Antimicrobial Peptides for Fire Blight Control - ACS Publications

Chapter 19

Antimicrobial Peptides for Fire Blight Control Downloaded by OHIO STATE UNIV LIBRARIES on May 29, 2012 | http://pubs.acs.org Publication Date (Web): April 4, 2012 | doi: 10.1021/bk-2012-1095.ch019

V. Sarojini* School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland Central, Private Bag 92019, Auckland 1142, New Zealand *E-mail: [email protected]

Fire blight, caused by the Gram negative bacterium Erwinia amylovora, is an economically devastating disease of pome fruit trees affecting North America, Europe, New Zealand and several Eastern Mediterranean countries (1, 2). Currently used chemical control options, except streptomycin, do not provide satisfactory results in serious outbreaks. The development and spread of streptomycin resistant strains of E. amylovora in countries like New Zealand, North America and Israel make it necessary to develop novel compounds, possibly having different biological targets, to control this disease. Antimicrobial peptides are attractive candidates as alternatives to conventional antibiotics, since they have less chance of resistance development and exhibit novel mechanism of action (3). Research from various laboratories around the world during the past two decades has identified several naturally occurring antimicrobial peptides and their synthetic analogues with potential for fire blight control. Even though these molecules are yet to find entry into the commercial sector for chemical control of fire blight, there is ample evidence of their potential to provide the necessary and desired alternative to antibiotics for fire blight control.

© 2012 American Chemical Society In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Downloaded by OHIO STATE UNIV LIBRARIES on May 29, 2012 | http://pubs.acs.org Publication Date (Web): April 4, 2012 | doi: 10.1021/bk-2012-1095.ch019

Introduction Antibiotic resistance is a growing public health problem the world over. The antibiotics that saved millions of lives since the discovery of Penicillin in the 19th century are now at risk, as high levels of drug resistance threaten their effectiveness. These multidrug resistant (MDR) microorganisms cling together to surfaces (biotic or abiotic) and use extracellular polymer matrices as a barrier to drug penetration. They are commonly known as biofilms. Biofilm formation by human, animal and plant pathogens lead to high health costs to humans and severe economic loss in the agricultural sector. Pseudomonas aeruginosa that inhabit the lung tissues of cystic fibrosis patients and Erwinia amylovora the causative agent of fire blight, are examples of bacterial biofilms that cause major concerns in different fields (4, 5). Antimicrobial peptides (AMPs) that form part of innate immunity in almost all forms of life are attractive candidates for the development of novel treatment options to tackle the multi drug resistant bacterial biofilms (6–8). This chapter will highlight the research from various laboratories on peptide based molecules that have shown antibacterial activity against E. amylovora and are hence promising candidates for fire blight control.

Fire Blight, Its Economic Importance, and Its Causative Agent Fire blight affects the entire plant causing five distinct types of infection known as blossom blight, shoot blight, canker blight, fruit blight and rootstock blight (9, 10). Severe outbreaks of fire blight necessitate the removal of whole orchards resulting in significant economic loss for fruit growers. Millions of dollars worth of damages have been reported from affected countries, especially U.S.A. and New Zealand (11, 12). Prevalence of fire blight in New Zealand has restricted market accessibility of New Zealand grown apples putting additional economic burden on farmers (13). Given the existing scenario with the rapid spread of fire blight into newer apple and pear growing regions, the economic impact caused by this disease is only going to continue to increase. E. amylovora, the causative agent of fire blight, is a Gram negative bacterium belonging to the Enterobacteriaceae family (10). Numerous factors are known to contribute to the pathogenicity of E. amylovora. These include the production of the exopolysaccharides, amylovoran and levan, the harpin proteins, the siderophore desferroxamine as well as the quorum sensing signalling molecules (10, 14, 15). More recently it has been shown that E. amylovora forms bioflms and that biofilm formation is crucial for the pathogenicity of this bacterium (4).

Fire Blight Management The options available for managing fire blight are limited as well as costly. The common methods available to farmers for minimizing the spread of the disease like removal of the source of infection, spraying chemicals to protect the plant against the pathogen, avoidance of susceptible cultivars etc. do not provide an effective management strategy. 398 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Existing Chemical Control Options and Their Drawbacks Spray applications of streptomycin and copper compounds are the two most widely used chemical control options registered for fire blight in most countries affected by this disease (16). However, these options have significant drawbacks of resistance development and phytotoxicity respectively. The use of streptomycin, for fire blight control, is becoming more and more limited because of the spread of resistant strains of the bacterium as well as the existence of regulatory measures in many countries in the EU where the use of clinically relevant antibiotics in horticulture is prohibited (17). Certain countries in Europe use other chemicals like Flumequin, Fosetyl-Al and oxolinicacid for fire blight control. None of these have entered the global scenario yet. Table 1 lists the chemical control options used for fire blight control in different countries.

Table 1. Chemicals registered for use against fire blight in different countries Trade Name

Country

Comments

FirestopTM FructilTM MBR10995

France, Belgium and Cyprus

Not approved in NZ, U.S.A. and Canada

Fosetyl-Aluminum (Fosetyl-Al)

AlietteTM

France and Turkey

Inconsistent results from year to year and trial to trial

Oxolinic acid

StarnerTM

Italy

Mostly used for preventive treatment

Copper Compounds

Available under various trade names

NZ, US, Netherlands, Italy,Canada, Belgium, Greece (under several different trade names)

Phytotoxicity

Streptomycin

AgrimycinR17, Plantomycinetc

NZ, US, Netherlands, Italy, Canada, Belgium, Greece (under several different trade names)

Resistant strains identified in U.S.A., Canada, New Zealand and Israel

Compound Flumequin

399 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Copper Compounds Bordeaux mixture containing a mixture of copper sulphate (CuSO4) and lime [Ca (OH)2] is a bactericide used for fire blight control since early 1900s (18). However, phytotoxicity is a major drawback of copper compounds (16, 19). Additionally, the fact that several other phytopathogenic bacteria have become resistant to copper raises the concern of a similar resistance developing in E. amylovora, further restrictng the use of copper as a desirable fire blight control in future (20, 21).


Antibiotics According to the original definition by Selman Waksman, ‘an antibiotic is a chemical substance produced by a microorganism that has the capacity to inhibit the growth and even destroy bacteria and other microorganisms’ (22). The restricted use of copper compounds because of their phytotoxicity resulted in the use of antibiotics in horticulture immediately after the wonder drug penicillin saved thousands of human lives during the time of World War II in the 1940s (23)–(25). Several antibiotics, such as penicillin, neomycin, erythromycin, chloramphenicol, chloromycetin, tetracycline, streptomycin etc. were tried for their ability to control E. amylovora infections in vitro as well as in the fields (26, 27). Amongst these, only streptomycin, oxytetracycline and kasugamycin satisfied the requirements for use in field trials (16). However, the use of kasugamycin is restricted because of its phytotoxicity (28, 29). Oxytetracycline is inferior to streptomycin in fire blight control and hence has been used in combination with streptomycin especially to minimise the development of streptomycin resistant strains of E. amylovora (30).

The Growing Problem of Antibiotic Resistance: Antimicrobial Peptides as a Potential Solution Antibiotic resistance is constantly on the rise threatening the effectiveness of several life-saving durgs. In order to raise awareness about this ever increasing problem, The World Health Organization chose ‘combat antimicrobial resistance’ as the theme for the World Health day 2011 and has prompted the scientific community to develolp novel compounds to treat multi drug resistant microorganisms. Antimicrobial peptides are desired alternatives to conventional antibiotics for the prevention and treatment of various microbial infections (3, 8). Antimicrobial peptides (AMPs) are produced by various organisms including plants, insects, microbes, amphibians and fish in order to battle against invading pathogens. AMPs are cationic in nature and contain 15 to 45 amino acids in their sequences (31, 32). Their secondary structures vary from amphipathic helices, two to four stranded β-sheets, extended structures and loops. Naturally occurring AMPs show broad spectrum activity against several microorganisms, exhibit drug synergism with other antibiotics and have very few side effects (3, 33). Despite the major advantages peptides have over conventional antibiotics, peptide research had a relatively slow growth until the Nobel prize winning work 400 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


by du Vigneaud on the synthesis of the peptide hormones oxytocin and vasopressin (34). Initial advances in protecting group chemistry through the contributions of Bergmann and Zervas, Bodanzky, Carpino and others followed by the Solid Phase Peptide Synthesis pioneered by Robert Bruce Merrifield made the chemical synthesis of relatively large polypeptides amenable (35–42). Currently, peptide drugs sold worldwide account for more than 12 billion dollars (43). Several AMPs are under various stages of clinical trial developments (44–48). However, the low bioavailability of peptides is still considered as a drawback in their marketability as pharmaceuticals, despite the fact that the peptide drugs insulin and erythropoietin are still available only as injections (33). The advances in the chemical synthesis of peptides make it possible to develop novel synthetic peptides with enhanced bioavailability incorporating the desirable properties of naturally occurring AMPs to treat various microbial infections.

Peptides for Fire Blight Control Protein/peptide based molecules reported in the literature for fire blight control belong to three categories – 1) large proteinaceous toxins like bacteriocins, 2) AMP genes expressed in transgenic plants to enhance resistance to fire blight and 3) smaller AMPs and their synthetic variations. The best example in the first category is Serracin P, a high molecular weight (HMW) bacteriocin isolated from Serratia plymithicum J7 culture supernatant that showed antibacterial activity against 24 pathogenic strains of E. amylovora from different countries (49). Examples of AMP genes expressed in plants include the work from Norelli’s group where the expression of the lytic protein gene attacin E in orchard grown apple trees has been shown to confer increased resistance to fire blight (50, 51). Another example in this category is the enhanced resistance to E. amylovora exhibited by Royal Gala apple shoots transformed by the MB39 gene which is a modification of the lytic protein cecropin SB37 (52, 53). This review will elaborate on the third category listed above – namely small antimicrobial peptides and their synthetic variations that have shown potential for fire blight control either in the laboratory or field trials or both. The main focus will be on peptide based molecules with potential for fire blight control reported from New Zealand. This will be followed by a brief mention of other peptide antibiotics produced by strains of Pantoea agglomerans and finally synthetic analogues of magainin, and the cecropin-mellitin hybrid peptides that have shown potential for fire blight control.

Antimicrobial Peptides for Fire Blight Control Reported from New Zealand Antimicrobial compounds that suppress the growth of the fire blight pathogen Erwinia amylovora have been discovered from a wide range of Pseudomonas and Pantoea agglomerans strains (syn. Erwinina herbicola) in New Zealand and elsewhere (54–64). These antimicrobial compounds, on their own or through synthetic manipulations, provide useful means for potential control of fire blight. 401 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Pyrazole-Conjugated Peptides Mitchell has done extensive screening of numerous strains of the Pseudomonas species in an effort to find novel chemical control options for fire blight. Tropolone and a peptide derivative isolated from liquid cultures of Pseudomonas species were found to inhibit E. amylovora in vitro and on immature pear fruits (57, 58). However, the level of infection on immature pear fruits treated with tropolone was raised to 100% after five days, but the tripeptide derivative provided complete protection from infection after five days. More recent work from Mitchell’s group which included extensive 1H and 13C nuclear magnetic resonace (NMR) and mass spectral (MS) investigations on the peptide derivative confirmed it to be L-alanyl-L-homoserinyl-L-aspartic acid with an unusually substituted pyrazole ring linked to the beta carboxylic acid group of the C-terminal aspartic acid, 1 (65).

The unusually substituted oxopyrazole ring was found to be critical for the observed antibacterial activity of this compound. The antibacterial activity of 1 was suppressed by L-glutamine, indicating glutamine synthesis pathway as the potential target of this peptide. The authors reported that several other bacterial species were susceptible to the oxopyrazole peptide and thus this pyrazole conjugated peptide had a much broader spectrum of antibacterial activity. Table 2 summarises the antibacterial activity reported for this peptide. The technical advances in computers and information technololgy during the past two decades has made computer-aided drug design an integral part of the modern drug discovery process. Today, the discovery and development of novel drugs, identification of novel biological targets for existing pathological conditions are all done with the aid of advanced computer technology (66). A combination of in vitro screening, synthetic chemistry and advanced spectroscopic techniques has led to the design, structure elucidation and discovery of novel peptides, in some cases with biological activities (65, 67, 68). We have recently extended our tool box to the use of advanced molecular modeling software to aid in the design of novel antimicrobial peptides. This approach was used to identify other potential targets for the oxopyrazole peptide as described below.


Table 2. Antibacterial activity of the oxopyrazole peptide (65)


Name of the bacterium

Activity of the oxopyrazole peptide*

Erwinia amylovora

+

Pectobacterium carotovorum (synonym: Erwinia carotovora)

+

Pectobacterium atrosepticum (synonym: Erwinia carotovora pv. atroseptica)

+

Pectobacterium carotovorum subsp. carotovorum (synonym: Erwinia carotovora pv. carotovora)

+

Pseudomonas syringae pv. syringae

+

Pseudomonas syringae pv. tomato

+

Xanthmonas capmpestris pv. pruni

+

Pseudomonas corrugata

-

Pseudomonas marginalis

-

+ = active; - = not active

Similarity of the oxopyrazole ring in 1 to the nucleoside antibiotic pyrazofurin, isolated from Streptomyces candidus, prompted De Zoysa et al to undertake molecular modelling and docking experiments to evaluate the possibility that these two molecules could have similar biological targets in the bacterium (69). Pyrazofurin inhibits Orotatephosphoribosyltransferase (OPRT) and orotidine-5-phosphate decarboxylase (ODC) that catalyze the final steps in the biosynthesis of uridine-monophosphate (UMP) the building block for DNA synthesis (70). The molecular modelling studies, by De Zoysa et al., showed that the oxopyrazole peptide completely overlaps the natural ligand (orotic acid) and binds more strongly to the active site of OPRT (69). Figure 1 shows the oxopyrazole peptide and orotic acid docked separately onto the binding site of the enzyme OPRT. The additional hydrogen bonding interactions of the peptide with the OPRT binding site that involve the side chains of residues Lys (27) and Lys (76) are circled. The oxopyrazole ring carrying different substituents were synthesized and tested for activity, on their own or coupled to form peptide linkages, against E. amylovora. Figure 2 shows the various synthetic analogues of 1 (2-6) investigated for in vitro activity against E. amylovora. (De Zoysa et al., manuscript). Only the brominated oxypyrazole and its peptide analogues inhibited the growth of E. amylovora. However, the antibacterial activity of these analogues was not as prominent as the natural product which further confirms that changes to the substitution pattern of the oxopyrazole ring can be detrimental for antibacterial activity. Further work on the modelling, synthesis and activity studies on these peptides is currently in progress in the author’s laboratory.



Figure 1. Hydrogen bonding interactions of co-crystalisedorotic acid (natural ligand of OPRT) and the docked oxopyrazole peptide with the binding pocket of OPRT.

Figure 2. Synthetic analogues of the oxopyrazole ring and its peptide conjugates.

Iminopyrrolidine- and Piperidine-Containing Peptides Two related compounds, with strong in vitro inhibitory activity against E. amylovora, were isolated by Mitchell and Teh from liquid cultures of Burkholderia plantarii (syn. Pseudomonas plantari) in 2005 (71). Extensive NMR and MS studies established the bioactive compounds to be 2-imino-3-methylene-5-L(carboxy-L-valyl)-pyrrolidine 7 and 2-imino-3methylene-5-L(carboxy-L-threonyl)-pyrrolidine 8. Both compounds, at 1 μg, resulted in clear inhibitory zones of approximately 20 mm diameter on agar plates overlaid with mid-log phase cultures of E. amylovora. The bioactivity of these compounds was found to be closely linked with the conjugated imino alkene functionality. All four stereomers of 7 were synthesised and tested for activity against E. amylovora, which confirmed that the biologically active natural product has the L,L stereochemistry (72). The 2-imino-3-methylene-pyrrolidine structure was also coupled to several hydrophobic amino acids, the synthetic peptides 404 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


(9-16) purified to homogeneity, structurally characterized and tested for activity against E. amylovora. All compounds showed strong in vitro activity against E. amylovora, even though compounds 9, 11 and 16 were 2.5, 5 and 20 fold less active than the natural products. This study also included the syntheses of the higher homologs – the six membered ring piperidines coupled to L-Val, L-Ile and L-Thr (17-19) all of which showed strong in vitro inhibitory activity, comparable to the natural products 7 and 8, against E. amylovora (72). The structures of the natural product and synthetic peptides containing iminopyrrolidine and piperidine that showed in vitro activity against E. amylovora reported by Mitchell are shown in Figure 3.

Figure 3. Iminopyrrolidine and piperidine containing peptides with activity against E. amylovora (72). 405 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Inhibiting Growth and Biofilm Formation in E. amylovora De Zoysa et al. reported two non-natural amino acids with strong in vitro activity and biofilm inhibitory properties against E. amylovora (73). Both compounds were more potent than streptomycin against E. amylovora strain 1501. The compounds also had strong in vitro activity against the streptomycin resistant strain (Str4 Ea) of E. amylovora. Figure 4 shows the in vitro antibacterial activity of the lead compound, referred hereto as A, against Ea 1501 and Str4 Ea strains of this bacterium detected as zones of no bacterial growth surrounding the point of application of the compounds on agar plates overlaid with mid-log phase cultures of the respective bacterial strains. EA medium which consisted of K2HPO4 11.5 g, KH2PO4 4.5 g, MgSO4 0.2 g per 1 litre and L-asparagine 0.3 g, L-nicotinic acid 0.05 g and D-glucose 20 g was used for growing E. amylovora for these experiments. The top left quadrant in figure 4 (a) corresponds to the anti bacterial activity of 10 μg of streptomycin and the bottom left quadrant corresponds to that of 10 μg of inhibitor A against E. amylovora 1501. The larger and smaller inhibitory zones in figure 4 (b) correspond to 10 and 1 μg respectively of inhibitor A against the streptomycin resistant strain of E. amylovora. Streptomycin was used as the control in these experiments. Since plate (b) was overlaid with the resistant strain of E. amylovora, as expected, no inhibitory zone was detected surrounding the point of application of streptomycin on this plate. The diameters of the zones of inhibition measured from these plates are summarised in table 3. The MIC of A towards both bacterial strains was below 18 μM. Inhibitor A was not phytotoxic to apple flowers or tobacco leaves at concentrations 40 times higher than the MIC value (De Zoysa et al, manuscript under preparation). An immature pear fruit assay (74) was used to evaluate the ability of the inhibitor to reduce the incidence of fire blight under a controlled environment. Several other potentially inhibitory compounds to the fire blight pathogen were also included in this experiment. Results obtained from this assay, plotted as the number of fruits infected under the different assay conditions, are shown in figure 5. As can be seen from this figure, the efficacy of inhibitor A, to prevent infection on immature pear fruits, is equal to streptomycin when 102to 103cfu/ml of E. amylovora was used (De Zoysa et al, manuscript under preparation). We are currently evaluating the ability of the inhibitor to control infection in immature pear fruits at higher E. amylovora concentrations. The results also indicate that the other compounds tested in this experiment are less efficacious than streptomycin for fire blight control. Our research group is also involved in the development of antimicrobial peptides for tackling biofilm forming human pathogens like Pseudomonas aeruginosa and Staphylococcus aureus. We extended our biofilm protocols to test the ability of inhibitor A to interfere with biofilm formation in streptomycin sensitive and resistant strains of E.amylovora. Representative microscopic images of E. amylovora 1501 biofilms stained with crystal violet in the absence (figure 6 a) and presence (figure 6 b) of the inhibitor are shown in figure 6. Comparison of these two images clearly indicates that inhibitor A prevents biofilm formation in Ea 1501. Similar results were obtained for the streptomycin resistant strain of 406 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


E. amylovora as well.Several replicates were used in these experiments and the results were also validated by different staining techniques.

Figure 4. Antibacterial activity of inhibitor A in comparison to streptomycin against (a) the streptomycin sensitive strain Ea 1501 and (b) the streptomycin resistant strain Str4 Ea.

Table 3. Diameter of the zones of inhibition of Ea 1501 and Str4 Ea Diameter of inhibitory zones in cm Bacterial strains used

Streptomycin

Inhibitor A

10 μg

1 μg

10 μg

1 μg

Ea 1501

2.7

1.7

4.3

3.0

Str4 Ea

0

0

4.2

3.0

These results indicate that the compound by itself or its synthetic variations has the potential to be developed as novel chemical control options for fire blight. The nature of the antibacterial activity of this inhibitor was investigated by co-inoculating different protein amino acids with A in the bioassays. Results showed that L-proline at a concentration of 100 mM prevents the antibacterial activity of A. Several peptides incorporating this inhibitor were synthesized and tested for activity against E. amylovora. All, except the proline containing peptide, showed strong activity against the bacterium, which is again indicative of the proline biosynthesis pathway as a potential target. This work is currently in progress in the author’s laboratory. 407 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 5. Number of cores of immature pear fruits showing signs of fire blight infection after treatment with different compounds and inoculation with E. amylovora [6.7 × 102 (experiment 1), 1.1 × 103 (experiment 2) and 6.3 × 102(experiment 3) cfu/ml].

Figure 6. Representative microscopic images of E.amylovora1501 biofilms stained with crystal violet (400× magnification) (a) control (b) in presence of inhibitor A. Antimicrobial Peptides Produced by Pantoea Pantoea vagans (syn. P. agglomerans, Erwinia herbicola) is a non-pathogenic bacterium closely related to Erwinia amylovora that colonises the same plant surfaces as E. amylovora. As mentioned above, there are numerous literature reports on the production of antibiotics by P. agglomerans, some of which have been shown to have activity against E. amylovora (55, 56, 60–63). Herbicolin O produced by Erwinia herbicola strain C9-1 is a broad spectrum antibiotic that inhibits the growth of several Gram negative bacterial species including 408 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


the fire blight pathogen (56). The chemical structure of Herbicolin O is not yet published. However, its similar molecular weight to Pantocin A and the fact that both compounds lose their antibacterial activity in the presence of L-histidine has led some researchers to speculate that Herbicolin O could be the same as Pantocin A, produced by P. agglomerans strain Eh318. Eh318 has been shown to produce another antibiotic, named Pantocin B with strong inhibitory activity against E. amylovora (60, 62). The structures of Pantocin A and B are shown in figure 7. Pantocin B inhibits arginine biosynthesis in the bacterium. Both have a peptidic nature to their structures. The authors reported that, in minimal media, Pantocin B has picomolar activity against E. amylovora.

Figure 7. Chemical structures of Pantocin A (20) and B (21).

Eh252 is another strain of P. agglomerans that has been shown to produce antibiotics, on a minimal medium, that inhibit the growth of E. amylovora (55). The production of antibiotics was found to be a crucial factor for the biological control of fire blight by Eh252. Comparison of Eh252 mutant strains that were not inhibitory to E. amylovora with wild type Eh252 showed marked differences in their ability to reduce the incidence of fire blight in immature pear fruit assays. The inhibition of E. amylovora by Eh252 was prevented in the presence of L-histidine as well as proteinase K indicating that this antibiotic has a peptidic nature and that its potential target lies in the histidine biosynthesis pathway. This antibiotic named MccEh252 is believed to belong to the category of microcins and has been shown to control fire blight in the laboratory and in the field (54). Antibiosis has also been shown to contribute to biological control of fire blight by P. agglomerans strain Eh252 in orchards (75). Eh1087 strain of P. agglomerans produces the broad-specturm phenazine peptide antibiotic D-alanylgriseoleutic acid (AGA) 22 (63). AGA has been shown to have in vitro activity against several Gram positive and Gram negative bacteria including E. amylovora (76). An Eh1087 strain (EhΔAGA) was much less effective than the strain producing AGA in preventing stigma colonization by E. amylovora (77). 409 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


An oxirane containing peptide, 2-amino-3-(oxirane-2,3dicarboxamido)propanoyl-valine, 23 was isolated from P. agglomerans strain 48b/90 (64). NMR and MS analysis was used to elucidate the structure of this peptide. The same antibiotic peptide is produced by three different strains of P. agglomerans.

Magainins and Cecropin-Melitting Hybrids against E. amylovora Several synthetic variants, including hybrids, of the naturally occurring AMPs like magainins, cecropins and melitins have been reported with strong in vitro activity against E. amylovora. ESF12, a synthetic antimicrobial peptide, mimicking the amphipathic α-helix of magainin, was found to inhibit the growth of E. amylovora in vitro with an MIC of 250 μM (78). Cecropin B from the silk moth (MW: 3832) and a synthetic analogue of the same named SB-37 (MW:4089) have shown bactericidal activity against E. amylovora with MIC values below 15 μM (79). BP76 is a cecropin-melittin hybrid linear undecapeptide with an MIC value of 2-5 μM against E. amylovora (80). BP76 was resistant to proteases and had ED50 of 2.5 μM. BPC194 and BPC198 are the first set of cyclic peptides reported to have strong antibacterial activity against E. amylovora (81). These peptides, designed and synthesized using a combinatorial chemistry approach, had an MIC of 6-12 μM (BPC194) and 12-25 μM (BPC198) against E. amylovora PMV6076 strain and displayed very low cytotoxicity against mammalian cells when used at approximately 100 times the MIC concentrations, implying that they are suitable candidates for field trials at the antibiotic concentrations currently used in such experiments. These peptides have been specifically designed to induce protease resistance by the judicious choice of D-amino acids at specific positions in their 410 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


sequences and by way of cyclization. Work on the combinatorially synthesized peptides was extended by the incorporation of D-amino acids which resulted in ten peptides with strong inhibitory activity against E. amylovora. Representative members from this combinatorial library (BP143 and BP145) containing one D-amino acid at the two and four positions in their sequences were also evaluated in immature pear fruits infected with the bacterium and in in planta assays for fire blight control in pear. Results of these assays showed that BP143 had comparable efficacy to streptomycin in controlling fire blight and hence has the potential to be developed as a plant protection product (82).

Conclusions Antimicrobial resistance is a problem that has existed ever since Sir Alexander Fleming discovered penicillin in the 1940s. Fleming did realize the seriousness of the issue which he addressed in his Nobel lecture where he said ‘Penicillin is to all intents and purposes non-poisonous and there is no need to worry about giving an overdose and poisoning the patient. There may be a danger though in an under dosage. … Moral: If you use penicillin, use enough’. In the decades that followed, several antibiotics, natural and semi-synthetic, became available that helped to contain these infectious diseases. By the 1980s there was this widespread notion that ‘infectious diseases is a thing of the past’. However, infectious diseases came back with a vengeance because of wide spread resistance to conventional antibiotics by the microbes. The message from the WHO director general on the World Health Day 2011 reads ‘“In the absence of urgent corrective and protective actions, the world is heading towards a post-antibiotic era, in which many common infections will no longer have a cure and, once again, kill unabated.” The solution to this public health problem is provided by nature itself in the form of antimicrobial pepitides (AMPs) that act as defense weapons to combat fatal microbial infections (83). The AMPs have found potential not only to combat human pathogens, but also as potential solutions in the animal world and to provide protection to infectious diseases of plants. This chapter has summarized the antimicrobial peptide based compounds reported from various laboratories around the world that have shown potential for chemical (and in some cases biological) control of fire blight. Some of these peptides have also shown broad spectrum antibacterial activity and the ability to inhibit multidrug resistant bacterial biofilms, not only in the fire blight pathogen, but also more problematic human pathogens as well. Even though peptide based molecules have shown promise to tackle the multidrug resistant bacterial biofilms that affect humans, their use in controlling plant pathogens is currently unheard of. Our research in this area has shown the potential of peptide based molecules to control bacterial biofilms amongst plant pathogens, using E. amyolovora as a test case. In our research we have used traditional biological screening and analog synthesis methods together with the more modern molecular modeling tools to identify potential targets and develop novel peptide based molecues with antimicrobial activity. The work presented in this chapter on ‘inhibiting biofilm formation in E. amylovora’ is in itself a preliminary account only and is one of the 411 In Small Wonders: Peptides for Disease Control; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

major ongoing research projects in the author’s laboratory currently. In future, this class of molecules have the potential to provide the desirable solution to tackle the wide spread public health problem of combating antimicrobial resistance.


Acknowledgments The author would like to thank Dr. Robin Mitchell for proof reading this chapter. The phytotoxicity and immature pear fruit assays were carried out in the laboratory of Dr. Joel Vanneste, Plant and Food Research Ltd., Hamilton, New Zealand. Part of the research from the author’s laboratory reported in this chapter was supported by an Auckland Uniservices grant No. 1078476.

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