Engineering bacteria to disperse bacterial biofilms

Abstract

Biofilms are aggregates of bacteria embedded in a self-produced matrix. In nature most bacteria exist in the form of biofilms. They are exceptionally resistant to environmental stress. They can survive harsh conditions such as starvation and desiccation, and can withstand conventional antimicrobial agents. Bacteria in the form of biofilms cause many diseases in humans, animals and plants. Formation of biofilms leads to corrosion of pipelines incurring huge losses in industries. The major focus of the research in microbiology is now shifting to biofilms. Researchers are exploring various strategies to combat biofilms. Among different methods of combating biofilms, biological methods offer some advantages over other methods. The biological methods make use of the existing signaling pathways in bacteria and bring about efficient disruption of the target biofilm without having to add any artificially synthesized compound. These methods are particularly attractive in treating biofilm-induced diseases. In this project I have proposed three different strategies of engineering Escherichia coli to disrupt the biofilms of E. coli and Staphylococcus epidermidis. The anti-biofilm agent used in all the strategies is Dispersin B, an enzyme that hydrolyzes poly-N-acetyl glucosamine found in the matrix of the target biofilms. Hydrolysis of this polymer leads to disruption of the biofilm. The first strategy makes use of the N-acetyl glucosamine signaling and chemotaxis pathway of E. coli to detect the target biofilm and to synthesize Dispersin B. The product of the action of Dispersin B is N-acetyl glucosamine, which acts as an inducer to elevate the synthesis of Dispersin B. The second strategy exploits the biofilm-specific pattern of gene expression in E. coli biofilms. The engineered bacterium expresses Dispersin B from the promoter of a gene that is multi-fold activated when the bacterium acquires the biofilm lifestyle. It incorporates itself in the target biofilm and disrupts the biofilm through the action of Dispersin B. The third strategy is specifically aimed at disrupting S. epidermidis biofilms. It uses the agr quorum-sensing system of S. epidermidis to detect the target biofilm and to synthesize Dispersin B. The strategies proposed in this project have been partially successful. I have demonstrated that E. coli can be engineered to express and secrete Dispersin B, which can disrupt the target biofilm efficiently. The strategies proposed in this project are versatile in application. They can be modified to engineer similar biological systems against biofilms of other species of bacteria. In the course of accomplishing the objective of this project I have explored some unknown aspects of the N-acetyl glucosamine signaling in E. coli. The analysis of the transcriptome of E. coli biofilms has revealed the up regulation of redox stress-associated genes in the attached cells of E. coli biofilms. These interesting findings can lead us to explore further into bacterial signaling and biofilm formation.