Template:NAU-CHINA/antifungal

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Respond Module

Introduction: Originally, two families of antimicrobial peptides are thought to be applied to mend wheat scab. Plant defensin and Cecropin, which can be synthesized by plants and insects respectively. We deliberately select MtDef4, PDC1 and Cecropin A as our candidates. Unlike current chemical interference measures, antimicrobial peptides are prone to have less side effects:

1.The half-life of the polypeptide is shorter, and its decomposition product is nitrogen-containing organic matter, which can be reused by the crop.
2.The above two families of peptides can specifically form pores in the surface of microbial cell membrane, so that the outflow of cell plasma contents cause cell death, this specific sterilization mechanism makes the microbial tough to mutate resistance, and they can also coordinate with other antimicrobial peptides, enzymes or even traditional antibiotics, because the antimicrobial peptide can increase the permeability of the cell membrane, it can play a synergistic effect without cross-resistance
3.They function on multiple targets within the cell. If the pathogen mutate any target within, eventually will not have a significant impact on antimicrobial peptides sterilization function.
4.Antibacterial peptides inhibitory concentration and sterilization concentration is very close, which makes them ideal fast fungicide. Moreover, plant defensins are generally considered non-toxic for plant and mammalian cells, thus make them promising candidates for advanced biological control against microbes and guidance for further development in novel antimicrobial agents.

Approach

We chose to examine 3 candidate antimicrobial peptides: Cecropin A, PDC1, and MtDef4. Our aim was to identify whether these candidates can exhibit a Fusarium graminearum killing activity that could be used for innovated therapy for wheat scab. These antifungal peptides are produced by inducer—methanol. Three different antimicrobial peptides shared the same method of construction (See Figure 1) and the chassis—Pichia. In these constructs, a gene inserted downstream under the control of a inducible promoter would be expressed in the presence of the methanol. This part allows us to achieve the goal of massive production for antimicrobial peptides in a yeast cell, by which we can effectively kill the Fusarium graminearum growing in the infected wheats

Figure 1: A schematic representation of our basic plasmid types, the response module. It includes the anti-fungal peptide coding gene (anti-fungal peptide) and the inducible promoter. This construct allows genes placed downstream of the inducible promoter to be transcribed when the yeast cells are induced with the appropriate inducer—methanol.

Film 1: A schematic film of our response module, after treating with methanol, the inducer, pAOX1 will be activated later on the downstream gene will be switched on. Under the guidance of secretion signal, the primary peptide with 6*His tag will be secreted outside the yeast cells. Certainly, in order to circumvent the effect of 6*His tag, processes are expected to cutting the 6*His tag.

Key Achievement

Successfully clone out the gene of Cecropin A, Plant Defensin Corn 1, and MtDef4. Successful eukaryotic expression and purification of Cecropin A, Plant Defensin Corn 1, and MtDef4. The antimicrobial activity of Cecropin A, Plant Defensin Corn 1, and MtDef4 was successfully verified

Results

The results displayed in Figure 3A show the gene amplification result of Cecropin A, Plant Defensin Corn 1, and MtDef4 and the eukaryotic expression of Cecropin A, Plant Defensin Corn 1, and MtDef4 by Tris-tricine SDS-PAGE. The graph from Figure 3B and Figure 3C represents the MASS spectrometry report and HPLC analysis of Cecropin A and MtDef4, respectively.

Figure 3A: (A) Amplification result of antifungal peptide gene, the molecular weight of Cecropin A, plant defensin corn 1, and MtDef4, are 177bp, 393bp, and 231bp respectively. These graphs also show the result of eukaryotic expression of Cecropin A, plant defensin corn 1, and MtDef4. Among them, the graph of PDC1 Tris-Tricine SDS-PAGE is before purification, while the other two Tris-Tricine SDS-PAGE graphs are showing the result after purification of nickel column and enterokinase cutting down the 6*HIs tag. The molecular weight of Cecropin A is 4kDa, the molecular weight of Plant Defensin Corn 1 is 11kDa, and the molecular weight of MtDef4 is 5kDa.


Figure 3B&3C: (B) The MASS spectrometry report and HPLC analysis of Cecropin A, from the graph we can get the weight measured by MASS spectrometry is 4003.78U and the amino acid sequence is Lys-Try-Lys-leu-phe-Lys-Lys-ile-Glu-Lys-val-Gly-Gln-Asn-Ile-Arg-Asp-Gly-Ile-Ile-Lys-Ala-Gly-pro-Ala-val-Ala-val-val-Gly-Gln-Ala-Thr-Gln-Ile-Ala-Lys-NH2 (C) The MASS spectrometry report and HPLC analysis of MtDef4, from the graph we can get the weight measured by MASS

◎ Through the graph of zone of inhibition experiment we can see that compared with negative control (sterile water) different concentrations of Cecropin A, Plant Defensin Corn 1, and MtDef4 and can show the inhibition of the growth of Fusarium graminearum spores, in higher concentrations of the Cecropin A, Plant Defensin Corn 1, and MtDef4, the antimicrobial peptides can show a similar or even more bactericidal activity compared with positive control—Carbendazim. The results were displayed in Figure 3D. It is noteworthy that since carbendazim needs to be dissolved in acetic acid, but the dissolution of antimicrobial peptides requires only PBS, so the bactericidal efficacy of carbendazim is largely due to the faster rate of diffusion of organic reagents on the surface of the culture medium and Largely dependent on the physiological toxicity of acetic acid. It is gratifying to note that under this condition the antimicrobial peptides still exhibit strong bactericidal activity. We further testify the antifungal activity of MtDef4 by measuring OD at 595nm wavelength after treated for 50h and Cecropin A by measuring the conductivity ratio of Fusarium graminearum spores treated with cecropin A for a various of time. The graphs and data suggest that Cecropin A, Plant Defensin Corn 1, and MtDef4 are promising tool for the bactericidal function in our circuit.

Figure 3D: The inhibition zone results show the three antifungal peptide can exhibit a great antifungal activity, and the area of the inhibition zone is dose dependent. We choose to utilize sterile water as our negative control and 100ppm carbendazim dissolved in acetic acid as positive control. All the three antifungal peptides are dissolved in sterile water.

Figure 3F: We test the cell viability under the action of a series of different concentrations of antifungal peptide. Among them we can conclude the MtDef4 possess the best antifungal activity while PDC1 possess a inferior antifungal activity. Significant differences versus vehicle were assessed by student’s t test: *=p<0.05, **=p<0.01, ***=p<0.001, ****=<0.0001.