Team:Groningen/Template/MODULE/Project/secretion/part2

Certain pathogens protect themselves by producing a extracellular polymeric substance. The bacteria encapsulate themselves with exopolysaccharides which can adhere to other cells with these extracellular “sugars”. The encapsulated pathogen can also become resistant against any type of antibiotics. If a patient becomes infected with these pathogens and the pathogens get the chance to adhere themselves to a surface, curing these infections can become very difficult. But before we try to solve this problem, how do biofilms actually form? Well, first certain individual bacteria must adhere to a surface and form micro colonies. These will be the basis of the biofilm. Then motile bacteria will try to migrate to these colonies and adhere themselves on top of the colonies. after a couple of cycles, these bacteria will have formed a mushroom kind of structure, which can release bacteria individuals. These individuals will in their turn create their own biofilm.

so we had to think about a strategy to battle these structures. After a lot of research, Dispersin B came to our minds. The enzyme is commonly used by Aggregatibacter actinomycetemcomitans, which is a periodontal pathogen. Dispersin B itself catalyses the hydrolysis of N-acetyl-D-glucosamine found in the biofilm. Therefor releasing certain bacteria, and which under the right circumstances become very susceptible for antibiotics. Therefor there has been a gigantic market opportunity for this enzyme as detergents or medicine.The enzyme is a 361 amino acids counting protein and is 40kDA big. It hydrolyses the β-1, 6-glycosidic link between the acetyl glucosamine polymers in the biofilm matrices. The active site contains 3 amino acids which have highly conserved acidic residues. 3 amino acids are D183, E184 and E322. E184 act as a Proton donator to the OR-site of the 1st carbon in the polymer. D183 assist the activation of the N-acetyl group, which gives of the electron to the 1st carbon of the polymer, then water can hydrolyse the polymer. ³

The problem with Pseudomonas aeruginosa and Staphylococcus aureus is that they also can produce a biofilm which can protect them against any antimicrobial. Because of the limitations on obtaining a gene from a ML-II bacteria, we decided to synthesize this gene, with the ssUSP45 and 6 times Histidine tag already attached. We decide to attach the HIS tag because of some logistic reasons concerning PCR and Gibson assembly.