CURRENT MICROBIOLOGY Vol. 55 (2007), pp. 36–41 DOI: 10.1007/s00284-006-0554-z
Current Microbiology An International Journal ª Springer Science+Business Media, LLC 2007
Effects of Cations on Antimicrobial Activity of Ostricacins-1 and 2 on E. coli O157:H7 and S. aureus 1056MRSA Haryadi Sugiarto, Pak-Lam Yu Biotechnology Group, Institute of Technology and Engineering, Massey University, Private Bag 11-222, Palmerston North, 5301, New Zealand Received: 2 November 2006 / Accepted: 9 March 2007
Abstract. Ostricacin-1 and ostricacin-2 (Osp-1 and Osp-2) were b-defensins antimicrobial peptides that were purified from ostrich leukocytes using a cation-exchange column and a semi-prep RP-HPLC column. Both ostricacins were subjected to increased concentrations of monovalent cations (K+ and Na+) and divalent cations (Ca2+ and Mg2+) in order to investigate the effect of cations on the activity of these ostricacins on Gram-negative bacteria and Gram-positive bacteria. The radial diffusion assay method showed that both ostricacins were sensitive to the presence of cations. The divalent cations showed more antagonized effect on the activity against Gram-negative bacteria than the monovalent cations, as the ostricacins lost ability to inhibit bacterial growth at very low concentration (5 mM). When viewed in the context of other defensins activity, our data support a hypothesis that defensinsÕ overall net positive charge determine the sensitivity to cations.
Defensins are small cationic antimicrobial peptides characterized with a triple-stranded b-sheet structure and widely found in insects, animals, plants, and humans [7, 12, 19]. These b-sheet structures are interconnected with three disulfide bridges formed by their six cysteines. Defensins have shown a wide range of antimicrobial activities against Gram-positive bacteria, Gram-negative bacteria, yeast, fungi, viruses, malignant, and nonmalignant cells. In spite of the broad spectrum of activity, the potency of defensins are affected by the presence of cations [5, 7, 21]. The presence of cations reduces the affinity of defensins to the negatively charged microbial membrane. In addition, divalent cations, such as magnesium ions, are cations that are usually present around specific binding sites of lipopolysaccharides (LPSs) of Gram-negative bacteria. Therefore, increasing concentration of cations would inhibit the interaction between defensins with the microbial binding sites. Because these cations are usually present in body fluids of animals and humans, 100–150 mM of sodium and potassium ions as well as 1–2 mM of magnesium and calcium ions, defensins are believed to be effective in Correspondence to: Pak-Lam Yu; email:
[email protected]
killing pathogens only at sites with low concentration of cations and serum such as in the phagocytic vacuoles of phagocytes and on the surfaces of skin and mucosal epithelium [22]. Four ostricacins, classified as b-defensins, have been previously purified and characterized from ostrich leukocytes [18]. Two of the ostricacins, ostricacin-1 and ostricacin-2 (Osp-1 and Osp-2), have shown the ability to inhibit bacterial growth with minimum inhibitory concentrations (MICs) of 1–12 lg/mL. Both peptides also share homology to chicken and turkey heterophil b-defensins. The molecular weights and the amino acid sequences of both peptides are shown in Table 1. In this study, the potency of Osp-1 and Osp-2 against Escherichia coli O157:H7 and Staphylococcus aureus 1056MRSA were investigated in the presence of increasing concentrations of cations using a widely accepted procedure developed by Lehrer to test antimicrobial activity [17]. Materials and Methods Materials. Underlay and overlay agar were made using 100 mM sodium phosphate buffer. This buffer was made by mixing a monobasic sodium phosphate and a dibasic sodium phosphate
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H. Sugiarto and P.-L. Yu: Effects of Cations Table 1. Amino acid sequence of peptides Peptide name
Weight (Dalton)
Amino acid sequence
Osp-1 Osp-2
4009.8 4704.9
LFCRKGTCHFGGCPAHLVKVGSCFGFRACCKWPWDV-NH2 APGNKAECEREKGYCGFLKCSFPFVVSGKCSRFFFCCKNIW-NH2
solution to pH 7.4 [17]. The underlay agar was made by mixing 100 mL of sodium phosphate buffer with 10 mL of Trypticase Soy Broth (TSB; Difco 0370-17-3, Sparks, MD, USA) and 10 g of Ultra PureTM Agarose (Invitrogen 15510-019, Carlsbad, CA, USA). The TSB broth was made according to manufacturerÕs direction. Deionized distilled water was added to bring the volume to 1 L. The overlay agar was made by mixing 60 g of TSB and 10 g of Ultra Pure Agaraose in 1 L of deionized distilled water. The underlay agar contained a limited amount of nutrients, as it would be used for pathogen inoculations, whereas the overlay agar contained the nutrients for the pathogen to grow. Because the plates were incubated overnight, the influence of the salt, in terms of delaying the cell growth, was considered minimal. Antimicrobial Peptides. The two ostrich peptides (Osp-1 and Osp-2) were isolated from ostrich neutrophils using cation-exchange chromatography and high-performance liquid chromatography (HPLC) as previously described [18]. To obtain sufficient materials for these characterization tests, a semi-prep HPLC column, Jupiter 10u Proteo 90A (Phenomenex OOG-4397-NO, Torrance, CA), was used with an injection volume of 200 lL. The peptides were separated using a linear 21–29% acetonitrile gradient with 0.1% trifluoroacetic acid (TFA). Elution of the antimicrobial peptides was carried out at 1 mL/min flow rate and monitored using absorbance at 230 nm. Each purified peptide was redissolved with 0.01% acetic acid after freeze-drying. The amino acid sequence of these peptides is shown in Table 1. Bacterial Strains and Growth Conditions. Escherichia coli O157:H7 and S. aureus 1056MRSA were used throughout this experiment. The E. coli O157:H7 was obtained from Communicable Disease Centre, New Zealand and the S. aureus 1056MRSA was obtained from Institute of Food Nutrition and Human Health, Massey University, New Zealand. Both micro-organisms were grown on brain heart infusion agar (BHI; Merck 113825, Darmstadt, Germany). These bacteria are well-known food pathogens. They were used to represent each class of bacteria. Radial Diffusion Plate Assay. This assay was used to determine the MIC of the diluted ostricacins [13]. E. coli O157:H7 and S. aureus 1056MRSA were grown to mid-log phase in TSB. The MIC of each purified peptide was determined using radial diffusion plate assays of diluted peptides. A graph of log peptide concentration against the size of clearing on the plate minus the size of the well was plotted and a best-fit straight-line was determined using regression. The MIC was calculated by finding the intersection of the line with the x axis, indicating the peptide concentration required to have a zero clearing. Cation Effects. There were two monovalent cations (Na+ and K+) and two divalent cations (Ca2+ and Mg2+), in the form of chloride salts, used in this assay. The NaCl or KCl was added to the underlay agar to the final concentrations of 0, 10, 50, 100, 200, and 500 mM, whereas the CaCl2 and MgCl2 was added to the underlay agar to the final concentrations of 0, 2, 5, and 10 mM.
Results Effect of Monovalent Cations. An example of raw data for the calculation of the Osp-1 MIC affected by 10 mM NaCl is shown on Table 2. From these raw data, a graph of log peptide concentration against the size of clearing on the plate was plotted and shown on Fig. 1. The MIC was subsequently calculated by finding the x-intercept, which indicated the minimum peptide concentration required to have a zero clearing. The concentrations of Osp-1 and Osp-2 obtained from the semi-prep HPLC column was 325 and 465 lg/mL, respectively. The results of the monovalent cation effect on the MIC of Osp-1 and Osp-2 against E. coli O157:H7 are shown in Fig. 2. When there were no cation present, the MIC values of Osp-1 and Osp-2 were 1.8 and 1.5 lg/ mL, respectively. These values would be referred to as the original MIC values. After the concentration of monovalent cations was increased, the ostricacinsÕ antimicrobial activity on the Gram-negative bacterium decreased, as indicated by the increase of both ostricacinsÕ MIC with increasing of the concentration of Na+/K+. The activities of both ostricacins were fully diminished at 500 mM. The results of the monovalent cation effect on Osp-1 and Osp-2 MIC against S. aureus 1056MRSA are summarized in Fig. 3. The original MIC values of Osp-1 and Osp-2 against the Gram-positive bacterium were 2.5 and 2.3 lg/mLl, respectively. The results of the effect of monovalent cations on S. aureus showed a similar trend to previous results, indicating that both ostricacins lost their potency with increasing cation concentrations. Both ostricacins also lost their activities at 500 mM. In addition, Fig. 3 demonstrated that the activity of Osp-2 was markedly affected by potassium ions because it completely lost its potency at 200 mM of potassium ions, whereas at the same concentration of sodium ions, this peptide still retained moderate antimicrobial activity. Effect of Divalent Cations. The results of the divalent cation effect on the activity of ostricacins against E. coli O157:H7 are shown in Fig. 4. The magnesium ions suggested a statistical significance on both ostricacinsÕ
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CURRENT MICROBIOLOGY Vol. 55 (2007)
Table 2. An example of Osp-1 data for MIC calculation affected by 10 mM NaCl Average inhibition (mm) Sample dilution
Concentration (lg/mL)
E. coli O157:H7
S. aureus 1056 MRSA
1 1/2 1/4 1/8 1/16
325.00 162.50 81.25 40.63 20.31
3.5 3 2.5 1 0
4 3 2.5 1.5 0
4.5 Osp-1
Inhibition (mm)
4
S. aureus y = 1.3706Ln(x) - 3.8271 2 R = 0.9704
3.5 3 2.5
E. coli y = 1.2984Ln(x) - 3.7099 2 R = 0.9529
2 1.5 1 0.5 0 1
10
100
1000
ln (peptide conc.)
Fig. 1. An example of Osp-1 MIC calculation affected by 10 mM NaCl.
antimicrobial activities. The MIC of both peptides increased rapidly, with Osp-1 losing antimicrobial activity at 10 mM and Osp-2 losing no antimicrobial activity at 5 mM. In addition, the calcium ions also affected the ostricacinsÕ antimicrobial activities. Unlike the effect of magnesium ions, Osp-1 and Osp-2 still retained their activities when the calcium ion concentration was increased from 0 to 10 mM. The results of divalent cation effect on S. aureus 1056MRSA are summarized in Fig. 5, showing that the magnesium ion also affected the ostricacinsÕ antimicrobial activities. However, the inhibitory effect was not as strong as the effect of divalent cations on the antimicrobial activities of ostricacins against E. coli O157:H7. Figure 5 indicates that the magnesium ions initially caused slight increase in the MIC of Osp-1 and Osp-2 and that these values had a slight change with increasing magnesium ion concentrations. A similar effect was also indicated by the calcium ions on Osp-1 antimicrobial activity. However, the MIC of Osp-2 rapidly increased with increasing calcium ion concentrations. The growth of bacterial lawn in most of the plates suggested that there was no influence of the salt on the bacterial growth. The salt, however, affected the ability of the peptide to inhibit the bacterial growth, as will be discussed in the following section.
Discussion Cationic peptides are known to interact with bacterial membrane through electrostatic interactions between the positively charged peptides and negatively charged bacterial membranes [4, 9, 23]. The interactions subsequently lead to inactivation of the bacteria. Because the interaction of peptide and the bacterial membrane is the critical step for the inactivation of the bacteria, the presence of cations can prevent the peptides from interacting with the membrane and, subsequently, disable the peptides capability to kill bacteria. In this study, the results indicated that the cations inhibited antimicrobial activities of the ostricacins. The results of the monovalent cation effects resemble other investigations of the effect of monovalent cations on other defensins. The study of salt effect (NaCl) on human b-defensins (HBD-1 and HBD-2) demonstrated that the potency against P. aeruginosa and E. coli decreased when the concentration of salt increased [3, 8]. Activities of other b-defensins, such as canine (cBDs) [2] and mouse (MBD-1) [16], against E. coli have also been reported to decrease with increasing sodium chloride concentrations. On the other hand, two animal b-defensins, king penguin spheniscin 2 (Sphe-2) and human b-defensins 3 (HBD-3), have demonstrated the ability to retain antimicrobial activity under high cation concentrations. Sphe-2 showed potency against E. coli and S. aureus under a high concentration of NaCl [11], whereas human HBD-3 showed a decrease of potency against S. aureus at 250 mM, but its activity was not affected at the physiological salt concentration (100–150 mM) [10]. The discrepancies were believed to be due to the differences in the net positive charge of the peptides. Sphe2 and HBD-3 have a higher net positive charge (+11 and +10, respectively) than Osp-1, Osp-2, HBD-1, HBD-2, cBDs, and MBD-1, which have a net positive charge of +3, +4, or +6. It seems possible that b-defensins with high net positive charges (‡+10) are less affected by the monovalent cations than b-defensins with low net positive charges (
