Results

Fig.1 The SDS-PAGE results for the amplification of the three proteins

As shown in Figure 1, the gel electrophoresis results display the three proteins extracted from colony 1, colony 2, and colony 3 after amplification. In the figure, “M” represents the marker, providing a reference for the molecular sizes of the three proteins, which helps us assess their sizes. "PVC15" refers to the protein translated from the PVC1-16 fragment with PVC13 knocked out (the part encoding the tail fiber protein), leaving only the outer shell of the PVC system, ensuring its structural integrity after the tail fiber protein is replaced. "GP20" is the tail fiber protein from T4 bacteriophage, which specifically recognizes Vibrio cholerae. "VgrG3" represents a toxin protein. Based on the gel results and the known molecular weights, the sizes of the extracted proteins match their theoretical values. In order to certify which structure mainly perform functions in the bacterial-killing procedure, we then conduct the Sterilisation experiments on checkerboard point panels by mixing specific proteins with EPI300, the target bacterium in our project.

Fig.2 The antibiotic test on EPI300

(Left: the experimental group; Middle: the control group with PVC 13 gene absent,; Right: the control group with payload VGRG3 gene absent. The concentration of the protein changed for 1.25 mg/ml to 10 mg/ml (from A to D).) Figure showed that the reconstructed PVC system can not perform its functions without either tail fiber or payload. The function would be sound only if the system contains tail fiber (for specific recognition) and payload (for toxic killing).

Fig.3 PVC caused the rupture of Vibrio cholerae

After preliminary determination of the killing effect through the growth of colonies on agar plates, we incubated PVC with Vibrio cholerae and captured electron micrographs during the killing process. These micrographs revealed ruptures in the cell membranes of the vibrios, with cellular contents leaking out. And various forms of PVC particles, both in pre-contraction and contraction states are observed. With this, we have confirmed that PVC indeed targets and binds to Vibrio cholerae, achieving bactericidal effects.

Fig.4 Electron micrographs after PVC completed the killing

(A: The PVC fiber under lower multiply;

B: The PVC fiber under higher multiply;

C: the pre-contraction step, the tail fiber was kept in and only spike was exposed;

D: the recognition step, the tail fiber recognized the receptor on the bacteria and fixed;

E: the contraction step, the sheath contracted to penetrate the spike into the cell.） The figure showed the complete process for a PVC fiber to function, which gave evidence to the antibiotics tests.

Discussion

In this study, we builds on previous research by utilizing the PVC injection system to experimentally address the infection caused by Vibrio cholerae. We successfully constructed specific plasmids and expressed PVC proteins in Escherichia coli, followed by conducting a killing assay using the dot plate method to verify the potential efficacy of the PVC system against Vibrio cholerae. The experimental results tentatively indicate that the PVC system has potential in combating infections by Vibrio cholerae. Further insights from transmission electron microscopy (TEM) observations revealed the destructive effect of PVC proteins on Vibrio cholerae, offering preliminary theoretical evidence for the application of the PVC system in medical treatment.

However, there are several limitations in the experimental design and execution that warrant consideration: 1. Limitations in sample size: The sample size used in the dot plate assay was relatively small, which may not fully represent the actual antibacterial effectiveness of PVC proteins. Future studies should increase the sample size to more accurately evaluate the bactericidal efficacy of PVC proteins. 2. Control of experimental conditions: The experimental process may have been influenced by external environmental factors such as temperature and humidity, which could impact the activity of PVC proteins. Subsequent experiments need to rigorously control these conditions to minimize potential interference with the experimental outcomes. 3. Targeted modifications of the PVC system: Although we made specific modifications to the tail fiber protein of the PVC system to enhance its targeted killing capability against Vibrio cholerae, the experimental results did not adequately validate the effectiveness of these modifications. Future research should focus on optimizing the modification strategies to enhance the PVC system's targeting of specific bacterial populations. 4. Research gap on bacterial resistance: This study did not investigate the potential for Vibrio cholerae to develop resistance to the PVC system. Given that resistance may emerge in real-world applications, this could affect the long-term therapeutic benefits of the PVC system. Future research should address this issue to provide comprehensive data support for the practical application of the PVC system. 5. Evaluation of biocompatibility: As a novel biotreatment approach, the biocompatibility of the PVC system must be thoroughly evaluated. A comprehensive biocompatibility assessment is essential before the widespread clinical implementation of the PVC system to ensure its safety for both humans and the environment.

In conclusion, this study provides initial experimental evidence for the application of the PVC system in the treatment of Vibrio cholerae infections. To widely apply the PVC system in clinical settings, further research is needed in areas such as increasing sample size, optimizing experimental conditions, refining modification strategies, investigating bacterial resistance, and conducting biocompatibility evaluations. Based on these advancements, the PVC system could potentially become an innovative and effective treatment modality for the prevention and treatment of bacterial infectious diseases like cholera.