Combating cariogenesis with aptamers
If hearing a drill gives you goosebumps, you know that caries can be one of the most traumatizing infections in childhood. With 60-90% of children and most adults being affected by it, it is the second most prevalent infection worldwide.¹’² For a long time, caries mostly affected the population of developed countries, but with an increase in sugar consumption the prevalence of this oral disease is increasing in developing countries as well.
Nowadays, the prevalence of dental lesions derived from caries has been declining in developed countries due to preventive means such as the use of fluoride in toothpaste and a better biofilm control through regular tooth care.²’³ However, there is still a need for better caries prevention to further diminish the number of those suffering from it. To find effective supplements for the common preventive methods, one needs to further understand the process of cariogenesis.
Dental caries describes the demineralization of the enamel (the hard tissue of teeth) due to organic acids that are being produced by a microbial biofilm. One of the most prominent and best studied cariogenic bacteria is the family of mutans streptococci. It is one of the first colonizers of the tooth and provides an extracellular polysaccharide matrix (EPS) that is indispensable for the further formation of a so called dental plaque that is composed of multiple microorganisms.⁴’⁵
The adherence of the bacteria on the tooth surface takes place in multiple phases.
At first, the bacteria may adhere to the teeth by weak Van-der-Waals forces.² For a permanent, stronger adherence to the teeth, natural colonizers have developed very specific pathways. The outer surface of our teeth is called the enamel and is mainly composed of hydroxyapatite. The enamel is covered with a so called “acquired pellicle”, a variety of proteins and glycoproteins that are constantly being secreted through the salivary glands.²’⁶ This pellicle is used as an anchor for primary colonizers that have developed specific adhesins to bind to those proteins.
For Streptococcus mutans, a well-studied example is the antigen I/II (also known as AgI/II, SpaP, Pac or AgB) that binds to the salivary pellicle.⁷
In a subsequent phase, the EPS is formed to induce an accumulation of bacteria. The formation of the EPS strongly depends on the presence of sucrose on the one hand, but also glycosyltransferases (GTFs) on the other hand. The GTFs hydrolyze sucrose to fructose and glucose. The latter is then used to synthesize glucans with different branch points.⁴ These GTFs are subdivided into three main types: GTFB, GTFC and GTFD. In addition to their catalytic activity to form soluble or insoluble glucans, these proteins possess glucan-binding sites that promote an interconnection between the glucans.⁷’⁸ Some of them may adhere to saliva-covered hydroxyapatite whereas others rather bind to bacterial surfaces.⁸’⁹
These three GTFs are thus responsible for the synthesis of the EPS on the surface of our teeth which may then serve as a matrix for an initial clustering of secondary colonizers and the establishment of a dental plaque.⁵’¹⁰
Once this dental plaque has formed, the acidic by-products of the microbial metabolism, such as lactic acid, may lead to a pH drop causing the demineralization of the enamel of our teeth.²
Taking into account the pivotal role of antigen I/II and the GTFs in the initial attachment of S. mutans to the pellicles and the production of the EPS, respectively, they seem to be a promising target to prevent early cariogenesis. Early studies showed that mutations in the genes encoding the different GTFs may indeed diminish the virulence of S. mutans.⁷’¹⁹
Once the exact sequences encoding those proteins was determined in the late 80s,¹¹’¹² the possibility to clone them accelerated the further characterization of their functional domains.¹³⁻¹⁸ The availability of bulk amounts of those proteins furthermore ushered in vaccination studies with GTFs and antigenI/II. These studies revealed very promising results in inhibiting the biofilm formation in vitro, but also in vivo. ⁷’²⁰⁻²⁶
In early vaccination studies, antibodies against whole S. mutans have been shown to have cross-reactivity with human and rabbit cardiac tissue. (Harris R. Vaccines for dental caries. Aust Dent J 1983;28:115-6) Even though these cross-reactivities have not been found in vaccination studies conducted with proteins like antigen I/II and GTFs²⁷, they may still prevent people from embracing a vaccination against caries. Moreover, the production of monoclonal antibodies as a passive immunization is quite cost-intensive and as vaccine hesitance is on the rise, this method does not seem to be the holy grale in caries prevention.²⁸
We thus came up with the idea to generate aptamers against GTFs and antigen I/II.
Aptamers are short, single-stranded DNA or RNA fragments that may form three-dimensional structures and bind to target molecules.
They are not only less expensive than monoclonal antibodies, but they may also exhibit a higher binding affinity and have no batch-to-batch variability as do antibodies.²⁹’³⁰ Another great advantage is that they mostly bind to exosites of proteins, which is why we hope to be able to target specific sites in our target proteins that are important for their function.
We hope to generate aptamers that may bind to the target proteins and inhibit the biofilm formation as it was observed in the vaccination studies, but which would be saver and cheaper in use.
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