Team:Grenoble Alpes/Description

PyoBuster - Description


Antibiotic resistances are one of the main global health problems worldwide. These resistances come from their massive use as preventive and therapeutic treatments. To overcome those resistances, new molecules or therapeutic approaches are needed such as phagotherapies or antimicrobial peptides to constantly challenge the emergence of new resistant strains. When it comes to resistant strain, Pseudomonas aeruginosa accounts for one of the most problematic/threatening. Due to its complex and massive genome on one side and its ability to form biofilm on the other , Pseudomonas aeruginosa has high adaptability capacities explaining why it is one of the most problematic strains worldwide.

Biofilm description.
Biofilm formation begins when highly motile planktonic cells adhere to a surface. The cells undergo physical changes that involve down regulation of motility apparatus and virulence factors whilst upregulating the production of an exopolysaccharide-rich extracellular matrix. The community matures into dense mushroom-shaped structures. In response to inducing conditions, cells become motile once again and disseminate to colonise new ecological niches.
Extracted from Matrix exopolysaccharides; the sticky side of biofilm formation. Eve Maunders and Martin Welch. Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.

Its biofilm constitutes an additional problem to the bacterial virulence as it favorsits development and proliferation. As bacteria settle on surfaces, they can switch from a motile to a sessile life. From this step, they can grow in micro-colonies and produce the biofilm matrix. Biofilm consists of a 3D protective extracellular matrix made of exopolysaccharides, nucleic acids, and proteins which will protect bacteria from oxidative stress, phagocytosis, and conventional antibiotics. Different layers of biofilm are composed of bacteria at different metabolic states creating a gradient of activity. Besides, this matrix is a place of exchange of metabolites and DNA, and can then lead to the apparition of mutations and resistances. Thus, biofilm could be an interesting therapeutic target to fight against P. aeruginosa’s infection as it would weaken its defense, increasing its vulnerability to different treatments and the immune system.

Biofilms are at the center of many health problems caused by P. aeruginosa, including nosocomial infections, wound and burn infections, otitis, or optical infections in both animals and humans. This pathogen is also known to be implicated in chronic lung infections in patients affected by cystic fibrosis promoted by the accumulation of a thick layer of mucus. Today the only accessible treatment for these bacterial infections is antibiotics. Unfortunately, this involves long and regular treatment with high doses of drugs leading to consequent secondary effects.

our bacteria modified

To counter this problem, we created biomedicine consisting of engineered E. coli that we called PyoBusters. Initially, our project was to create a biotreatment that would specifically sense and eradicate the biofilm of P. aeruginosa in the lung environment of cystic fibrosis patients but we then expanded the applications to other infections. This biomedicine would have complemented the antibiotic treatments by destroying the biofilm, thereby increasing their efficiency in severe infections. This could reduce the frequency, duration, and posology of treatment. With lower doses of antibiotics, the development of bacterial resistances would be reduced.

To ensure a secure treatment, it was important for us to make sure that our engineered bacteria would not colonize off-target sites but would spread only on P. aeruginosa biofilm. Through our research in the literature, we decided to add the specific bactericidal capacity against P. aeruginosa to our genetically engineered bacteria and to finely control it. This way, we aim to create a specific and efficient treatment that could eventually replace the use of antibiotics.

This is why we incorporated a genetically engineered safety system that would be activated by the absence of the targeted pathogen triggering the autodestruction of our PyoBusters and thus preventing their proliferation. We also include a delivery system that will release therapeutic molecules when the population of our PyoBusters reaches a given threshold.

our incubator

It was important for us to test the efficiency of our system in real situations. To do so, we decided to elaborate a testing bench that will emulate the lung environment by adapting the humidity and temperature conditions. This device will be closed to ensure sterile conditions to grow P. aeruginosa’s biofilm. A camera will be installed in the regulated enclosure to follow by fluorescence the evolution of the biofilm in the presence and absence of our PyoBusters bacteria.

As we further continued our research, we decided to extend our project to treat any possible infection by P. aeruginosa in both humans and animals. Our testing bench allows us to control environmental conditions to mimic any infection site and test the efficiency of our PyoBusters in other health problems caused by P. aeruginosa.

Our system was designed using the quorum sensing of both our bacteria E. coli and P. aeruginosa. Quorum sensing is widely used in bacterial communities and therefore in biofilms. We can compare it to a complex bacterial communication system between each other. Thanks to this system, bacteria can sense their environment and population density, modify the expression of their genes to survive, and proliferate by modulating their metabolism, motility, or forming a biofilm.

Schemas representing the cycle of life of PyoBusters
  1. First, our engineered E. coli will be sent to the infection’s zone. Then, PyoBusters will sense P. aeruginosa’s biofilm employing its specific quorum sensing molecule : BHL (C4-HSL). The absence of BHL in our PyoBusters’ environment will lead to the activation of a safety system that will cause its death. If BHL is indeed present, indicating the presence of P. aeruginosa at the biofilm life stage, our PyoBusters will survive and proliferate. (Step 1)

  2. As the population grows, PyoBusters will constitutively produce and accumulate in its cytoplasm anti-biofilm as well as bactericidal therapeutic molecules specific to the pathogen (Step 2).

  3. At a given PyoBusters population level, the AI-2 concentration in environnement will reach a threshold and activate the delivery system . As the bacteria will be lysed, all therapeutic molecules accumulated will be released on the biofilm. Therefore, the biofilm and P. aeruginosa will be eliminated (Step 3 and 4).

  4. The massive release of the therapeutic molecules will destroy the biofilm and P. aeruginosa. However, it is possible that the amount of anti-biofilm molecules may not be sufficient to destroy the entire biofilm. In this case, the remaining BHL on the biofilm will allow PyoBuster to survive and restart a life cycle. Therefore, PyoBusters can be effective until there is no more biofilm and P. aeruginosa in the infection site (step 4 and 1).