QBlock

Abstract

Chronic wounds are wounds that take longer than three months to heal and inflict a disproportionate burden on patients and the healthcare system. They are frequently subsequent complications of more persistent health issues. Biofilms contribute to the slower healing of around 80% of chronic wounds. Our project, QBlock, aims to limit biofilm formation by targeting bacterial communication molecules (i.e. AIPs) that contribute to biofilm formation in Staphylococcus epidermidis by inhibiting bacterial communication with DARPins (designed Ankyrin Repeat Proteins), which are synthetic proteins that mimic antibodies. Through the manipulation and randomization of amino acid locations, a DNA library of 42 DARPins was created. AlphaFold2 predicted the structure of these DARPins, and their affinity for AIP was determined experimentally by ribosomal display. In control tests, a DARPin with a known sequence was used to bind GFP. Not only did structural predictions and simulations validate a strong binding with AIP, but they also emphasized the uniqueness and diversity of DARPins.

Chronic Wounds - "A Silent Epidemic"

Chronic wounds are a burden for both the patient and the healthcare system as a whole. They are frequently caused by other underlying chronic diseases and are therefore known as a "silent epidemic" (Benitez et al., 2019). As a result, we decided to address the issue and bring it to a wider audience through our initiative. The biggest difference between chronic wounds and other wounds is that they have a significantly reduced healing time, and can stay open for three months or even more. In the EU, between 27 and 50 % of acute hospital beds are occupied by patients with wounds (Moore et al., 2014). In affluent nations, chronic wounds affect an estimated 1% to 2% of the population (Järbrink et al., 2016). Moreover, chronic wounds account for 3 to 5% of healthcare expenditures in developed nations (Posnett and Franks, 2008; Järbrink et al., 2017). 15% to 25% of diabetic individuals develop diabetic foot ulcers as a result of underlying illnesses (Huang and Kyriakides, 2020). These patients are seen to have a higher incidence of morbidity and mortality (Moulik et al., 2003), a lower quality of life, and a higher risk of amputation. As the incidence of chronic wounds rises as a result of the aging and obesity of the population both in Finland and globally, it is not envisaged that the problem will become less difficult in the future. With our project, we aimed to show that ribosome display can be an effective method for selecting the most suitable DARPin for the target signalling molecule, by testing the system with a known GFP-binding DARPin. We also built a bioreporter for detecting the effect our DARPins have on blocking the signalling between the bacteria. We tested the baseline signal of GFP from the bioreporter, however, due to time restrictions, we have time to test the bioreporter with DARPins included. More optimizing is however needed with the bioreporter promoters, to get a reliable result of the activation of the P2 promoter in the bioreporter construction.

Biofilms Are Slowing Down Wound Healing

Biofilms, which are structured communities of microorganisms, are present in around 80% of chronic wounds (Malone et al., 2017) and impede their recovery (IWII, 2022). Due to the hypoxic environment, it is challenging for the immune system to cleanse the wound site of bacteria. This reduces immune function and renders patients more prone to infection (Wang et al., 2020). Biofilms are known to be tolerant to antimicrobials, antiseptics, and host defenses, rendering most therapeutic techniques ineffective (Høiby et al., 2010). The establishment of biofilms is divided into four general stages (Madigan et al., 2009). First the planktonic bacteria become sessile and settle at the site of infection. This stage is called attachment. It is followed by the stage of reproduction, maturation and finally shedding. Bacteria cluster together early in the attachment phase due to a process called quorum sensing.

Quorum Sensing - Talking Bacteria

Quorum sensing is defined as population density measurement and bacteria can utilize this information in order to “communicate” with each other (Madigan et al., 2009). This allows them to know their population density, which is very important for the onset of infection for bacteria. The process of quorum sensing is either dependent on chemical signal molecules or peptides. The process that Staphylococcus epidermidis utilizes is the AIP (autoinducing peptide) system, which as the name implies is a peptide based system (Olson et al., 2014). In our project we will focus on S. epidermidis, because bacterium is a biosafety level 1 organism. During the process of quorum sensing, bacteria not only measure their bacterial density, but also induce downstream gene activity (Olson et al., 2014). This downstream gene activity also includes virulence genes such as RNAIII in S. epidermidis. Bacteria utilizing quorum sensing use communication circuits to regulate a variety of physiological processes. This involves symbiosis, pathogenicity, competence, conjugation, antibiotic synthesis, motility, sporulation, and biofilm formation (Miller & Bassler, 2001).

Introducing QBlock to Prevent Biofilm Formation

Our product, QBLOCK, is designed to prevent biofilm development by targeting quorum sensing. We intend to prevent the ability of bacterial autoinducing peptides (AIPs) to induce either other bacteria or themselves by binding them to DARPins. DARPin stands for designed ankyrin repeat protein and refers to peptides that resemble antibodies (Zahnd et al., 2017). DARPins consists of a N- and C-cap with three or more repeats in between (Seeger et al., 2013). The amino acid sequence is derived from a common consensus sequence, where we will be modifying some amino acids to find the best construct for the DARPin to bind to our target AIP molecule (Hansen et al., 2017; Shilling et al., 2014; Seeger et al., 2013). Simply expressed, we aim to suppress quorum sensing via DARPins, which can then be used as a treatment in combination with other antibiotic treatments to heal the wounds faster.

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