What is "biofilm"?

Biofilm is a densely woven network-like material that is synthesized by certain bacteria as a means of withstanding adverse environmental conditions. Furthermore, it is worth noting that biofilm formation is a prevalent occurrence in harmful bacteria, including Pseudomonas aeruginosa.

P. aeruginosa exhibits the ability to withstand the conditions within the human body by forming biofilms and adhering to diverse tissue surfaces, thereby persisting until an opportunity arises for infection. Our objective was to specifically target P. aeruginosa, a bacterium that attaches to the gastrointestinal tract by forming biofilms.

How do we want to deal with biofilm?

A biofilm is composed of various components, including extracellular DNA (eDNA), proteins, and polysaccharides. In order to achieve comprehensive disruption of biofilm, our objective was to target both the protein and polysaccharide components of the biofilm structure.

The initial aspect to consider is the protein's dimension. The protein DNABII is known to have a significant impact on the process of biofilm formation in P. aeruginosa. The DNABII protein is an secreted protein that serves as a fundamental constituent of biofilm structures. The primary interaction of the DNABII protein is with extracellular DNA (eDNA), whereby it facilitates the formation of a grid-like arrangement by inducing the bending of the eDNA molecules. Additionally, the DNABII protein is capable of converting the conformation of eDNA from the canonical B-form to the alternative Z-form. Nevertheless, it is important to note that the human body lacks DNase enzymes that specifically recognize and degrade DNA in its Z-form conformation. As a result, extracellular DNA (eDNA) present in biofilms exhibits a high degree of durability. Considering the DNA-binding properties exhibited by the DNABII protein, we have developed a protein known as HMGB1, which is obtained from humans. Over the past two years, there has been emerging evidence demonstrating the disruptive potential of HMGB1 in biofilm disruption. HMGB1 is a nuclear protein that has the ability to interact with DNA within the nucleus of mammalian organisms. The dimeric DNABII protein is involved in biofilm formation and maintenance, and its binding and bending features are similar to those of HMGB1 protein. Thus, HMGB1 protein has the ability to competitively interact with DNABII protein, resulting in HMGB1 protein binding to eDNA. This interaction leads to decreased eDNA stability and ultimately to the disintegration of biofilm structures.

In the present study, the investigation focused on the disruptive potential of several forms of HMGB1, including HMGB1_FL, HMGB1_AB box, HMGB1_A box, and HMGB1_B box, on biofilm development. It is worth noting that HMGB1 consists of two structurally comparable domains, namely the A box and B box.

Additionally, there exists a dimension that is peculiar to polysaccharides. The major polysaccharides found in P. aeruginosa are primarily composed of Psl. Our objective was to induce hydrolysis of Psl by expressing PslG. PslG is an extracellular protein that is produced by P. aeruginosa. Its primary function is to catalyze the hydrolysis of Psl, a key component of the biofilm matrix. Through this enzymatic activity, PslG plays a crucial role in the regulation of biofilm formation and degradation. Furthermore, PslG facilitates the release of P. aeruginosa from the biofilm structure under favorable environmental conditions. Through the utilization of modified bacteria, our objective is to induce the expression of PslG in order to interfere with the regulatory mechanisms governing biofilm formation, ultimately leading to the disruption of P. aeruginosa's biofilm formation.

How to specifically destroy the biofilm?

This is the core part of our project. In order to specifically destroy the biofilm produced by Pseudomonas aeruginosa in human body without affecting the normal cells of the patients, we found two specific proteins (enzymes) to construct the secreting system: recombinant protein HMGB1 and polysaccharide enzyme PslG. The recombinant HMGB1 protein targets DNA in biofilm, destabilizing it and make it easy to be decomposed, and the polysaccharide enzyme PslG targets polysaccharide, which can effectively inhibit the formation of biofilm.

For both proteins above, we designed two feasible pathways of action.

The first one is the secretory pathway. First, the engineered bacteria we constructed will continuously express population-sensing effector molecules that can detect Pseudomonas aeruginosa, which, when combined with them, will cause the expression of downstream genes. For the expression of the enzymes that actually destroy the biological periplasm, we use to add linker co-expression, or separately. At the same time, we add signaling sequences upstream of these gene sequences so that effector proteins can be secreted through type II secretion system.

The second is the lysis pathway. In order to prevent the concentration of the target protein from being too low, we can enrich the effector protein in the engineered bacteria without adding the signal sequence. At the same time, we add an alkaline phosphatase into the cells of the engineered bacteria, when the concentration reaches a certain high level, it will lyse the engineered bacteria, so that the target protein in the body will be released rapidly, and reach a very high concentration in the microenvironment, resulting in a very strong killing effect.

To successfully accomplish the project and guarantee safety, our primary focus was on conducting tests pertaining to protein function integrity, direct observation of biofilm, design of the quorum sensing system, cytotoxicity testing, and cell lysis testing. Please consult our pages on "engineering," "experiment," "notebook," and "result" for further information.