Content

Background

At present, there are three mainstream drugs on the market, namely small molecule drugs, biologics, and peptide drugs. Among them, peptide drugs are increasingly favored and have become a focus in drug design due to their relatively low immunogenicity, high efficiency, and good security. However, it is regrettable that peptide drugs are easily degraded by proteases in the body's environment due to their inherent properties, resulting in a low half-life. At the same time, peptide drugs struggle to cross the cell membrane and exert their functions within target cells. Traditional methods of drug administration—such as oral and injectable routes—cannot solve the aforementioned problems of peptide drugs and also limit the types of drugs and therapeutic effects, preventing peptide drugs, a type of drug with immense potential, from replacing traditional hormone drugs on the stage.

Our project, based on bacterial secretion systems for drug delivery, attempts to solve this dilemma. Bacterial secretion systems are diverse, and their primary role is to help bacteria cope with survival pressures, thus obtaining ecological advantages. Amongst many secretion systems, we pay particular attention to the Type VI secretion system (T6SS) of bacteria. This is a secretion system unique to Gram-negative bacteria, capable of targeting prokaryotic and eukaryotic cells and directly injecting the bacteria's effector proteins into the cytoplasm of target cells. This excellent characteristic can be artificially transformed into a secretion system that carries peptide drugs, and it has an effective structure even before secretion. We hope to use Vibrio cholerae and Pseudomonas aeruginosa with toxicity genes knocked out as engineering bacteria. Through genetic engineering and other technologies, we will modify their original effector proteins to realize drug loading, expression, and protection of peptide drugs, and activate the secretion system when conditions are right to deliver drugs to the target cells, thereby enabling the drugs to function more efficiently.

Figure 1. How T6SS carries effectors [1]

Aims

The use of drugs has promoted the development of modern medicine, but traditional drug administration methods—such as oral administration, injection, etc.—limit the types of drugs and therapeutic effects. To improve treatment efficiency and save labor costs, we want to explore a new method of drug delivery.

Peptide drugs have become a focus in drug design due to their high specificity, high efficacy, and low immunogenicity. Unfortunately, the nature of peptide drugs themselves causes them to be easily degraded in the body’s environment and to have difficulty crossing the cell membrane to function within target cells. Not until the Type VI secretion system (T6SS) was discovered, did we utilize this bacteria-specific secretion system, seeking detoxified bacteria that can carry effective peptide drugs, to achieve the aim of continuous, stable, and precise drug delivery.

Experiment Design

We select strains with Type VI secretion systems (T6SS) and knock out their major toxicity proteins using homologous recombination. Next, we choose suitable peptide drugs to construct expression vectors, splicing the gene sequence of the peptide fragments into the Type VI secretion system gene cluster, thereby expressing the corresponding fusion proteins. Then, we utilize Western Blot for protein expression activity verification, ultimately realizing the delivery of peptide drugs using the Type VI secretion system.

Figure 2. Designing and engineering workflow

Engineering Overview

We have selected two bacteria carrying the Type VI secretion system (T6SS) as model carriers: Vibrio cholerae rhh strain and Pseudomonas aeruginosa DUEC strain. The Vibrio cholerae rhh strain is a new strain formed by knocking out some pathogenic genes of the Vibrio cholerae V52 strain. The V52 strain is a non-O1/O139 Vibrio cholerae, considered to be a low-pathogenic type within the Vibrio cholerae family. Therefore, the Rhh strain is not further detoxified. The DUEC strain is a variant of the Pseudomonas aeruginosa PAO1 strain, whose toxicity to the human body primarily originates from the Type III secretion system (T3SS) (reference) and is quite harmful, necessitating knockout.

Thus, we employed Gibson Assembly technology and, through the principle of homologous recombination, proceeded with the knockout of toxic protein genes. We constructed the pEXG 2.0 plasmid and introduced it into the WM6026 E. coli strain. pEXG2.0 is a commonly used vector, while the WM6026 strain is a good plasmid donor, often used in conjugation experiments. The WM6026 strain conveyed the empty vector pEXG 2.0 plasmid into the DUEC strain and knocked out the vfr, exoS and exoT genes of DUEC.

Subsequently, we observed and confirmed the normal activity of the Type VI secretion systems of PAO1 under a microscope, as we wanted to observe if our attenuation affects the activity of T6SS. We added fluorescence to the tail of the tssB1 protein (a T6SS protein); if the cells emit fluorescence, it is considered normal.

Next, we screened potential peptide drugs and used Gibson Assembly technology to connect the gene coding of peptides inside the bacterial T6SS gene cluster, constructing a fusion protein expression vector, and introducing it into model strains via electroporation or chemical transformation.

Finally, we checked whether the target peptide was successfully expressed, whether it was secreted and whether it remained active after secretion. We verified the normal expression and activity of the fusion protein through Western Blot.

Expected results

The toxicity has been essentially removed from the two model strains, and they can express and deliver suitable peptide drug proteins.

Limitation

Due to the ethical risk of conducting animal experiments, and to minimize bacteria release possibility, we have not conducted any animal cytotoxic experiment, but the graphs provided in the safety and engineering parts should be enough to prove that the strains we use pose minimal damage.