not contain the pPOW2-mpisAHN recombinant plasmid. The two preparations should therefore only differ in the presence of P126.
Radial diffusion assay
The L. monocytogenes 4A strain used in this study was isolated from contaminated meat and has been confirmed to cause listeriosis, resulting in mortality, in mice and chickens (data not shown). In vitro usage has been described previously.16 Bacteriocin activity was assayed as inhibition of bacterial growth on agar plates through radial diffusion assays. Dilutions of L. monocytogenes containing 1 x 106 cfu/mL were spread across the surface of Trypticase-Soy Broth (TSB) agar (BBL, Becton Dickinson). This level of inoculum produces confluent growth on the agar surface following overnight incubation. Wells were punched into the TSB agar layer and 50 µL of each P126 solution was added to a well. P126 (40 µg/mL) was added neat and then in two-fold dilutions, to the lowest concentration of 0.625 µg/mL, using sterile water as the diluent. Plates were incubated at 37°C overnight and examined for areas of growth inhibition surrounding the wells indicating antimicrobial activity. When confluent growth occurs to the edge of the well, the strain is regarded as resistant to the test solution.
Liquid killing assay
L. monocytogenes was grown in 10 mL of TSB until OD600 reached the range of 0.20.3 equivalent to 5 x 107 cfu/mL. Aliquots (50 µL) of the culture were taken and mixed with either 50 µL of P126 (40 µg/mL) resulting in a final concentration of (20 µg/mL), 50 µL of PBS, 50 µL defibrinated horse blood or 50 µL defibrinated horse blood plus 50 µL P126 resulting in a final concentration of (13.3 µg/mL). The mixtures were incubated at 37°C for 60 min and the remaining number of viable L. monocytogenes was determined by plating dilutions of the mixture onto TSB agar at 15 min intervals.
Zymogram
Concentrated supernatant and column eluate, containing P126, from the P126 purification protocol were run on 1020% Tricine gels according to the manufacturers instructions (Invitrogen). Pre-stained markers (Bio-Rad, Broad Range) were run as size controls. Gels were run in duplicate. One gel was stained according to instructions (Invitrogen, SilverQuest) and the other was washed, overlaid with sensitive L. monocytogenes, and incubated as previously described.20
Evaluating toxicity of P126
Groups of four mice received either 50 µL (2 µg) of purified P126 intravenously (iv) or 500 µL (20 µg) intraperitoneally and were observed for signs of toxicity for 72 h post-injection. The following were used as markers of a toxic response: localized inflammation surrounding the site of injection, vomiting, diarrhoea, respiratory distress, depression and listlessness.
Preparation of log phase L. monocytogenes for challenge
L. monocytogenes was grown in 1 mL TSB overnight at 37°C, in a shaking incubator. Then 100 µL of this culture was added to 10 mL fresh TSB and the culture grown until the OD600 was in the range of 0.20.3. At this optical density, the viable count of L. monocytogenes was 5 x 107 cfu/mL. The culture was diluted in TSB to a level where 50 µL contained 1 x 104 cfu and this suspension was used to challenge mice in order to produce a sublethal infective dose.
Animals
Adult female BALB/c mice were used in all experiments. All animals had access to commercial mouse maintenance feed (Ridley AgriProducts, Australia) and water ad libitum. Cardboard cylinders were supplied for environmental enrichment. All experimentation was approved and conducted in accordance with guidelines set out by the Animal Ethics Committee of the Australian Animal Health Laboratories.
Treatment protocol
The experimental outline of the animal trial is listed in Table 1. All solutions were administered iv into the tail vein. Mice were observed for 72 h following challenge, clinical signs of disease were recorded and all mice were euthanized at the 72-h end point.
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Mice challenged with a sublethal dose of L. monocytogenes survive for 72 h post-challenge during which time colonization of both the liver and spleen occurs. Mice begin to display clinical symptoms of listeriosis 24 h post-challenge and these are exhibited as huddling, reduced activity and panting and they take on a scruffy appearance due to reduced preening. All mice were observed for these signs throughout the 72 h post-challenge.
Evaluation of treatment
Following euthanasia or mortality, mice were necropsied and the liver and spleen were removed. The organs were dunked in a 70% ethanol solution to kill any surface bacterial contamination and then ground through a strainer into 5 mL TSB broth to release the bacteria. Ten-fold dilutions of the solution were prepared in saline and dilutions of the ground organ were plated onto Palcam agar (Difco, Bacto Laboratories, Australia), selective for L. monocytogenes, in order to determine the number of viable L. monocytogenes colonizing the organ.
Statistical analysis
Following the trial, the median viable count of L. monocytogenes inhabiting either the liver or spleen was determined in both treatment groups. The P126 treatment group was then compared with the challenge only control, using the unpaired t-test, Welch corrected, to determine whether a statistically significant difference in the number of colonizing L. monocytogenes existed as a result of P126 administration.
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Results |
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After expression, concentration and ion exchange column purification the purified P126 solution was found to contain 40 µg protein per mL. The MocBac preparation contained less than 5 µg protein per mL. Radial diffusion assays were used to determine the in vitro antilisterial activity of these solutions. The purified P126 was active against L. monocytogenes at concentrations ranging from 40 µg/mL to a minimum inhibitory concentration of 1.25 µg/mL. When tested in an identical fashion, the MocBac preparation produced no detectable activity.
A sample of the P126 solution was run through a polyacrylamide gel in order to separate the constituent proteins and overlaid with L. monocytogenes to determine which fraction of the P126 solution produced the antilisterial activity. As shown in Figure 1 the activity can be attributed to a 4.4 kDa peptide corresponding to the expected size of P126. The figure also shows the purity of the sample with relatively few contaminating bands present.
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Groups of four mice received either 2 µg of purified P126 iv or 20 µg intraperitoneally and were observed for signs of toxicity for 72 h post-injection. None of the mice showed any signs of localized inflammation around the site of injection, vomiting, depression or other behavioural signs of toxicity.
L. monocytogenes challenge
A dose of 1 x 104 cfu L. monocytogenes delivered by iv injection was chosen for the challenge trial. Mice were administered 2 µg of P126 at varying times before or following L. monocytogenes challenge and the development of listeriosis was recorded. As a control, Group 5 was administered the MocBac preparation. Average resulting numbers of L. monocytogenes isolated from the liver and spleen and the percentage of mice displaying clinical signs of listeriosis are recorded in Table 2.
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Discussion |
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A purified solution of P126 was produced and confirmed to have in vitro antilisterial activity. A MocBac solution, containing no P126, was prepared at the same time and in the same manner for use as a control. When tested in an identical fashion to P126 the MocBac preparation showed no in vitro antibacterial activity. Liquid killing assays demonstrated that the activity of P126 was bactericidal, resulting in a greater than 90% population decrease, and that this killing was rapid, occurring within 15 min. This rapid killing may be required for an antilisterial effect within this model as L. monocytogenes become internalized within hepatocytes over a period of 10 min to 6 h following entry into the bloodstream.21 The addition of blood to the assay did not compromise activity, if anything apparent activity was higher. We had been concerned that because of the hydrophobic nature of the peptides they may have been selectively absorbed out by the blood cells. Activity suggests this is not the case or that P126 remains active even when associated with other cells.
The non-toxic nature of a bacteriocin pediocin AcH, a related Class IIA bacteriocin, has been reported previously.15 We have made a similar observation here with P126 as no toxic effects could be detected in mice even when dosed with 10 times the amount delivered therapeutically, as a part of the challenge trial. The lack of toxicity suggests that this molecule may be safely administered to mice at this level.
The standard challenge used here involved injection of 1 x 104 cfu of L. monocytogenes into the tail vein. This was found to be the highest number of bacteria that could be delivered to the mice resulting in clinical signs without mortality (data not shown). This method of challenge was chosen over the natural oral route so as to provide an artificially high number of L. monocytogenes within the blood during a narrow time frame. By using this approach we were biasing the infection process to provide the best opportunity for detecting in vivo activity of P126 against systemic L. monocytogenes. Analysis of mice in Group 6, 72 h post-challenge, showed high numbers of L. monocytogenes colonizing both the spleen and liver. In all Groups, clinical signs of listeriosis appeared to correlate well with the number of bacteria isolated from the liver and spleen. It would seem that as the number of colonizing L. monocytogenes approaches 1 x 106 cfu per organ the mouse displays obvious clinical signs of listeriosis.
Following in vivo testing, mice administered P126 15 min before challenge showed a significant reduction in the clinical signs of listeriosis and number of L. monocytogenes cultivable from either the liver or spleen when compared with those challenged only with L. monocytogenes. This treatment group contained a larger number of mice than other groups, as we believed it to be the most likely for detection of antimicrobial activity. A similar trend was seen in the group dosed 30 min post-challenge although the small group numbers make statistical analysis difficult. These results indicate that purified P126 retains antimicrobial activity in vivo and in conjunction with the liquid killing assays, that this peptide is not degraded by blood proteases within this time frame. This is an important observation as it could be postulated that rapid removal of these antimicrobial peptides by blood proteases would significantly reduce their function. Further work could focus on determining the half-life of this and related peptides within blood, tissue culture or systemically.
Mice in Group 5 treated with the MocBac preparation differ only slightly from those in the challenge-only Group 6. This result is important and allows the effects of P126 administration to be attributed to P126.
It is also interesting to note that listerial load reduction only resulted when P126 was administered at the very early stages of infection when high numbers of L. monocytogenes may be found in the bloodstream. A similar result was shown with the bacteriocin, mersacidin. In these works, mersacidin was shown to have in vivo activity against systemic methicillin-resistant Staphylococcus aureus10 yet did not inhibit intracellular multiplication of L. monocytogenes.11 One possible explanation for these results is that P126, like mersacidin, is active when faced with bacteria that are free in the bloodstream, but not likely to be effective at crossing the eukaryotic cell membrane to gain access to internalized cells. Uptake of peptides of this size by eukaryotic cells is likely to require specific receptors and we believe it unlikely that P126 would fortuitously activate such a receptor. The short time frame of activity displayed by P126 in this study limits the practical benefit of this type of molecule. However, novel antibiotics are needed to address the problem of antibiotic resistance and future studies might target modification of the bacteriocin in a manner to optimize uptake for activity.
Overall, this work has shown that the Class IIA bacteriocin, P126, retains antimicrobial activity to systemic L. monocytogenes and does not induce any toxicity after injection into an animal. These results have not been shown previously for Class IIA bacteriocins and are fundamental to the development of this class of antimicrobial peptides for use as in vivo therapeutic agents. Discovery of novel classes of antibiotics could be vital for the control of infectious bacterial diseases, especially those caused by antibiotic-resistant bacterial species. One limitation to this development is that P126 appears unable to kill intracellular bacteria, although it may be possible in future to modify P126 for uptake into eukaryotic cells. Other modifications to the amino acid sequence could potentially develop molecules with extended half-lives, increased activity or altered bacterial target range. Finally, considerable effort should go into future experimentation varying parameters such as the timing of dosing, number of doses and concentration of dose so as to better define the therapeutic effectiveness of these molecules.
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Acknowledgements |
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Footnotes |
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References |
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