Team:Tec-Chihuahua/Demonstrate
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Overview
With the intention of finding an effective treatment for Verticillium dahliae we tested the inhibition capabality of the following peptides:
- BBa_K2959005: Expressible Pinus sylvestris Defensin 1 with Erv1p
- BBa_K2959007: Expressible Wheat antimicrobial peptide 1b
- BBa_K2959010:Expressible Arabidopsis thaliana Profilin 1
Antifungal Assay
In order to test the antifungal activity of our peptides as well as prove their viability as a mechanism to inhibit Verticillium dahliae, an antifungal susceptibility test on a 96 well plate was carried out by measuring absorbance at 405 nm, wavelength used in standardized protocols to measure growth of filamentous fungi.1 Due to a lack of time, we couldn’t reach the experimental stage of the project of peptide purification, so experiments were made using soluble protein extracts from our transformed cells’ lysates. Different dilutions of the extracts were prepared which were applied to a spore suspension of V. dahliae. Dilutions of the extracts of untransformed cells were used as controls to prove that inhibition was the result of the peptides and not any other protein contained within the extract. Soluble proteins from each chassis was used, BL21 (DE3) were as control for the AtPFN1 extract, and SHuffle extract as control for both PsDef1 and WAMP1b. Concentrations of extracts with peptides were equalized to their controls. The tables 1 and 2 detail the concentrations of each dilution.
Table 1. Protein concentration at mg/mL of soluble protein extract of transformed cells induced to produce AtPFN1 and untransformed BL21 (DE3) control.
| Dilution | AtPFN1 (mg/mL) | BL21 (DE3) control (mg/mL) |
|---|---|---|
| 1 (undiluted) | 3.7402 | 3.74 |
| 3:4 | 2.8052 | 2.805 |
| 1:2 | 1.8701 | 1.87 |
| 1:4 | 0.9351 |
|
Table 2. Protein concentration at mg/mL of soluble protein extract of transformed cells induced to produce PsDef1 and WAMP1b and untransformed SHuffle®️ T7 Express control.
| Dilution | PsDef1 (mg/mL) | WAMP1b (mg/mL) | Shuffle control (mg/mL) |
|---|---|---|---|
| 1 | 4.2699 | 4.2699 | 4.27 |
| 3:4 | 3.2024 | 3.2024 | 3.2025 |
| 1:2 | 2.1350 | 2.1350 | 2.135 |
| 1:4 | 1.0675 | 1.0675 | 1.0675 |
96 well plates were prepared as shown in Figure 1. A final volume of 200 μL was completed in each well by mixing protein extract, sterile distilled water (to achieve desired concentrations), potato dextrose broth, and a spore suspension of V. dahliae with a final concentration of 2x104 spores/mL per well. Plates were incubated at 25°C. For more information visit Experiments Page
Figure 1. 96 well plate distribution.
Absorbance readings at 405 nm were performed in a Varioskan Lux 3020-231 microplate reader 1 and 24 hours after plate preparation. After analyzing the results, a growth rate percentage was estimated for every evaluated sample by using the following formula:
Growth rate =(( A1 - A0)/A0) x 100
where:
A1= Absorbance after 24 hours.
A0= Absorbance after 1 hour.
Results
Results generated by the previous formula correspond to the percentage in which absorbance increased in each well compared to the initial reading. The results are summarized in figures 2, 3, and 4. Through a careful interpretation, a pattern was observed for all peptides tested. As the figures show, an outstanding difference in growth values exists among the positive growth controls without any kind of protein extract, the untransformed strain extract controls, and the growth of the fungus with different peptides’ concentrations, was noted. The positive control was estimated to grow at a rate of 40.36% after 24 h, on the other hand, the lowest concentrations of added protein extracts clearly showed a decreasing behavior in growth rates. In comparison, growth rates from wells containing protein extracts of untransformed cells do not behave this way, meaning that all three peptides had a degree of inhibition against V. dahliae, even in crude extract. In order to better comprehend the results, growth rates were used to estimate the percentage of inhibition using the following formula:
I = 100 - (Gp / Gv) x 100
where:
I = inhibition %
Gp = Growth rate of V. dahliae treated with extract with peptides.
Gv = Growth rate of V. dahliae positive control
Untransformed E. coli BL21 (DE3) protein extracts seemed to increase the fungus’ growth in contrast with the extract containing the AtPFN1 peptide, which demonstrated inhibition in all three dilutions with the exception of the undiluted extract. The inhibition percentages for this peptide were of 30.40%, 76.96%, and 98.32% for dilutions 3:4, 1:2, and 1:4 respectively in comparison with the positive growth control. This assay was able to demonstrate the peptide’s reported ability to permeabilize the cell wall and membrane of fungal spores5 by showing growth reduction in almost all dilutions and close to total inhibition in the last one, as shown in figure 2.
Figure 2. Antifungal activity of AtPFN1 against V. dahliae. V. dahliae growth rate (%) challenged with different concentrations of soluble protein extract from transformed E. coli BL21 (DE3) with AtPFN1 and untransformed E. coli BL21 (DE3).
During the evaluation of PsDef1, untransformed protein extracts seemed to also increase the fungus’ growth in contrast with the extract containing the peptide, even though the control strain was changed to E. coli SHuffle. In contrast, in figure 3, inhibition caused by PsDef1 can be observable in the two lowest dilutions, presenting high inhibition percentages of 82.23% and 70.81% for dilutions 1:2 and 1:4, respectively. These reductions in growth rate helped to characterize the peptide’s reported ability to halt fungal growth6.
Figure 3. Antifungal activity of PsDef1 against V. dahliae. V. dahliae growth rate (%) challenged with different concentrations of soluble protein extract from transformed E. coli SHuffle with PsDef1 and untransformed E. coli SHuffle.
Similar results were obtained with WAMP1b, which was also compared to E. coli SHuffle strain and which again showed and increasing growth rate. As seen in figure 4, the trend of observing inhibition in dilutions 1:2 and 1:4 is repeated. In this case, the peptide showed an inhibition percentage of 97.52% and 81.54% for this dilutions respectively. Given that this peptide is reported to suppress V. dahliae's defense mechanism7, it is believed that this effect helped to inhibit the fungus’ growth as reported below.
Figure 4. Antifungal activity of WAMP1b against V. dahliae. V. dahliae growth rate (%) challenged with different concentrations of soluble protein extract from transformed E. coli SHuffle with WAMP1b and untransformed E. coli SHuffle.
Discussion
In comparison, the two highest concentrations of extracts with peptides (1 & 3:4) seemed to show a greater growth than the positive controls, even though previous results showed that the extracts were capable of inhibiting the fungus growth. Analyzing this lead to the opportunity of identifying error sources and troubleshooting this protocol.The remarkable difference from the positive control wells and those with our protein extract dilutions opens their interpretation based on several hypotheses.
Due to the time, equipment, and material limitations we encountered, the antifungal assays presented show preliminary results. Delving further into this, we concluded that a factor we could have overlooked in the design of this protocol was the possibility of compound precipitation. A common occurrence in microplates assays is the precipitation of the inhibitory components, which leads to inconsistent inhibition values.2 Peptides tend to precipitate at certain concentrations, depending on their composition, solubility and even the buffer or medium they’re diluted in.2,3 Given that we have almost no concrete information about our peptides, and that we saw ourselves forced to use crude extract, precipitation at the highest concentrations we used is a feasible possibility. It has been remarked in literature that this incidence makes it almost impossible to determine the MIC (Minimal Inhibitory Concentration) through this method.2
The precipitation of our peptides could have affected the optic density measurements causing variations that altered the overall results.3 Or, most likely, the precipitation altered the inhibitory effect of our peptides at high concentrations. This is a common occurrence, and recommendations can be found through literature, research papers, and protocol manuals.4 One of them is keeping the plates in agitation to avoid or minimize this effect, but in other cases agitating the plates lead to confusing outcomes.2,3 Since we never considered agitation necessary for our protocol, this is a potential modification to consider for future tests. Another option is trying different media or buffers, to decrease or completely avoid precipitation.4
References
- Schwalbe, R., Steele-Moore, L., & Goodwin, A. C. (2007). Antimicrobial susceptibility testing protocols. Crc Press.
- Eloff, J. (1998). A Sensitive and Quick Microplate Method to Determine the Minimal Inhibitory Concentration of Plant Extracts for Bacteria. Planta Medica, 64(08), 711–713. doi:10.1055/s-2006-957563
- Morishige, H., Mano, Y., Oguri, T., & Furuya, N. (2012). Comparison of four reading methods of broth microdilution based on the Clinical and Laboratory Standards Institute M27-A3 method for Candida spp. THE JAPANESE JOURNAL OF ANTIBIOTICS , 65(5), 335–347. Retrieved from http://jja-contents.wdc-jp.com/pdf/JJA65/65-5/65-5_335-347.pdf
- Berditsch, M. (2012). Two-fold Broth Microdilution Method for Determination of MIC. Institute for Bio and Geosciences. Retrieved from http://www.ibg.kit.edu/nmr/downloads/MICprotocoll_30_Jan2012.pdf
- Park, S.-C., Kim, I. R., Kim, J.-Y., Lee, Y., Kim, E.-J., Jung, J. H., … Lee, J. R. (2018). Molecular mechanism of Arabidopsis thaliana profilins as antifungal proteins. Biochimica et Biophysica Acta (BBA) - General Subjects, 1862(12), 2545–2554. doi:10.1016/j.bbagen.2018.07.028
- Khairutdinov, B. I., Ermakova, E. A., Yusypovych, Y. M., Bessolicina, E. K., Tarasova, N. B., Toporkova, Y. Y., … Nesmelova, I. V. (2017). NMR structure, conformational dynamics, and biological activity of Ps Def1 defensin from Pinus sylvestris. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1865(8), 1085–1094. doi:10.1016/j.bbapap.2017.05.012
- Naumann, T. A., & Wicklow, D. T. (2013). Chitinase modifying proteins from phylogenetically distinct lineages of Brassica pathogens. Physiological and molecular plant pathology, 82, 1-9.doi: https://doi.org/10.1016/j.pmpp.2012.12.001