Team:Groningen/Template/MODULE/Project/secretion

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Project > Secretion
 
 
 
The secreting system
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.
Signal peptide of USP45
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.
The anti-quorum sensing molecule
AiiA
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.
The construct
In order to synchronize the secretion of infection preventing molecules with the detection of the certain pathogens, a few conditions must been set.

  • The construct should be regulated through detection.
  • It must secrete DspB when L. lactis senses both P. aeruginosa and S. aureus
  • It must secrete Aiia when it detect P. aeruginosa
  • It must secrete Nisin when it detects S. aureus

After designing the systems, two constructs came out: The anti-Pseudomonas aeruginosa system:
Figure 6
 
Figure 6: The DspB and AiiA construct
 
 
The anti-Staphylococcus aureus secretion system:
Figure 7
 
Figure 7: The DspB and nisin construct
 
 
Nisin
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
Figure 3
 
Figure 3: Nisin binding to lipidII in the cell wall, causing pore formation
 
 
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.
 
 
 

 
 
 
 
Dispersin B
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 become 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 in their turn create their own biofilm.
Figure 4
 
Figure 4: The creation of the 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 catalyzes the hydrolysis of N-acetyl-D-glucosamine found in the biofilm. Thereby releasing certain bacteria, which under the right circumstances become very susceptible for antibiotics. Therefore, 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 40 kDa big. It hydrolyzes the β-1,6-glycosidic link between the acetylglucosamine polymers in the biofilm matrices. The active site contains 3 amino acids which have highly conserved acidic residues. The three amino acids are D183, E184 and E322. E184 act as a proton donator to the OR-site of the first carbon in the polymer. D183 assists the activation of the N-acetyl group, which gives of the electron to the first carbon of the polymer, so water can then hydrolyze the polymer. 3
Figure 5
 
Figure 5: The reaction between Dispersin B and the poly saccharides.
 
 
The problem with Pseudomonas aeruginosa and Staphylococcus aureus is that they also can produce a biofilm which can protect them against any antimicrobials. Because of the limitations on obtaining a gene from a ML-II bacteria, we decided to synthesize this gene, with the ssUSP45 and 6xhistidine tag already attached. We decide to attach the HIS tag because of some logistic reasons concerning PCR and Gibson assembly.
The construct
In order to synchronize the secretion of infection preventing molecules with the detection of the certain pathogens, a few conditions must been set.

  • The construct should be regulated through detection.
  • It must secrete DspB when L. lactis senses both P. aeruginosa and S. aureus
  • It must secrete Aiia when it detect P. aeruginosa
  • It must secrete Nisin when it detects S. aureus

After designing the systems, two constructs came out: The anti-Pseudomonas aeruginosa system:
Figure 6
 
Figure 6: The DspB and AiiA construct
 
 
The anti-Staphylococcus aureus secretion system:
Figure 7
 
Figure 7: The DspB and nisin construct
 
 
References
 
1. Bjarnsholt, T. (2013) The role of bacterial biofilms in chronic infections. APMIS 121: 1-58
 
2. Davis, D. (2007) Looking for chinks in the armor of bacterial biofilms. PLoS Biology 5
 
3.Mark, B.L. et al. (2001) Crystallographic evidence for substrate-assisted catalysis in a bacterial beta-hexosaminidase. J. Biol. Chem. 276: 10330–10337
 
4. Asseldonk, M., De Vos, W.M., and Simons, G. (1993) Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous a-amylase. Mol. Gen. Genet. 240: 428-434.
 
5. Natale, P. et al. (2008) Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane— Distinct translocases and mechanism. Bioch. Bioph. Acta 1778:1735–1756
 
6. Todar, K. Lactococcus lactis: nominated as the Wisconsin State Microbe. Online textbook of microbiology, UW Department of Bacteriology
 
7. Wiedemann, I. (2001) Specific binding of nisin to the peptidoglycan precursor Lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. J. Biol. Chem. 276: 1772-1779
 
8. Dong, Y.H. et al. (2000) AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. PNAS 97: 3526-3531
 
9. Kim, M.H. et al. (2005) The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase. PNAS 102
 
10. Rasmussen, T.B. and Givskov, M. (2006) Quorum sensing inhibitors: a bargain of effects. Microbiol. 152: 895-904
 
11. Reimmann, C. et al. (2002) Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1. Microbiol. 148: 923-932