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PROJECT

.DESCRIPTION

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

.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 thegene 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.

.MATH MODELLING

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 thegene 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.

.VISUAL MODELLING

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 thegene 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.

.RESULTS

.CONCLUSION

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.

.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. <http://www.ncbi.nlm.nih.gov/pubmed/22050223>.
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 College London. "Parasight." IGEM. N.p., 2010. Web. 12 June 2014. <https://2010.igem.org/Team:Imperial_College_London/Parts>.

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