Team:Groningen:Project:Secretion

In order to exterminate the Pseudomonas aeruginosa and Staphylococcus aureus, the genetically engineered Lactococcus lactis NZ9800 needs to secrete a couple of infection preventing molecules. For P. aeruginosa these molecules will be Dispersin B and AHL lactonase, coded by the genes dspB and aiiA. For S. aureus these molecules will be Dispersin B and nisin coded by the genes dspB and nisA. We chose these enzymes to create a hostile environment for these pathogens. Whilst the effects of these molecules differ from each other, a regulated release of these proteins will cause the pathogens to lose their virulence and even kill some of them. Also, the regulated response against these pathogens will reduce the chance of P. aeruginosa and S. aureus to become resistant against these infection preventing molecules. Controlling the virulence with these molecules is key to a good treatment of infected burn wounds.

AiiA is probably an intracellular enzyme, which makes secreting it kind of hard. Also, it is not quite sure that Dispersin B is secreted and if so, it would have a signal peptide from its host. These are some problems which can sincerely hurt our secretion system. The solution is a signal peptide from the gene USP45 which is secreted out of Lactococcus lactis very efficiently. This became clear when scientist fused a couple of genes together with the signal coding sequence of USP45. This resulted in an efficient way of secreting the coded proteins into the media.⁴ So how are these enzymes secreted out of the cell when the spUSP45 is fused to the enzymes? Well, at first this coding sequence of ssUSP45 should be fused together with the genes. Then after translation, the coded enzyme will meet a chaperone protein , called SecB, which bring the secreting enzyme to a protein conducting gate. This gate cleaves the signal peptide and frees the enzyme from the cell.⁵ Therefore, we have chosen to fuse all our coding sequences from the enzymes with ssUSP45.

Bacteria use certain specific molecules to communicate with each other, also known as quorum sensing molecules. For Pseudomonas aeruginosa one of these molecules is the acylhomoserine lactone (AHL) molecule. This is important because communication between bacteria with these molecules can up-regulate virulence genes⁸, thereby worsening the infection.

AiiA is a molecule that can enzymatically degrade the AHL molecules and thereby inhibit the quorum sensing between the pathogens. The AiiA molecule is part of the lactonase group. These enzymes can cut the ring in the AHL molecules, thereby inactivating the quorum sensing molecules⁹. Although quorum sensing is inhibited, these molecules do not interfere with basal life processes such as general RNA and protein synthesis of the bacteria, and growth, therefore it is conceivable that the selective pressure for development of resistance is minimized. Inhibiting the quorum sensing between the pathogens has several benefits. One of these benefits is the down regulation of virulence genes. Another benefit is that AiiA will decrease the ability of Pseudomonas aeruginosa to form biofilms, making them more susceptible to conventional antibiotics and the action of the host immune system¹⁰. The inhibition of AHL molecules also disturbs some of the motility of Pseudomonas aeruginosa¹¹.

By secreting this molecule through our bandage, the Gram-negative pathogens can be battled. Although we can not kill the pathogens, they will become less virulent, less motile, decrease the ability to form biofilms an thus make them more susceptible to the human immune system.

In order to synchronize the secretion of infection preventing molecules with the detection of the certain pathogens, a few conditions must been set.

After designing the systems, two constructs came out: The anti-Pseudomonas aeruginosa system: The anti-Staphylococcus aureus secretion system:

Lactococcus lactis is capable of producing nisin, an antibiotic-like substance, called a bacteriocin. It is a natural antimicrobial agent with activity against a wide variety of Gram-positive bacteria, including food-borne pathogens such as Listeria, Staphylococcus and Clostridium by targeting the cell membrane⁶. The activity of nisin towards Gram-positive bacteria is based on binding of nisin to the lipidII molecule in the cell wall. In this way nisin can inhibit growth of Gram-positive cells in two ways. First it will inhibit cell wall synthesis by binding of the lipidII molecule in the membrane. Secondly, nisin molecules can span across the lipid bilayer, creating a pore in the cell wall which causes the cell to lyse. Both mechanisms are shown in figure 1.⁷

By first detecting Staphylococcus aureus, in order to induce the nisin production we will reduce the chance at nisin-resistance. Overall the risk at pathogens becoming resistant to nisin is very low, because lipidII is an essential cell wall protein.