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- Materials and methods
Small antimicrobial peptides are excellent candidates for inclusion in self-processing proteins that could be used to confer pathogen resistance in transgenic plants. Antimicrobial peptides as small as 22 amino acids in length have been designed to incorporate the residual amino acids left from protein processing by the tobacco etch virus'(TEVs') NIa protease. Also, by minimizing the length of these peptides and the number of highly hydrophobic residues, haemolytic activity was reduced without affecting the peptide's antimicrobial activity.
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- Materials and methods
There has been an extensive amount of research on antimicrobial peptides in the medical area to develop peptides as topical antimicrobial agents, antitumour agents, and for enhanced wound healing ( Maloy and Kari 1995). Also, significant progress is being made to develop these types of peptides for use in the area of plant protection ( Rao 1995). Previously, we designed magainin-like, synthetic peptides that demonstrated growth inhibitory effects on both fungal and bacterial plant pathogens ( Powell et al. 1995 ). Our current interests lie in designing genes that can deliver multiple resistance products that would enhance sustainable plant pathogen resistance in long-lived, woody plant species ( Powell and Maynard 1997). In this report, antimicrobial peptide designs are described that could be used in self-processing protein precursors. Transgenic plants containing genes encoding these precursor proteins could be made to release a ‘cocktail’ of antimicrobial peptides, possibly with antimicrobial enzymes, that could act synergistically against the pathogen. Using a single self-processing gene has an advantage over standard multiple-gene pyramiding because the gene products would be produced under the control of a single gene promoter, therefore their expression controlled as a unit. Also, when a single gene is used to deliver multiple antimicrobial products, there is no possibility of segregation during plant breeding as there is when using multiple genes for pathogen resistance. A single self-processing polyprotein product also simplifies the technical problems with designing and building large constructs with multiple promoters and terminators flanking the coding regions. One way to design this type of gene system is to utilize the self-processing protein mechanisms found in potyviruses. The NIa proteases from two of these viruses have been shown to process cloned gene products in vitro ( Marcos and Beachy 1994) and in vivo ( von Bodman et al. 1995 ; Ceriani et al. 1998 ). One potential problem with using this system is that the proteases need a recognition site for cleavage that leave amino acid sequences on the resulting protein or peptide products. On a large protein, these small amino acid ‘tails’ are usually trivial and often have little or no effect on the protein's activity. However, for small antimicrobial peptides, like our ESF designs ( Powell et al. 1995 ), these residual sequences would account for about a third of the peptide's total amino acid sequence and therefore could significantly effect the peptide's activity. Our hypothesis is that residual amino acid residues retained on a self-processed peptide can be incorporated into the overall peptide design so that it retains its high antimicrobial activity and low haemolytic activity. To test this hypothesis, we initially determined the lower size limit of active ESF peptides in order to keep gene design as compact as possible. Then selected amino acid changes assessed for their effect the haemolytic and antimicrobial activities of the peptides. Then, the self-processing NIa protease system from tobacco etch virus (TEV) was chosen as a model for modifying the ESF peptides. Using the known protease recognition sequence from this system ( Carrington and Dougherty 1988; Dougherty et al. 1988 ), it was determined if residual amino acid sequences from the protease left on the final peptide products would change their antimicrobial and haemolytic activities.
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- Materials and methods
To determine if size effects the ESF peptide designs, peptides were synthesized containing 22 (ESF22A and B), 20 (ESF1B), 15 (ESF15), 13 (ESF13A) and nine (ESF9A) amino acids ( Table 1). These were compared with ESF12 (18 amino acids), ESF17 (17 amino acids), and a control (Ala8,13,18) magainin 2 which have previously been studied ( Powell et al. 1995 ). MICs were determined as described in the methods section and reported as an average of four or more repeats ( Table 2). Average MICs within a column containing the same superscript letter were not significantly different as determined by the Tukey's Studentized Range Test. Haemolytic bioassays ( Table 3) were performed with human red blood cells and report the average lysis with respect to the triton X-100 control.
Table 1. Antimicrobial peptides
|Peptide||AA sequence||a *||charge||H †|
|ESF22A||SRAAGLAARLARLALRELRYAQ||0·56||+ 3·91||− 0·12|
|ESF22B||SRAAGLARRLARLARRELRYAQ||0·68||+ 5·91||− 0·42|
|ESF1B||MVSRAAGLAARLARLALRAL||0·56||+ 3·91||+ 0·07|
|ESF6||MAARAAGLAARLAALALRAL||0·41||+ 2·91||+ 0·24|
|ESF5||MASRAAGLARRLARLARRAL||0·67||+ 5·91||− 0·25|
|ESF12||MASRAAGLAARLARLALR||0·53||+ 3·91||+ 0·01|
|ESF17||ASRAAGLAARLARLALA||0·45||+ 2·91||+ 0·10|
|ESF15||SRAAGLAARLARLAL||0·47||+ 2·91||+ 0·06|
|ESF13A||SRAAGLAARLARL||0·46||+ 2·91||− 0·01|
|ESF9A||SRLARLAAR||0·62||+ 2·91||− 0·23|
|(Ala8,13,18) magainin 2 ||GIGKFLHAAKKFAKAFVAEIMNS||0·46||+ 3·07||+ 0·20|
Table 2. Average minimum inhibitory concentration (MIC), in micromoles per litre (µmol l−1), of ESF peptides and control for four plant pathogens, Fusarium oxysporum, Cryphonectria parasitica, Agrobacterium tumefaciens and Pseudomonas syringae. The same superscript letter within a column indicates no significant difference in the average MIC as determined by Tukey's Studentized Range Test
|Peptide||No. AA||Fusarium oxysporum||Cryphonectria parasitica||Agrobacterium tumefaciens||Pseudomonas syringae|
|ESF22A||22||2·8 d||11 b||6·3c, d||88a, b|
|ESF22B||22||5·0c, d||13 b||3·8 d||50a, b|
|ESF1B||20||1·9 d||6·3 b||3·1 d||100a, b|
|ESF12||18||7·9c, d||8·8 b||25 c||150a, b|
|ESF17 *||17||> 100||> 100||> 100||> 100|
|ESF15 *||15||35 a||36 a||50 b||23 b|
|ESF13A *||13||> 100||> 100||> 100||> 100|
|ESF9A *||9||> 100||> 100||> 100||> 100|
|(Ala8,13,18) magainin 2 ||23||13b, c||13 b||100 a||250 a|
Table 3. Haemolytic activity, as a percentage of haemolysis caused by 0·1% triton X-100, for 250 µmol l−1 of ESF peptides and controls. The same superscript letter within the haemolysis column indicates no significant difference in the average MIC as determined by Tukey's Studentized Range Test
|Peptide||No. AA||#Leu or Val||Haemolysis *|
|(Ala8,13,18) magainin 2 ||23||N/A||10 d,e|
|PBS buffer|| || ||0·32f|
|0·1% Triton-X 100|| || ||100 a|
The results of the MIC bioassays indicate a significant loss of antimicrobial activity when reducing the ESF peptide size from 18 to 17 amino acids for all four of the plant pathogens tested. Interestingly, antimicrobial activity of the 15 amino acid derivative was significantly higher than that of the 17 amino acid form, but still significantly lower than the 18 amino acid form. The one exception to these results was with the bacterial assay using Ps. syringae, in which the 15 amino acid form's MIC was not statistically different than the 18 amino acid form. In this assay it was still more active than the 17 amino acid peptide. The only difference between ESF17 and ESF15 is the loss of an alanine from the amino and carboxyl ends of the ESF17 peptide to form ESF15 ( Table 1). The decrease in activity of ESF17 and regaining of some activity in ESF15 as the size suggests that the smaller peptide's activity could be due to a different mechanism as has been reported for other synthetic peptides ( Bessalle et al. 1993 ). There was little difference in the MICs of the peptides varying in size between 18 and 22 amino acids, with the exception of ESF12 in the Ag. tumefaciens bioassays where ESF12 was significantly less active ( Table 2). The relative consistency of antimicrobial activity among the different peptide sequences indicate some flexibility in design within this size range without loss of antimicrobial activity.
All the ESF peptides which were 18 amino acids in length or larger had similar MICs as the (Ala8,13,18) magainin 2 ( Chen et al. 1988 ) control in the fungal and Ps. syringae bioassays. In the Ag. tumefaciens bioassays, the ESF peptides were significantly more inhibitory than (Ala8,13,18) magainin 2. ESF15 was less active than (Ala8,13,18) magainin 2 in the fungal bioassays but more active than (Ala8,13,18) magainin 2 in the bacterial bioassays.
Differences in peptide size did cause a statistically significant change in haemolytic activity ( Table 3). ESF peptides of 18 amino acids or less were not significantly different in haemolytic activity than the PBS buffer control. ESF peptides of 20 amino acids or larger, including the (Ala8,13,18) magainin 2 control, were significantly more haemolytic than the PBS buffer control. Therefore, size effects haemolytic activity in general. However, by examining other differences among ESF1B, ESF6 and ESF5, which were all 20 amino acids in length, other influencing factors could be discerned. The most consistent factor among these three peptides was the number of highly hydrophobic amino acids in the design. By manipulating these amino acid sequences larger peptides can be designed with lower haemolytic activities than smaller peptides, as demonstrated by comparing ESF22B and ESF1B ( Table 3).
ESF22A and ESF22B were designed to contain amino acid residues of the TEV NIa protease's recognition sequence that would be retained by the resulting peptide if digested with this protease. The antimicrobial activity of both these peptides grouped with the most active peptides tested ( Table 2). ESF22A had a significantly higher haemolytic activity than ESF22B.
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- Materials and methods
The primary hypothesis tested was that the residual amino acids retained on a self-processed peptide could be incorporated into a peptide design so that it retains a high antimicrobial activity yet poses a low haemolytic activity. Consideration of both these activities is important to target the peptides to microbial pathogens while having a minimal effect on higher organisms that might consume plant products containing these peptides. In testing this hypothesis, we first determined the lower size limit of active ESF peptide before adding the residual amino acid sequences which would be left after cleavage by the self-processing NIa protease system from TEV ( Carrington and Dougherty 1988; Dougherty et al. 1988 ). The lower size limit for antimicrobial activity of the ESF peptide design was determined to be 18 amino acids. The exception was ESF15, which had measurable antimicrobial activity but was only 15 amino acids in length. The antimicrobial activity of ESF15 is likely due to a different mechanism than the larger ESF peptides. Changes in activity due to size reductions have been reported with other synthetic peptide designs ( Bessalle et al. 1993 ). However, there were no significant structural differences in ESF15 predicted using the Garnier-Robson and Chou-Fasman structural analysis functions of Protean computer software (DNASTAR).
After determining a minimal size that would retain antimicrobial activity, a peptide sequence was designed that would prevent amino terminal methionine cleavage during peptide expression in transgenic plants. ESF1B was designed by replacing the alanine at position two in ESF1 ( Powell et al. 1995 ), with a valine ( Table 1). This change did not significantly change the MIC as compared with the other most active ESF peptides in this study ( Table 2). However, ESF1B did have one of the highest haemolytic activities of the peptides tested ( Table 3). The haemolytic activity of ESF1B was compared with two other 20-amino acid peptides, ESF5 and ESF6. ESF1B had the highest haemolytic activity, followed by ESF6 and ESF5. The hydrophobicity, alpha moment or overall charge ( Table 1) of the three peptides does not appear to be associated with these haemolytic differences. The only discernible difference in these three peptide designs that follow the change in haemolytic activity was the total number of highly hydrophobic residues. ESF1B has six (1 Val, 5 Leu) as compared with ESF6 that has five (5 Leu) and ESF5 which has four (4 Leu). In other studies of synthetic antimicrobial peptides, increases in hydrophobicity have been associated with increases in haemolytic activity ( Dathe et al. 1997 ; Weiprecht et al. 1997 ). However, our data show that in some cases a more hydrophobic peptide of equal size can be less haemolytic. For example, ESF6 is more hydrophobic than ESF1B, yet it is less haemolytic. ESF6 is more hydrophobic because it contains more alanines (H = 0·62) in place of some hydrophilic amino acids present in ESF1B. But ESF1B has a greater number of highly hydrophobic amino acids such as valine (H = 1·10) even though its overall hydrophobicity is less then ESF6. Therefore, from these results we find it is more important to limit the number of highly hydrophobic amino acids than the overall hydrophobicity when trying to reduce haemolytic activity in antimicrobial peptide designs.
ESF1B had a relatively high haemolytic activity (73% as lytic as the 0·1% triton X-100 control) when tested at a concentration of 250 µmol l−1. However, when the concentration of ESF1B was reduced to 25 µmol l−1, the relative haemolytic activity was not significantly different from PBS buffer control. This concentration (25 µmol l−1) is approximately a magnitude higher than ESF1B's average MIC (1·9–6·3 µmol l−1 for the fungal pathogens) and therefore indicates this might still be a useful antimicrobial peptide in some applications.
To employ antimicrobial peptides in self-processing polypeptide systems in plants, they must be designed to be active despite containing residual amino acids left by the processing reaction. As a model system, we chose to design peptides containing residues from NIa protease recognition site Glu-X-X-Tyr-X-Gln/(Gly or Ser)( Carrington and Dougherty 1988; Dougherty et al. 1988 ). This recognition sequence has the advantage of placing a glutamic acid on the hydrophobic side of the amphipathic alpha helix, similar to the natural magainins. This configuration has been suggested to influence the magainin's differential lytic activity between animal and bacterial cells, possibly due to interaction with cholesterol ( Tytler et al. 1995 ). Our first peptide design to incorporate these residues was ESF22A ( Table 1). The design incorporated all the amino acids of ESF12 except the amino terminal methionine and alanine. In addition, amino acids were added to the carboxyl end that fit the recognition site pattern while still maintaining the amphipathic alpha helical structure of the overall peptide. ESF22A retained a high antimicrobial activity ( Table 2), indicating that a NIa protease recognition site could be incorporated with no significant effect on this activity. However, the haemolytic activity of ESF22 was among the highest activities of the peptides tested in this study ( Table 3). To reduce this activity, ESF22B ( Table 1) was designed by changing the eighth and fifteenth amino acids from an alanine and leucine, respectively, to arginines. This increased the positive charge, decreased the overall hydrophobicity, and lowered the number of highly hydrophobic amino acids. With these changes, ESF22B retained its antimicrobial activity to all of the plant pathogens tested and its haemolytic activity decreased significantly ( Tables 2 and 3). This demonstrated that if a new peptide design has a high haemolytic activity, small changes in its sequence could be made to reduce the haemolytic activity without compromising its antimicrobial activity.
From these experiments, we conclude that small antimicrobial peptides can be designed that incorporate self-processing recognition site residues. When designing these peptides, there is flexibility in the size and amino acid sequence used with respect to antimicrobial activity. However, to minimize haemolytic activity, the size and number of highly hydrophobic amino acids should be kept to the minimum needed to maintain antimicrobial activity. Other factors, as yet unidentified, must also contribute to the haemolytic activity because the control peptide (Ala8,13,18) magainin 2, had a longer amino acid sequence and also had more highly hydrophobic amino acids than the ESF peptides and yet had only a moderate haemolytic activity. These factors could not be identified in this study due to many differences in the amino acid sequence. Our next step is to design a gene encoding an antimicrobial peptide precursor using the ESF22 peptide design and test its ability to be processed in vitro and in vivo by the TEV NIa protease.