PROJECT

1.BACKGROUND ANG GOALS

Our project is to construct a biological device that can be used to detect the existence of Streptococcus Pyogenes (S. Pyogenes) in a cheaper and faster way in comparison to currently used techniques. The construct can be used as a detecting device for bacteria or virus secreting protease by modifying the cleavage sequence in the array (for more information see parts). The name of the project comes from ‘ "Diagnosing S. Pyogenes".

Streptococcus Pyogenes, also known as group A streptococci (GAS), is a Gram-positive pathogen responsible for a wider variety of human disease than any other bacterial species including pharyngitis (streptococcal sore throat), scarlet fever, impetigo, erysipelas, cellulitis, septicemia, toxic shock syndrome, necrotizing fasciitis (flesh-eating disease) and the sequelae, rheumatic fever and acute glomerulonephritis.(1)  The complications of current GAS infections are severe; bacteremia associated with aggressive soft tissue infection, shock, adult respiratory distress syndrome and renal failure are common; 30% to 70% of patients die in spite of aggressive modern treatments.(2) Patients with symptomatic pharyngitis rarely develop streptococcal toxic shock syndrome, though such cases have been reported, especially in the last year. Numerous cases have developed within 24 to 72 hours of minor nonpenetrating trauma, resulting in hematoma, deep bruise to the calf, or even muscle strain.(3)

There are currently several test methods to detect the existence of S. Pyogenes. TheStrep A Rapid Test Device (SARTD) is considered to be fastest one with detecting antigen in 5 minutes with the accuracy of 72%.(4) However SARTD is an expensive device that most health institutions have difficulty affording. Therefore blood agar plate culture is prepared which requires a long time interval (one to two days) to show the results.(4) Testing on the same day is important to reduce unnecessary antibiotic use and to prevent possible complications caused by S. Pyogenes. In our project we aimed to shorten the amount of time needed to detect S. Pyogenes while making the higher speed test more affordable.

3D computer-generated image of a Streptococcus Pyogenes. Content Provider(s): Center for Disease  Control and Prevention/ Melissa Brower

 

2.PARTS

-OmpA-SpeB_Cleavage_Site

 

 

We accomplish the task of detecting the existence of S. Pyogenes with the base part OmpA-SpeB_Cleavage_Site.Research was conducted to discover the proteins secreted by Streptococcus Pyogenes; results showed that SpeB is one of the main virulence factor of S. Pyogenes, which is a cysteine proteinase functioning protein secreted by the bacteria.(5) After further investigation on the mechanism of SpeB, we discovered that the amino acid sequence is cleaved by SpeB.(6) If there is S. Pyogenes in the medium, SpeBs secreted by S. Pyogenes split the amino acid sequence including SpeB cleavage site, into two. By using this for our benefit, construct consisting of a cell wall protein, linker sequence and SpeB cleavage site is designed. This is the base part designed for S. Pyogenes detecting devices. To the end of this part, the protein, which will be used to show that the part is cleaved, should be added. After the SpeB cleavage site is cleaved, the amino acid sequence placed after the cleavage site can become free and start action. (see part OmpA-SpeB_Cleavage_Site-xylE).
The cell wall protein keeps the construct on the cell wall, which helps to prevent unnecessary transmission between the protein coming after the SpeB cleavage site and the protein’s reactants, if designed. The cell wall protein OmpA is preferred, due to its abundant usage.(7) The linker segregates the OmpA and SpeB cleavage site and thus creating space for the SpeB to access the cleavage site; the sequence of the linker is taken from the Imperial College 2010 project.(8) The SpeB cleavage site is broken down in the existence of the SpeB and splits the amino acids, thus unchaining the substance from the cell wall.

 

-OmpA-SpeB_Cleavage_Site-xyIE

 

 

This part is constructed upon the base part OmpA-SpeB_Cleavage_Site by adding Catechol 2,3-dioxygenase to the end of the sequence. xylE is the gene encoding the enzyme catechol-2,3-dioxygenase, which converts catechol, a cheap colorless substance, to the bright yellow product 2-hydroxy-cis,cis-muconic semialdehyde, if provided with oxygen. The sequence of xylE is taken from the partsregistry.(9) In the existence of S. Pyogenes, SpeB secreted by the bacteria splits the amino acid sequence from the cleavage site, monomers of catechol-2,3-dioxygenase become liberated. Free monomers come together and form the tetramer form to start activation. By using this part, detecting organisms will be a lot easier.

 

Image: OmpA-SpeB_Cleavage_Site-xylE

 

List of our parts

 

3.KINETIC MODELLING

Cascade reactions: By using the SimBiology toolbox for MATLAB we created a diagram of the enzyme kinetic modelling(Figure 1).

Figure1: Diagram of the reactions involved in converting the Cathecol monomer to tetramer and degredation of cathecol. (Yellow colour is indicator of the reaction.)This was created in the SimBiology toolbox for MATLAB.

 

 

Table1: Mathematical representation of enzyme kinetic reactions.

 

Table 2: Values assigned to kinetic parameters described in Table 1. All reacrtion are irreversible

 

 

We have three reactions that are linked. According to our literature researches reactant and product balance in the reaction is as shown. In practice SpeB is activated by 2-Mercaptoethanol (reducing agent), which may blur our understanding in theory. However, our results showed that the uncertainty is not exceedingly high.

4.VISUAL MODELLING

In the visual modelling below 3D molecules of SpeB and tetramer of Catechol-2,3-dioxygenase are depicted. SpeB, is the protease secreted by S. Pyogenes In our design SpeB cleaves the SpeB cleavage site in between  Catechol-2,3-dioxygenase and the linker, which is tied to the cell wall protein OmpA. When Catechol-2,3-dioxygenases is liberated from the bacteria, they become together and forms the tetramer, which is the enzyme required for the reaction between catechol and oxygen. As a result of this reaction yellow color is produced. If there is S. Pyogenes in the medium, yellow color is produced. Hopefully, with our project we will be able to detect the bacteria cheaper and faster. 

 

5.CONCLUSION AND FUTURE PLANS

In the end, we hope to produce a working OmpA-SpeB_Cleavage_Site-xylE construct along with our base part OmpA-SpeB_Cleavage_Site. If we manage to show that the SpeB cleavage part is working, we can easily detect S. Pyogenes. It is our hope that our construct will be able to reduce the cost of rapid detecting systems and allow fast detections. In the future, researches can use the benefits of our system to develop more effective and faster methods for detection, helping patients to have more comfortable treatment.

 

As it can be seen from the results, we tested our construct with SpeB, the enzyme secreted by S. Pyogenes. However when the other proteins secreted by S. Pyogenes and other conditions are took into consideration, outcome may differ from our results. On the other hand, we are not allowed to conduct our researches with viruses due to the safety regulations of the lab that we use. Therefore in the future, we are going to test our construct with real S. Pyogenes bacteria. We are also making our plans on other detection modules instead of Catechol-2,3-dioxygenase to come up with the one, which is the most accurate. For instance one of the enzyme that we will use is Glucose oxidase. It catalyses the oxidation of glucose to hydrogen peroxide and D-glucono-δ- lactone. As a result of this reaction color change is observed. Hopefully we will be able to solve the problems of patients!

6.REFERENCES

1. Ferretti, Joseph J. "Complete Genome Sequence of an M1 Strain of Streptococcus Pyogenes." PNAS 98.8 (2001): 4658-663. Print.
2. Stevens DL, Tanner MH, Winship J, Swarts R, Reis KM, Schlievert PM, et al. Reappearance of scarlet fever toxin A among streptococci in the Rocky Mountain West: severe group A streptococcal infections associated with a toxic shock-like syndrome. N Engl J Med 1989; 321:1-7.
3. Dennis L. Stevens, Ph.D, M.D. "Streptococcal Toxic-Shock Syndrome: Spectrum of Disease, Pathogenesis, and New Concepts in Treatment." Emerging Infectious Diseases 1.3 (1995): 69-78. Print.
4. Forward, Kevin R., David Haldane, Duncan Webster, Carolyn Mills, and Diane Aylward. "A Comparison between the Strep A Rapid Test Device and Conventional Culture for the Diagnosis of Streptococcal Pharyngitis." Can J Infect Dis Med Microbiol 17.4 (2006): 221-23. Print.
5. Nelson, DC, J. Garbe, and M. Collin. "Cysteine Proteinase SpeB from Streptococcus Pyogenes - a Potent Modifier of Immunologically Important Host and Bacterial Proteins." (2011): n. pag. PubMed. Web. 12 June 2014.
6. Ender, Miriam, Federica Andreoni, Annelies Sophie Zinkernagel, and Reto Andreas Schuepbach. "Streptococcal SpeB Cleaved PAR-1 Suppresses ERK Phosphorylation and Blunts Thrombin-Induced Platelet Aggregation." (2013): n. pag. PLOS. Web. 12 June 2014.
7. "P0A910 (OMPA_ECOLI)." (2014): n. pag. UniProt. Web. 12 June 2014.

8. Imperial Collage London. "Parasight." IGEM. N.p, 2010. Web.12 June 2014.

 

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