*Committed Listener - Alison Cabrera Ontaneda*
Biofilms are high-density bacterial communities that adhere to surfaces and are difficult to remove, making them responsible for numerous problems, from persistent clinical infections to corrosion in industrial settings. This project proposes the production of a hydrolase enzyme with the ability to target the biofilm matrix, Dispersin B, through the use of various molecular and synthetic biology tools. The hypothesis is based on the recombinant production of this enzyme using a cell-free system. So that it can be applied as an effective treatment against bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. For this, a plasmid will be assembled with a version of the Dispersin B gene together with structural elements such as a peptide signal, GFP, and a His-tag. Then, the protein will be expressed in a cell-free system, including the secretion signal to obtain it in solution. Finally, the antibiofilm activity will be evaluated, and a prototype atomizer will be designed for a possible practical application. Tools such as in silico design for the plasmid, restriction enzyme digestion, cell-free expression, and biofilm eradication assays will be used to validate this proposal.
Keywords: Dispersin B, biofilms, recombinant, S. aureus, P. aeruginosa, cell-free system.
Main methods/tools
Main methods/tools
Main methods/tools
Over the years it has been shown that biofilms are one of the main challenges in the field of health. These bacterial structures give microorganisms resistance to antibiotics and the immune system (Flemming et al., 2016), which makes them difficult to control. Although the risk of formation increases even more in the presence of medical equipment such as probes or catheters, which implies a significant clinical challenge, the presence of these structures prevails even in the simplest spaces such as surfaces in the different work areas. In medical settings such as Latin America, where resources can be limited, alternative biotechnology-based solutions represent promising mitigation strategies. For this reason, the recombinant production of dispersin B, an enzyme that degrades these biofilms and can be efficiently generated in a cell-free system, is proposed. This idea stems from the interest in merging clinical microbiology with synthetic biology to generate solutions to current problems.
Dispersin B (DspB) is a glycosyl hydrolase enzyme secreted by Aggregatibacter actinomycetemcomitans, which specifically degrades the polymer poly-β(1,6)-N-acetylglucosamine (PNAG), a key component of biofilms (Kaplan et al., 2003) formed by many pathogens. This makes it a tool with broad-spectrum action potential, as numerous studies have shown that dispersin B exhibits broad-spectrum antibiofilm activity against more than 25 bacterial species (Kaplan et al., 2024).
Despite its potential, the production of this enzyme still presents a significant gap in terms of scalability and efficiency, especially in non-cellular systems. The literature presents some cases in which its production was tested by exploring variants such as dispersin 624 and in which the results showed that it could modulate the spatial organization of cells within biofilms (Stacy et al., 2014). There have also been successful cases of its production in E. coli (Gökçen et al., 2013) and studies in which it was shown that recombinant expression of Dispersin B in Escherichia coli allowed the disruption of Staphylococcus epidermidis biofilms. In addition, recent research has highlighted the versatility of Dispersin B as an anti-biofilm agent, showing its ability to inhibit biofilm formation, disintegrate preformed biofilms, and increase the susceptibility of bacteria to antibiotics and other antimicrobial agents (Kapñan et al., 2024). However, little has been done to optimize this process in cell-free systems.
In recent years, protein production in cell-free systems has proven to be an efficient and flexible platform, especially useful when rapid, controlled, and cell contaminant-free expression is sought. Therefore, this project also proposes to develop a protocol that allows the recombinant production of Dispersin B for the control and prevention of biofilms such as those formed by Staphylococcus aureus and Pseudomonas aeruginosa. This would employ cell-free systems technology, combined with the use of optimized genetic elements to improve its performance, such as codon optimization. Unlike traditional strategies involving bacterial culture and purification, this approach reduces costs, accelerates production time, and avoids contamination risks, which is essential in hospital settings. In addition, it could facilitate decentralized local production, allowing rapid access in areas with limited infrastructure.
The idea for this project incorporates the construction of a plasmid using a novel design to achieve recombinant production of dspB. Thus, in addition to the optimization of the gene of interest, a number of key components such as the E. coli-specific ompA signal peptide have been added. An innovative alternative that has not been reported before and that allows the secretion of this enzyme in the supernatant and offers greater ease in the recovery processes compared to purification by chromatography.
This also makes it possible to dispense with costly techniques, since one of the visions of this project focuses on its accessibility. Additionally, the project seeks to take advantage of the advantages of cell-free systems to achieve production at scale to overcome the barriers subject to the use of traditional methods. In this way, this project not only aligns with existing technologies but also seeks to extend its applications to decentralized and novel solutions by making use of the emerging tools of synthetic biology.
Biofilm formation is a global problem that causes billions of dollars in annual losses, with the most affected industries being the healthcare and food industries. These are a recurrent contamination problem, with annual losses of up to 368.8 and 324 billion respectively (Cámara et al., 2022). These in turn are associated with persistent infections and the growing problem of bacterial antibiotic resistance. Therefore, this project proposes the development of an enzyme capable of dissolving the biofilm matrix and facilitating its elimination. Through its optimized process, it seeks to develop an accessible, portable, and economical biotechnological tool compared to the existing alternatives in the market. The focus of this project seeks to respond to the needs of Latin American countries, where monetary restrictions and the lack of technology transfer hinder access to innovative solutions. This protocol can also be performed locally, which will help to reduce the access gap to advanced antimicrobial solutions in the region. It is an initiative that promotes technological autonomy and sets a precedent for the scientific community in adjacent areas to seek to develop technology that aligns with their local realities and needs.
The scheme for the development of this type of tool would not be complete without considering its impact on ethics. In this case, the production of an enzyme intended to intervene and alter a biological process in an industrial and sanitary environment must be considered under a series of principles. First, the principle of non-maleficence is considered, since damage to the environment and human beings must be avoided when the product is released. This implies adequate handling and experimental validation before, during, and after the testing stages. The principle of justice is also considered since it is sought that this technology is subject to equitable access. And finally, the principle of transparency ensures an informed decision by consumers.
In the case of the first scenario, it is necessary to establish a series of containment and regulatory barriers to ensure that this is a process that does not generate damage. It is necessary to follow the guidelines established in the regulations of each country. In the case of Ecuador, this part is somewhat complex, because the Constitution remains somewhat ambiguous for technologies derived from genetic engineering. But even so, the necessary biosafety measures must be taken and the necessary experimental validation must be carried out before going to market. In addition, this could lead to talks with public institutions to establish a clearer legal framework for these technologies. About the second and third principles, the equal availability of this product should be regulated and ensured, and personnel who wish to acquire it and those responsible for supplying it should be informed and trained. Despite these measures, unintended consequences such as inappropriate use and release into the environment must be considered. This should be mitigated by production under controlled conditions and adequate training of personnel.