Exploration of Using Antisense Peptide Nucleic Acid (PNA)-cell Penetrating Peptide (CPP) as a Novel Bactericide against Fire Blight Pathogen Erwinia amylovora

Patel, Ravi R.; Sundin, George W.; Yang, Ching Hong; Wang, Jie; Huntley, Regan B.; Yuan, Xiaochen; Zeng, Quan

doi:10.3389/fmicb.2017.00687

Cited by 32 publications

(14 citation statements)

References 41 publications

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“…Amylovoran and LPS are major virulence factors for E. amylovora and, as such, represent attractive potential targets for disruption to counteract fire blight disease development. It might be possible to deliver YibD into the E. amylovora cell to disrupt LPS and amylovoran production, perhaps using the cell-penetrating peptide approach already used for antisense oligonucleotide delivery into E. amylovora (63). Such an inhibitor could be applied to host flowers to counteract blossom blight and possibly injected into the plant to counteract established infections (64).…”

Section: Resultsmentioning

confidence: 99%

Virulence Genetics of anErwinia amylovoraPutative Polysaccharide Transporter Family Member

et al. 2020

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The Gram-negative enterobacterium Erwinia amylovora causes fire blight disease in apple and pear trees. Lipopolysaccharides and the exopolysaccharide amylovoran are essential E. amylovora virulence factors. We found that mutations to rfbX disrupted amylovoran production and virulence in apple fruits and tree shoots, and that deletion of yibD suppressed the rfbX mutant phenotype. Expression of yibD was about ten-fold higher in ΔrfbX compared to wild-type. A forward genetic suppressor screen in the ΔrfbX mutant uncovered multiple mutations in yibD and supported the conclusion that the virulence defect of rfbX mutants is due to reduced amylovoran production. The yibD and rfbX genes are expressed as a two-gene operon, yibDrfbX. The rfbX gene encodes a previously-uncharacterized putative polysaccharide subunit transporter, while yibD encodes a predicted glycosyltransferase. Mutation of rfbX did not have a detectable effect on lipopolysaccharide patterns; however, overexpression of yibD in both the wild-type and ΔyibDrfbX genetic backgrounds disrupted both amylovoran and lipopolysaccharide production. Additionally, overexpression of yibD in ΔyibDrfbX inhibited bacterial growth in amylovoran-inducing medium. This growth inhibition phenotype was used in a forward genetic suppressor screen and reverse genetics tests to identify several genes involved in lipopolysaccharide production which, when mutated, restored the ability of ΔyibDrfbX overexpressing yibD to grow in amylovoran-inducing medium. Remarkably, all the lipopolysaccharide gene mutants tested were defective in lipopolysaccharide and amylovoran production. These results reveal a genetic connection between amylovoran and lipopolysaccharide production in E. amylovora. IMPORTANCE This study discovered previously unknown, genetic connections between exopolysaccharide and lipopolysaccharide production in the fire blight pathogen Erwinia amylovora. This represents a step forward in our understanding of the biology underlying the production of these two macromolecules. Fire blight is an economically important disease that impacts the production of apples and pears worldwide. Few fire blight control measures are available, and growers rely heavily on antibiotic applications at bloom time. Both exopolysaccharide and lipopolysaccharide are E. amylovora virulence factors. Our results indicate that overexpression of the yibD gene in E. amylovora disrupts both lipopolysaccharide production and exopolysaccharide production. This effect could potentially be used as the basis for development of an antivirulence treatment for the prevention of fire blight disease.

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Section: Resultsmentioning

confidence: 99%

Virulence Genetics of anErwinia amylovoraPutative Polysaccharide Transporter Family Member

et al. 2020

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“…However, as the development of antibiotic resistance in E. amylovora populations is a major concern related to the use of antibiotics for fire blight management (60,61), there is considerable interest in the development of alternatives to antibiotics for fire blight management. Some promising new approaches include bacteriophage (62) and antisense nucleotide delivery systems (63). Application of auxotrophic E. amylovora cells to apple blossoms might have potential as a biocontrol measure for fire blight and could represent an additional novel mode of action for fire blight management.…”

Section: Resultsmentioning

confidence: 99%

Erwinia amylovora Auxotrophic Mutant Exometabolomics and Virulence on Apples

Klee

Sinn

Finley

et al. 2019

Appl Environ Microbiol

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The Gram-negative bacterium Erwinia amylovora causes fire blight disease of apples and pears. While the virulence systems of E. amylovora have been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophic E. amylovora mutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-type E. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenic E. amylovora auxotrophs could have utility as fire blight biocontrol agents. IMPORTANCE This study has revealed the availability of a range of host metabolites to E. amylovora cells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterial de novo synthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophic E. amylovora mutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulent E. amylovora on flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenic E. amylovora auxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.

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“…Here, the concentration of (KFF) 3 K-FAM used during incubation was 0.4 μM. Also note that, with the exception of Erwinia, Pantoea, and Pectobacterium (Patel et al, 2017), seven of the bacteria tested come from genera that have not been treated with CPP. Overall, we observed that (KFF) 3 K-FAM could permeate into all the bacterial strains at conditions before optimization but the efficiency was very low (Figure 6A).…”

Section: Resultsmentioning

confidence: 99%

Abiotic Factors Promote Cell Penetrating Peptide Permeability in Enterobacteriaceae Models

et al. 2019

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Conventionally, the delivery of biomolecules into bacteria for the generation of characterized or functional mutants has relied greatly on horizontal gene transfer techniques. However, the low compatibility of these techniques with novel or hard-to-transform bacteria currently serves as a challenge to the bioengineering field. Here, we explored the use of cell penetrating peptides (CPPs) as an alternative biomolecule delivery approach by investigating the effects of the abiotic factors during CPP permeation. Using the (KFF)3K-FAM conjugate and Escherichia coli as models, we evaluated four abiotic factors where two of these factors, temperature and solution tonicity, promoted (KFF)3K-FAM permeation efficiency. Our data show that optimal (KFF)3K-FAM permeation efficiency was achieved for E. coli at approximately 98.1% under conditions of 37°C (growth optimal temperature) and 50% PBS concentration. Based on these conditions, we subsequently tested the applicability of CPP permeation in various bacterial strains by treating 10 bacterial strains from the Enterobacteriaceae family among which seven strains have no CPP permeation records with (KFF)3K-FAM. Interestingly, when compared with non-optimized conditions, all 10 strains showed a marked increase in CPP permeation ranging between 20 and 90% efficiency. Although using strains within Enterobacteriaceae that are phylogenetically close, our results hinted on the possibility that with proper optimization of the abiotic factors, CPPs could be compatible with a broad range of bacterial strains. Our efforts suggest that CPP could serve as an effective alternative approach for mutant generation and for biomolecule delivery into novel or hard-to-transform bacteria.

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Exploration of Using Antisense Peptide Nucleic Acid (PNA)-cell Penetrating Peptide (CPP) as a Novel Bactericide against Fire Blight Pathogen Erwinia amylovora

Cited by 32 publications

References 41 publications

Virulence Genetics of anErwinia amylovoraPutative Polysaccharide Transporter Family Member

Virulence Genetics of anErwinia amylovoraPutative Polysaccharide Transporter Family Member

Erwinia amylovora Auxotrophic Mutant Exometabolomics and Virulence on Apples

Abiotic Factors Promote Cell Penetrating Peptide Permeability in Enterobacteriaceae Models

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