JAC Advance Access published online on January 9, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkl495
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The spectrum of antimicrobial activity of the bacteriocin subtilosin A
1 Department of Biologic and Materials Sciences, The University of Michigan School of Dentistry, 1210 Eisenhower Place, Ann Arbor, MI 48108, USA 2 Department of Chemistry and Biophysics Research Division, The University of Michigan, Ann Arbor, MI 48109, USA
* Corresponding author. Tel: +1-734-975-0946; Fax: +1-734-975-9329; E-mail: ceshelbu{at}umich.edu
Received 14 August 2006; returned 21 September 2006; revised 9 November 2006; accepted 12 November 2006
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Abstract |
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BACKGROUND: Bacterocins are antimicrobial peptides produced by bacteria with a relatively narrow range of activity against closely related organisms. Subtilosin A is a bacteriocin produced by Bacillus subtilis that has activity against Listeria monocytogenes, which might indicate antimicrobial activity unusual for bacteriocins.
OBJECTIVES: To examine the antimicrobial activity and factors affecting the activity of subtilosin A against a range of potentially pathogenic bacteria.
METHODS: The peptide was purified from cultures of B. subtilis and the MIC determined for 18 species of bacteria using a microdilution methodology. The extent of capsule formation was determined using microscopic examination of cells mounted in India ink. Protease mutants of a susceptible bacteria and mild heat shock were used to examine the effect of environmental stress on subtilosin A activity.
RESULTS: Subtilosin A proved to have antimicrobial activity against a wide range of bacteria including Gram-positive and Gram-negative bacteria and both aerobes and anaerobes. The peptide was less effective against capsulated forms of two Gram-negative bacteria than the non-capsulated strains of either. Heat shock but not protease activity also altered the effectiveness of the bacteriocin.
CONCLUSIONS: Subtilosin A has limited antimicrobial activity against a number of human pathogens which, combined with its relative ineffectiveness against some capsulated pathogens, may limit its usefulness as a human therapeutic.
Key Words: antimicrobial peptides , capsules , heat shock
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Introduction |
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Bacterocins are a group of antimicrobial peptides produced by bacteria with a relatively narrow range of activity against closely related organisms. Subtilosin A (subtilosin) is one of several antimicrobial peptides produced by Bacillus subtilis.1,2 A complete three-dimensional structure of subtilosin has recently been determined; the 35 amino acids form a circular structure with three cross-links between sulphurs of cysteine and the
-carbon of the two phenylalanines and the threonine. This distinctive post-translationally generated structure probably indicates that subtilosin belongs to a unique class of bacteriocins.3 The mechanism by which anionic peptides kill bacteria is not understood. Since anionic peptides require zinc for maximal activity it has been suggested4 that zinc may form a cationic salt bridge which attracts these peptides to the negative charge of the microbial surface. Alternatively, the antimicrobial activity of subtilosin may be a function of interaction with membrane-associated receptors, similar to that seen with the lantibiotic, nisin.5 NMR and fluorescence experiments on model membranes suggest that binding to the outer cell membrane, resulting in membrane permeabilization, is also a possible mechanism for bacterial killing.6 This is unlike the mechanism of most cationic antimicrobial peptides that interact directly with the cell membrane and cause membrane disruption leading to bacterial cell death.7
Little is known about the breadth of subtilosin antimicrobial properties, although it has consistently been reported to be effective against Listeria monocytogenes.2 Here we describe an expanded evaluation of the antimicrobial activity of subtilosin and describe some environmental factors that might alter that activity.
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Materials and methods |
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Bacterial strains and growth
American Type Culture Collection strains were obtained directly from the ATCC or from MicroBiologics, St Cloud, Minnesota. Porphyromonas gingivalis ATCC 33277 gingipain mutants KDP129 (kgp–) and KDP133 (rgp1– and rgp2–) were a gift from Dr Koji Nakayama, Kyushu University, Fukuoka, Japan.8 Enterococcus faecalis and Streptococcus gordonii Challis ATCC 49818 cultures were a gift from Dr Donald B. Clewell, University of Michigan. Clinical isolates of Klebsiella pneumoniae were obtained from the University of Minnesota. Anaerobic bacteria were maintained in an anaerobe chamber (Coy Manufacturing, Grass Lake, MI, USA) at 37°C on PRAS brucella agar plates (Anaerobe Systems, Morgan Hill, CA, USA) in an atmosphere of 5% hydrogen, 10% carbon dioxide and 85% nitrogen. Broth cultures were grown in brucella broth supplemented with 5 mg/L haemin and 5 µg/L vitamin K. Aerobic species were maintained on Trypticase soy agar and broth cultures grown directly from individual colonies in Trypticase soy broth.
A sterile, 384-well microtitre plate (NUNC) was used as the platform for the assay. Overnight cultures of each bacterium were diluted to 106/mL in sterile phosphate-buffered saline and seeded into each well (10 µL) of the plate. Doubling dilutions (final concentrations of 200–0.39 mg/L in 2 x brucella broth) of subtilosin (10 µL) in replicates of 8 were then added and the cultures covered with a sterile adhesive plastic film. For heat shock experiments the plates were then placed in a 45°C incubator for 30 min. After centrifugation for 1 min at 800 g to collect droplets from the walls of the plate it was incubated at 37°C for 12–48 h in the anaerobic chamber or overnight in air. Growth was determined by measuring the optical density at 595 nm (OD595) of each well using a microwell plate reader. The MIC was determined as the lowest dilution of subtilosin without significant growth above the original inoculum (P
0.01, t-test).
Subtilosin was produced as described previously.2 B. subtilis ATCC 6633 was grown anaerobically at 37°C in Trypticase soy broth, the cells removed by centrifugation at 7000 g at 4°C for 20 min and the supernatant made to 65% ammonium sulphate with the solid salt. The precipitate was dissolved in 0.1% (v/v) trifluoroacetic acid and 20% (v/v) acetonitrile in water (buffer A). The peptides were separated by reversed-phase HPLC (ODS 80T; 5 µm particles; 2 mm x 25 cm column; TOSOH Biosciences, Montgomeryville, PA, USA). Buffer B was 0.1% (v/v) trifluoroacetic acid in acetonitrile. Samples were applied with 100% buffer A and eluted with a linear gradient of buffer B (2–100%). Fractions were tested for antimicrobial activity against Kocuria rhizophila (ATCC 9341) using an agar diffusion assay as described previously.2 Three separate HPLC fractions with antimicrobial activity against K. rhizophila (1 µL) were mixed with 1 µL of matrix solution (20 µg/µL of -cyano-ß-hydroxycinnamic acid in buffer A) on a MALDI-TOF target and dried in air. MALDI-TOF mass spectral analysis confirmed the identity of the third peptide as subtilosin A with a signal at m/z 3400.7. Expansion of the chromatogram also showed minor species at m/z 3422.7 and m/z 3438.6, the sodium and potassium adducts, respectively.
Measurement of bacterial capsule formation
Strains of bacteria were tested for evidence of capsule formation by suspension in India ink. Cells pellets were resuspended in India ink. Capsule size was measured at magnification of 1000x. At least three different fields were randomly chosen and photographed. The capsule size (distance from the edge of the capsule to the cell wall) of 20 cells from each strain was measured and averaged.
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Results |
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Subtilosin has effective antimicrobial activity against a number of bacteria
The peptide was most effective (MICs between 1 and 12.5 mg/L) against E. faecalis OGX-1, L. monocytogenes ATCC 19115, P. gingivalis ATCC 33277, K. rhizophila ATCC 9341, Enterobacter aerogenes ATCC 13408, Streptococcus pyogenes ATCC 19615 and Shigella sonnei ATCC 25931. This group contained both Gram-negative and Gram-positive bacteria and anaerobes and aerobes. A second group of bacteria demonstrated moderate susceptibility (25–100 mg/L) including Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, S. gordonii Challis ATCC 49818 and Staphylococcus aureus ATCC 6538. All other test strains were resistant (MIC >100 mg/L) to subtilosin (Table 1).
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Subtilosin antimicrobial activity is blocked by capsule polysaccharide of some bacteria
The antimicrobial activity of the peptide was substantially reduced in capsulated strains of P. gingivalis and K. pneumoniae. P. gingivalis ATCC 33277 has little or no capsule and was susceptible (MIC 3.125 mg/L) to subtilosin. However, the MIC for the capsulated strain of P. gingivalis W83 was >100 mg/L. Strains of K. pneumoniae with either reduced or no observable capsule were susceptible to subtilosin concentrations between 1.25 and 25 mg/L. All other strains were resistant to the peptide and had relatively larger amounts of capsule as detected by the India ink staining (Table 1).
Porphyromonas gingivalis serine proteases do not affect subtilosin activity
To determine whether P. gingivalis proteases might degrade subtilosin activity we compared P. gingivalis 33277 with two mutants lacking the lysine-specific or arginine-specific enzymes. We expected that if the lysine-specific enzyme was able to degrade subtilosin, the lysine disruption mutant would have a lower MIC value than the parent. There are no arginine residues in subtilosin, so the arginine-specific mutant was not expected to be different from the parent strain. The MIC values for both mutants, however, were not different from the parent strain (Table 1).
Heat shock increases susceptibility of selected strains to subtilosin
Seven strains of bacteria found to be resistant to subtilosin were tested after a 45ºC heat shock. There was a reduction in the MIC for all the Gram-negative species, but no significant change in either of the Gram-positive organisms tested (Table 2).
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Discussion |
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The ability of subtilosin to inhibit growth of L monocytogenes prompted investigation into its affect on other bacteria. It demonstrated only moderate effectiveness against many pathogenic bacteria, but remains a viable candidate for other uses. We also investigated three environmental effects that have been shown to modify the effectiveness of other antimicrobial peptides.
The capsule-based resistance to subtilosin we observed also suggests a limited therapeutic potential since capsule formation is an important virulence mechanism for many bacteria, including K. pneumoniae. There was a relationship between the level of capsule formation in that organism and its resistance to subtilosin. The mechanism may simply be physical exclusion by capsulated species or the bacteriocins might be bound by the capsules before they can reach the bacterial cell surface. Either mechanism would protect the bacterium and allow persistence in the face of competition with bacteriocin-producing species.
Stress by rapid change in temperature results in activation of a large number of genes in all bacteria, some of which are important in resistance to antimicrobial peptides. In this study heat stress increased the effectiveness of subtilosin against selected Gram-negative bacteria, similarly to that reported with nisin and Gram-negative bacteria.9 This increased susceptibility could reduce the survival of these pathogens in the presence of subtilosin.
Lastly, we examined the effect of proteolytic enzymes on the activity of subtilosin. P. gingivalis has both arginine- and lysine- specific activities important in virulence.10 We hypothesized that if the lysine-specific enzyme could degrade subtilosin that it might indicate a possible role for this gene in resistance to bacteriocins in the oral cavity. This was apparently not the case although the reason is not clear from these experiments. It is possible that the lack of protease susceptibility is the result of the three cross-links between sulphurs of cysteine and the -carbon of the two phenylalanines and the threonine, similar to that of lantibiotics which are also protease resistant.
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None to declare.
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Present address. The Centre for Cellular and Molecular Biology, Hyderabad, India. 
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Acknowledgements |
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We thank Dr Dominica G. Sweier for critical reading of this manuscript. We also thank Professor J. C. Vederas, Department of Chemistry, University of Alberta, Edmonton, Canada, for providing subtilosin A that was used in our preliminary experiments. This study was supported by funds from NIH DE11117 (to D. E. L.), NIH DE 07256 (to M. S. L.) and AI054515 [GenBank] (to A. R.).
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References |
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