Team:Estonia TUIT/Description



Caries is one of the most widespread dental diseases in the world, affecting permanent teeth of around 2.3 billion people (WHO). Some food particles remain on the tooth surface and between the teeth after eating, creating a suitable environment for bacterial growth, especially when consuming sugar-containing food (Dental Caries: Symptoms Causes and Treatment of Tooth Caries| Oral-B India). Without good oral hygiene and proper tooth brushing, bacteria can attach to the tooth surface and use food remains as a growth substrate. As a result of microbial metabolism, different organic acids are produced. They might damage hard tooth tissues and cause their degradation. These processes lead to caries -- tooth decay (Young et al., 2015).

The most common bacterial species that causes caries in humans is Streptococcus mutans . This bacteria binds to a specific protein called SALSA (The Salivary Scavenger and Agglutinin) on the tooth surface. This interaction helps S. mutans to attach to the tooth surface and form a biofilm (Nakai et al., 1993).

SALSA description and structure

SALSA protein is an innate immune system protein that has diverse functions. For instance, SALSA regulates inflammation and tissue repair (Lee et al., 2019). Upon pathogen invasion, SALSA is secreted from mucosal surfaces and regulates the activation of the complement system (Gunput et al., 2016a; Reichhardt et al., 2014). Complement is a group of innate immune proteins (Gunput et al., 2016b) responsible for enhancing antibody and phagocytic cell function (Mortensen et al., 2017a) through binding directly to mannose-binding lectin (a pattern recognition molecule that activates the complement system by means of attaching to foreign invaders) (Turner, 2003). Besides that, SALSA also binds and agglutinates the cariogenic bacterium S. mutans (Prakobphol et al., 2000). SRCRP2 is the only SRCR peptide found to bind a number of bacteria, including S. mutans (Bikker et al., 2002).

In humans, SALSA consists of three protein domains and is structured the following way: a stretch of 13 highly conserved scavenger receptor cysteine-rich (SRCR) domains, separated by SRCR interspersed domains (SIDs), followed by two C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein-1 (CUB) domains, the 14th SRCR domain and a zona pellucida (ZP) domain in the C-terminus (Reichhardt & Meri, 2016) (Figure 1). The SRCR and CUB domains are involved in ligand binding, the ZP domain functions in protein polymerization (Lee et al., 2019). The attachment of SALSA to teeth is mediated via the SRCR domains (Bikker et al., 2002).

Figure 1. Scheme illustrating the domain structure of SALSA protein. SRCR domain - Scavenger Receptor Cysteine-Rich (SRCR) domain. SID domain - SRCR-interspersed domain. CUB domain - C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein-1 domain. ZP domain - Zona Pellucida domain.

After examining the SALSA structure, we came up with an idea to eliminate the possibility for cariogenic bacteria to use SALSA as an anchor for the attachment to teeth. For this purpose, we aimed to use a protease that will cleave the SALSA protein, leaving no sites for the bacteria to bind on the tooth surface.


Proteases are a class of degradative enzymes that break proteins into small peptides or single amino acids. They catalyze the reaction of peptide bond hydrolysis between two amino acids in the protein (Razzaq et al., 2019). Proteases are classified as exo- and endonucleases by the place where they cleave the protein. Exonucleases cleave at the terminus of a protein, while endonucleases potentially cleave peptide bonds along the whole length of the protein (Laskar & Chatterjee, 2009). Another classification of proteases is based on their catalytic mechanism. This classification is determined by the amino acid residue in the catalytic center (e.g., serine proteases, aspartate proteases, etc.).

We studied research literature to determine potential proteases capable of cleaving the SALSA protein. We aimed to choose the protease with the highest specificity for the SALSA protein and the lowest toxicity. For our experiments, we selected trypsin and prolyl peptidase. It was shown that the residues in the C-terminus of the Streptococcus antigen I/II are the primary place that binds the salivary agglutinins. To increase the affinity of our target proteases, we used a part of the adhesin sequence of S. mutans as a SALSA-binding sequence (Kelly et al., 1999). Additionally, we plan to implement error-prone PCR for creating a library of proteases with mutations that alter the protease specificity.

Target site for cleavage

We aimed to find amino acid sequences in SALSA protein that potentially can be targeted by proteases. Moreover, these sites should be accessible for proteases (Bikker et al., 2002). SRCR domains are involved in ligand binding (bacteria binding) and were considered to be protease targets. Nonetheless, according to the three-dimensional structure of the SALSA protein, the SRCR domains are hidden in the core of the globule and are not easily accessible to proteases (Figure 2). The linkers, or SRCR-interspersed domains (SIDs), however, are located on the protein surface and can be easily cleaved. SIDs are roughly 20-amino-acid-long threonine-serine-proline-rich stretches consisting of a number of glycosylation sites that proposedly render the conformation of the linkers extended to 7 nm (Reichhardt et al., 2020; Turenchalk & Xu, 2001).

Figure 2. The structural model of SALSA, predicted by AlphaFold. SRCR - Scavenger Receptor Cysteine-Rich (SRCR) domains. SID - SRCR-interspersed domains. CUB - C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein-1 domain.

Candidate proteases

We have used three proteases for our experiments: TEV protease, trypsin, and prolyl peptidase. TEV (Tobacco Etch Virus) protease is a highly sequence-specific cysteine protease. TEV protease cleaves protein substrates between Gln and Gly/Ser residues. Optimal activity of this protease can be observed in the pH range from 6.0 to 9.0 (Nam et al., 2020).

Since TEV is highly specific and well-studied, we have selected it as our model and control enzyme. As a proof-of-principle, we introduced a 6-nucleotide-sequence specific for TEV cleavage in the middle of the SID sequence embedded in the testing system so as to ensure successful cleavage of the linker. If the cleavage does not occur, we are notified that the problem lies in the system design but not in the inability of the protease to cut specific amino acid sequence. This approach enables us to determine whether our test system meets our purposes (RB et al., 2002).

Trypsin is a pancreatic serine protease that participates in digesting food proteins and other processes occurring in living organisms. This medium-sized globular protein is produced as a zymogen, trypsinogen, an inactive precursor of the enzyme. It can be activated by the removal of a terminal hexapeptide to yield single-chain β-trypsin, which by autolysis yields α-trypsin. Trypsin cleaves peptide bonds from the C-terminal side of positively charged lysine and arginine residues (Trypsin - Worthington Enzyme Manual).

Prolyl peptidase is a large enzyme that belongs to a class of serine peptidases. It is widespread in mammals, bacteria, fungi, and plants. Prolyl peptidase functions in immune and inflammatory responses and nutrient digestion. Prolyl peptidase cleaves C-terminally of prolyl residues, and its optimal pH is 4-5 (Shan et al., 2005). In the human organism, prolyl peptidase is believed to be responsible for the maturation and degradation of peptide hormones and neuropeptides. The reason behind choosing prolyl peptidase for our project is its abundance, low price, and the absence of harmful effects to human cells. Additionally, target site for cleavage in the SALSA protein is proline-rich, which makes prolyl peptidase the first candidate for our project (Shan et al., 2005).

Some of our selected proteases cause mild side effects or even may have detrimental effects on the cells when used as medical treatments (Huijghebaert et al., 2021). In the case of trypsin, these issues can be overcome by using a neutralizer alpha-1 antitrypsin immediately after using the protease. Alpha-1 antitrypsin is a human protein that accumulates in human serum and it accounts for more than 90% of antiprotease activity in serum (Serres & Blanco, 2014).

Development of a method to engineer specific proteases

As there are no known proteases that specifically cleave SALSA, we looked at possible options to engineer proteases with distinct specificity. Driven by this, we set up a novel method that allows high-throughput screening of proteases based on their specificity for a desired target sequence. The screen is carried out in yeast, where the protease cleavage efficiency is translated into a growth rate as a simple readout. This allows pooled screening of libraries of either different proteases or mutated versions of a protease. This system includes three transcriptional units integrated into the yeast genome (Figure 3):

1. A protease
2. An inhibitor of a cell cycle progression (sic1ΔN)
3. SRCR-SID domain (protease substrate) flanked by activation and binding domain of the transcription factor.

Figure 3. Transcriptional units in the yeast protease activity assay. TU1 - Transcriptional Unit 1 consists of sic1ΔN under the control of LexA binding promoter. TU2 - Transcriptional Unit 2 includes LexA binding domain (LexA BD) and activation VP16 domain separated by SRCR-SID linker from SALSA protein (under control of GAL1 promoter). TU3 - transcriptional unit 3 consists of protease fused to ligand facilitating protease binding to SALSA (under control of GAL1 promoter).

The protease gene is fused to a ligand that binds SRCR (Bikker et al., 2002). Once bound, the protease cuts the SID that causes dissociation of transcription factor’s activation and binding domains (Figure 4), normal cell growth serves as an indicator of the high proteolytic activity of the protease.

Figure 4. Schematic representation of the assay developed to assess protease ability to cut the target sequence. sic1ΔN (an inhibitor of cell cycle progression) expression depends on the activation by a transcription factor that consists of two domains: LexA binding domain and VP16 activation domain that are bound by the linker to be cut by the protease. If the protease is unable to cleave the peptide, the transcription factor initiates sic1∆N expression that leads to cell cycle arrest, stopping yeast growth. In case of efficient cleavage, the two domains of the transcription factor dissociate, and sic1∆N expression does not occur, allowing normal cell growth. In the case of non-efficient cutting, sic1∆N expression is low, resulting in slow yeast growth.

In our project, we aim to use this approach to improve the specificity of native proteases towards SALSA. In addition, we fuse the protease with a peptide from S. mutans , the most abundant cariogenic bacteria, that binds strongly to SALSA. This leads to increased local concentration, promoting specific targeting of SALSA.

Almitairi, J. O. M., Venkatraman Girija, U., Furze, C. M., Simpson-Gray, X., Badakshi, F., Marshall, J. E., Schwaeble, W. J., Mitchell, D. A., Moody, P. C. E., & Wallis, R. (2018a). Structure of the C1r–C1s interaction of the C1 complex of complement activation. Proceedings of the National Academy of Sciences, 115(4), 768.

Almitairi, J. O. M., Venkatraman Girija, U., Furze, C. M., Simpson-Gray, X., Badakshi, F., Marshall, J. E., Schwaeble, W. J., Mitchell, D. A., Moody, P. C. E., & Wallis, R. (2018b). Structure of the C1r–C1s interaction of the C1 complex of complement activation. Proceedings of the National Academy of Sciences, 115(4), 768.

Bikker, F. J., Ligtenberg, A. J. M., Nazmi, K., Veerman, E. C. I., van’t Hof, W., Bolscher, J. G. M., Poustka, A., Amerongen, A. V. N., & Mollenhauer, J. (2002). Identification of the Bacteria-binding Peptide Domain on Salivary Agglutinin (gp-340/DMBT1), a Member of the Scavenger Receptor Cysteine-rich Superfamily*. Journal of Biological Chemistry, 277(35), 32109–32115.

Dental Caries: Symptoms Causes and Treatment of Tooth Caries| Oral-B India. (n.d.). Retrieved October 16, 2021, from

Gunput, S. T. G., Wouters, D., Nazmi, K., Cukkemane, N., Brouwer, M., Veerman, E. C. I., & Ligtenberg, A. J. M. (2016a). Salivary agglutinin is the major component in human saliva that modulates the lectin pathway of the complement system. Innate Immunity, 22(4), 257–265.

Gunput, S. T. G., Wouters, D., Nazmi, K., Cukkemane, N., Brouwer, M., Veerman, E. C. I., & Ligtenberg, A. J. M. (2016b). Salivary agglutinin is the major component in human saliva that modulates the lectin pathway of the complement system. Innate Immunity, 22(4), 257–265.

Huijghebaert, S., Vanham, G., Winckel, M. van, & Allegaert, K. (2021). Does Trypsin Oral Spray (Viruprotect®/ColdZyme®) Protect against COVID-19 and Common Colds or Induce Mutation? Caveats in Medical Device Regulations in the European Union. International Journal of Environmental Research and Public Health, 18(10).

Kelly, C. G., Younson, J. S., Hikmat, B. Y., Todryk, S. M., Czisch, M., Haris, P. I., Flindall, I. R., Newby, C., Mallet, A. I., Ma, J. K.-C., & Lehner, T. (1999). A synthetic peptide adhesion epitope as a novel antimicrobial agent. Nature Biotechnology, 17(1), 42–47.

Laskar, A., & Chatterjee, A. (2009). Protease – Revisting the Types and Potential. Online Journal of Biotechnology Research (OJBR), 1(1), 55–61.

Lee, G. K. C., Tessier, L., & Bienzle, D. (2019). Salivary Scavenger and Agglutinin (SALSA) Is Expressed in Mucosal Epithelial Cells and Decreased in Bronchial Epithelium of Asthmatic Horses. Frontiers in Veterinary Science, 6, 418.

Mortensen, S. A., Sander, B., Jensen, R. K., Pedersen, J. S., Golas, M. M., Jensenius, J. C., Hansen, A. G., Thiel, S., & Andersen, G. R. (2017a). Structure and activation of C1, the complex initiating the classical pathway of the complement cascade. Proceedings of the National Academy of Sciences of the United States of America, 114(5), 986–991.

Mortensen, S. A., Sander, B., Jensen, R. K., Pedersen, J. S., Golas, M. M., Jensenius, J. C., Hansen, A. G., Thiel, S., & Andersen, G. R. (2017b). Structure and activation of C1, the complex initiating the classical pathway of the complement cascade. Proceedings of the National Academy of Sciences, 114(5), 986.

Nakai, M., Okahashi, N., Ohta, H., & Koga, T. (1993). Saliva-binding region of Streptococcus mutans surface protein antigen. Infection and Immunity, 61(10), 4344–4349.

Nam, H., Hwang, B. J., Choi, D., Shin, S., & Choi, M. (2020). Tobacco etch virus (TEV) protease with multiple mutations to improve solubility and reduce self‐cleavage exhibits enhanced enzymatic activity. FEBS Open Bio, 10(4), 619.

Prakobphol, A., Xu, F., Hoang, V. M., Larsson, T., Bergstrom, J., Johansson, I., Frängsmyr, L., Holmskov, U., Leffler, H., Nilsson, C., Borén, T., Wright, J. R., Strömberg, N., & Fisher, S. J. (2000). Salivary Agglutinin, Which Binds Streptococcus mutans and Helicobacter pylori, Is the Lung Scavenger Receptor Cysteine-rich Protein gp-340*. Journal of Biological Chemistry, 275(51), 39860–39866.

Razzaq, A., Shamsi, S., Ali, A., Ali, Q., Sajjad, M., Malik, A., & Ashraf, M. (2019). Microbial Proteases Applications. Frontiers in Bioengineering and Biotechnology, 7, 110.

RB, K., J, T., TD, C., & DS, W. (2002). The P1’ specificity of tobacco etch virus protease. Biochemical and Biophysical Research Communications, 294(5), 949–955.

Reichhardt, M. P., Jarva, H., de Been, M., Rodriguez, J. M., Jimenez Quintana, E., Loimaranta, V., Meindert de Vos, W., & Meri, S. (2014). The Salivary Scavenger and Agglutinin in Early Life: Diverse Roles in Amniotic Fluid and in the Infant Intestine. The Journal of Immunology, 193(10), 5240.

Reichhardt, M. P., Loimaranta, V., Lea, S. M., & Johnson, S. (2020). Structures of SALSA/DMBT1 SRCR domains reveal the conserved ligand-binding mechanism of the ancient SRCR fold. Life Science Alliance, 3(4).

Reichhardt, M. P., & Meri, S. (2016). SALSA: A Regulator of the Early Steps of Complement Activation on Mucosal Surfaces. Frontiers in Immunology, 7, 85.

Serres, F. de, & Blanco, I. (2014). Role of alpha-1 antitrypsin in human health and disease. Journal of Internal Medicine, 276(4), 311–335. Shan, L., Mathews, I. I., & Khosla, C. (2005). Structural and mechanistic analysis of two prolyl endopeptidases: Role of interdomain dynamics in catalysis and specificity. PNAS March, 8(10), 2021.

Trypsin - Worthington Enzyme Manual. (n.d.). Retrieved October 16, 2021, from

Turenchalk, G. S., & Xu, T. (2001). Lats in Cell-cycle Regulation and Tumorigenesis BT - Encyclopedic Reference of Cancer. Encyclopedic Reference of Cancer, 491–496.

Turner, M. W. (2003). The role of mannose-binding lectin in health and disease. Molecular Immunology, 40(7), 423–429.

Young, D. A., Nový, B. B., Zeller, G. G., Hale, R., Hart, T. C., Truelove, E. L., Ekstrand, K. R., Featherstone, J. D. B., Fontana, M., Ismail, A., Kuehne, J., Longbottom, C., Pitts, N., Sarrett, D. C., Wright, T., Mark, A. M., & Beltran-Aguilar, E. (2015). The American Dental Association Caries Classification System for Clinical Practice: A report of the American Dental Association Council on Scientific Affairs. The Journal of the American Dental Association, 146(2), 79–86.

Zhang, X.-L., & Ali, M. A. M. (2008). Ficolins: Structure, Function and Associated Diseases. In J. D. Lambris (Ed.), Current Topics in Complement II (pp. 99–109). Springer US.

Poor dental health is a massive issue in today's society (Dye and Thornton-Evans, 2010; HEALTH, 2014; Mackenzie and Sanjai). According to the World Health Organization, around 2.3 billion people suffer from tooth decay, a health issue that is four times more common than asthma among adolescents (CDC, 2020). One of the most frequent diagnoses of people going to the dentist is dental caries. One could describe caries as the breakdown of the tooth enamel that is the result of bacteria producing acid in the process of food decomposition (CDC, 2020). Starting from the enamel and spreading deep into the dentin, this process can lead to purulent inflammation and damage the pulp (neurovascular bundle), bone tissue, and periosteum. If not treated properly, this often results in tooth loss.

With the rising costs of dental services, It is our responsibility to ensure the cleanliness of our teeth to prevent ourselves from getting caries. In order to avoid cavities, some standard and easy preventative measures against the formation of plaque are carried out; however, they have limited efficacy . The most common methods for teeth protection are: reducing the frequency of consuming sugary and starchy foods, oral hygiene, fluoride application, pit and fissure sealants (Stop Caries NOW for a Cavity-Free Future). However, teeth brushing does not ensure full protection from plaque. Moreover, it can contribute to enamel degradation as over brushing is a common cause of enamel loss (Attin and Hornecker, 2005; Mackenzie and Sanjai). Modern medicine offers many drugs that strengthen teeth enamel: Fluoride varnish, Belagel, Remodent, and others (Baehni and Guggenheim, 1996; Periodontitis; Stop Caries NOW for a Cavity-Free Future). However, these medications are used only under the supervision of a specialist and are not suitable for routine use.

In order to diminish the occurrence of cavities and increase accessible oral hygiene, our team has uncovered an outstanding and innovative solution to the global dental health concern. Scientists from the University of Tartu’s iGem team have studied a protein called SALSA that is attached to the tooth surface and acts as an anchor for bacteria. By binding SALSA protein, bacteria accumulate on the teeth' surface and create an acidic environment, causing cavities. We have developed a genetically engineered enzyme - a specific protease that cleaves SALSA and releases it from the tooth surface alongside the harmful bacteria attached to it, decreasing the chance of caries.

Our product, which could be in the form of a mouthwash, spray, or chewing gum, is a powerful asset in avoiding cavities and your shortcut to healthier teeth.

Attin, T., and Hornecker, E. (2005). Tooth brushing and oral health: how frequently and when should tooth brushing be performed? Oral Health Prev. Dent. 3, 135–140.

Baehni, P.C., and Guggenheim, B. Potential of diagnostic microbiology for treatment and prognosis of dental caries and periodontal diseases.

CareCredit Dental Costs & Prices | CareCredit.

CDC (2020). Hygiene-related Diseases | Hygiene-related Diseases | Hygiene | Healthy Water | CDC. Cent. Dis. Control Prev.

Dye, B.A., and Thornton-Evans, G. (2010). Trends in oral health by poverty status as measured by Healthy People 2010 objectives. Public Health Rep. 125, 817–830.

HEALTH, S.O.O. (2014). Maintaining and Improving the Oral Health of Young Children. Pediatrics 134, 1224–1229.

Mackenzie, N.G., and Sanjai, S. Dental Myths, Debunked | Everyday Health.

Periodontitis Periodontitis - Dental Disorders - MSD Manual Professional Edition.

Stop Caries NOW for a Cavity-Free Future What is Caries? - The Alliance for a Cavity-Free Future.

Contact Us: