Introduction
Peptides, by biological definition, are short chains of amino acids that are fundamental to many physiological processes. Their relatively small size, compared to larger proteins, makes them versatile tools in therapeutic and research applications. This project focuses on two specific lytic peptides—Hecate and Melittin—which possess the ability to disrupt cell membranes. Hecate is a synthesized lytic peptide known for its antimicrobial effects. While it can be engineered into a precision tool by combining it with a specific ligand to target particular cell receptors (like the FSH receptor in cancer research), our project will utilize its core lytic function in a general manner to conduct a preliminary toxicity assessment. In contrast, Melittin is a natural lytic peptide and the primary toxic component in European honeybee venom, also renowned for its potent, non-specific ability to rupture cell membranes.
The real-world applications of these peptides highlight their distinct mechanisms. Hecate is typically designed for targeted disruption, making it a valuable asset in pre-clinical studies for diseases like cancer. Melittin, however, acts as a powerful, broad-spectrum blowtorch against cells. The purpose of employing these lytic peptides in our project is not to directly kill termites. Instead, we aim to leverage their membrane-disrupting properties to target and impair the symbiotic protozoa that reside within the termite's gut. These protozoa are essential for the termite's ability to digest cellulose, its main food source. By disrupting this critical symbiosis, we can explore a novel approach to termite control.
Plasmid Construction
We constructed the plasmids with pET28a backbone, using the inducible anaerobic promoter FNR promoter to control the expression in low oxygen conditions where glucose is present; and added the kanamycin resistance gene for later transformation screening. Our Hecate and Melittin sequence is fused with a secretion signal NSP4. Important to mention that the NSP4 sequence is modified, from M-K-K-I-T-A-A-A-G-L-L-L-L-A-A-Q-P-A-M-A to M-K-K-I-T-A-A-A-G-L-L-L-L-A-A-Q-P-A-M-K, last A changed to K, and one more K is added in the C-terminus, to create the cleavage site for trypsin. Besides of creating a inducible gene expression, this allows the peptide to be secreted outside the cell, and in case of need to obtain pure peptide, it is possible to cut off the signal peptide by using enzymatic digestion.
The plasmid was custom-synthesized by VectorBuilder (a commercial gene synthesis and plasmid construction service) and extracted using a standard plasmid isolation kit (Medium-Speed Plasmid Extraction Kit (Universal Type), Beyotime D0020). It was quantified via UV spectrophotometry (dsDNA setting) to ensure suitability for downstream experiments.
Figure 1.1: pET28a-FNR-mCherry
Figure 1.2: pET28a-FNR-Hecate
Figure 1.3: pET28a-FNR-Melittin
After extracting the plasmids, we introduced the plasmid into competent cell E. coli strain MG1655, which is compatible with FNR promoter. The competent cell is cultured in LB agar plate with Kanamycin at 37°C overnight. Then single colonies are picked, culture in liquid LB broth medium with Kanamycin, cultured overnight again. The bacterial culture obtained will be made into stocks by adding glycerol, making 50% glycerol in the final mixture; the stocks are kept in -80°C.
The cultures we kept:
- MG1655 without plasmid (no need to go through transformation)
- MG1655 with pET28a-FNR-Hecate (11.2ng/μL)
- MG1655 with pET28a-FNR-Melittin (23.8ng/μL)
Experimental Design
1. Preparation of bacterial supernatant:
a. Obtain the stocks and culture the bacteria
- Control: MG1655 strain without plasmid
- Hecate: MG1655 strain carrying the pET28a-FNR-Hecate
- Melittin: MG1655 strain carrying the pET28a-FNR-Melittin
b. Induce expression in anaerobic condition
When the OD600 of the cultures reach to 0.4~0.6; then the lid is closed tightly to induce expression by activating anaerobic FNR promoter; the oxygen will eventually be used up. The cultures are collected at different time points: 4h, 6h and overnight (O/N). After collection, the culture is centrifuged at 5000 rpm for 5min, then the supernatant is transferred to clean EP tubes and stored under -80°C.
2. Preparation of the pure peptides:
a. Stock solutions
- Control: Pure DMSO
- Hecate: Pure DMSO is added to 1mg of peptide to make up 1mg/mL stock solution.
- Melittin: Pure DMSO is added to 1mg of peptide to make up 1mg/mL stock solution.
3. Toxin assay
We performed testing on E. coli to confirm the functionality of lytic peptides, Hecate and Melittin, which are known to have antimicrobial effects that suppress bacterial growth. The general procedure involved mixing both the bacteria-synthesized peptides and pure peptides with a culture of the E. coli strain DH5α. Bacterial growth was then monitored by measuring the optical density at 600 nm (OD600) over a 12-hour period, with an additional data point collected at 24 hours.
Second, we assessed the effect of the lytic peptides on the multicellular organism C. elegans. This worm is a suitable model for preliminary toxicity assessment due to the ease of observing morphological and behavioral changes. We first tested the peptides' effect on motility by exposing the worms to the peptides and counting the number of thrashes within a 30-second interval in M9 buffer. Furthermore, we used the act-5p::GFP strain to enable direct observation of the gut morphology following peptide exposure.
The detailed protocols for these experiments are provided in the LAB PROTOCOL document.
Results
Pure Peptide Melittin Shows Higher Toxicity to E. coli strain DH5α compared to Pure Hecate
Figure 2.1: 介绍介绍介绍介绍介绍介绍介绍介绍
Figure 2.2: 介绍介绍介绍介绍介绍介绍介绍介绍
In both graphs we can see that the pure peptides has inhibitory effects on the E. coli growth. Hecate does not show significant difference using either 1μg/mL or 10μg/mL. Meanwhile, Melittin at the concentration of 10μg/mL even inhibits more compared to positive control kanamycin.
Hecate Bacteria Supernatant shows Impact on C. elegans Thrashing Ability
Figure 3: 介绍介绍介绍介绍介绍介绍介绍介绍
A Bonferroni's post-hoc analysis performed by using the Prism 10 software revealed that treatment with Hecate resulted in a statistically significant reduction in thrashing rate compared to the control group (mean difference = 16.05, 95% CI [3.263, 28.84], p=0.0124). In contrast, treatment with Melittin did not produce a significant effect on motility (p > 0.9999). These results suggest that Hecate, but not Melittin, has a paralytic or inhibitory effect on C. elegans.
| Comparison | Mean Difference | 95% Confidence Interval | Significance | Adjusted P Value |
|---|---|---|---|---|
| Control vs. Hecate | 16.05 | 3.26 to 28.84 | * | 0.012 |
| Control vs. Melittin | 2.21 | -11.88 to 16.30 | ns | >0.99 |
Footnote: ns = not significant; * = statistically significant (p < 0.05 after Bonferroni adjustment for 2 comparisons).
Bacterial Supernatant Shows No Acute Toxicity to C. elegans After Two-Hour Exposure
Figure 4: 介绍介绍介绍介绍介绍介绍介绍介绍
To explore the effect of toxins on C. elegans, we exposed the worms to the bacterial supernatant for 2 hours. The mechanism of the hecate and melittin peptide mainly functions to disrupt the cell membrane, and since they are ingested into the gut, then we hypothesized these peptides have potential to cause harm in the worm gut. However, by observing the animals under normal brightfiled, there is no clear phenotypic change. When looking under the Green-Fluorescent Protein (GFP) Fluorescence, we observe that the gut is linear and smooth, shows no twist, indicating that the worm status is healthy after 2h exposure to the toxins.
This might also indicate that these peptides shows selectivity for the cell membrane, because the membrane from eukaryotes is different from the protozoa cell membrane.
References
- 参考文献一
- 参考文献二
- 参考文献三
- 参考文献四
- 参考文献五