destroy the quorum sense

Today I propose to look at plaque from a slightly different angle. I think most people know that plaque is bad; it contains bacteria that will eventually destroy the enamel, cause tooth decay and gum inflammation. I need to brush my teeth. Everything is quite transparent here.

But I want to talk about the less obvious and hidden life of bacteria in the oral cavity. Let us reveal the dramatic mutual assistance in a multispecies bacterial colony and the interactions are often no less complex than in large multicellular organisms. And at the same time, I’ll tell you how quorum sensing helps Porphyromonas eat your periodontium (i.e., the periodontal tissues that hold the tooth in the alveolus), and on top of all these troubles, also cause Alzheimer’s disease. Well, at the same time, let’s figure out why chewing dolphin feces can help us a lot.

Pellicle

You've just brushed your teeth and are leisurely getting ready for work with the pleasant feeling of smooth, polished enamel. Well, or with the feeling of plaque residue in hard-to-reach places, but we strongly disapprove of this. From the moment the soft abrasive polishes and exposes clean tooth enamel, the process of salivary pellicle formation begins.

Amount of deposited pellicle on hydrophilic and hydrophobic silicon substrates over time. In the second group of experiments, the substrates were additionally washed with various buffers and detergents

The process proceeds extremely quickly – within a few minutes the enamel will be covered with a thin film 10–20 nm thick – a pellicle. After another 10–15 minutes, the thickness will already be up to 1 micron. The film deposition mechanism depends mainly on electrostatic interactions and to a lesser extent on van der Waals forces. Precipitation begins with small protein molecules that are acidic and rich in the amino acid proline. A little later, larger proteins, such as mucins, begin to precipitate onto the prepared substrate from the molecules that precipitated first.

All these proteins fall onto the surface of the enamel from the secretion of the salivary glands. One of the most important functions of saliva is the remineralization of enamel. The process of diffusion of calcium and phosphorus ions goes in both directions. In an acidic environment, calcium ions fly into the oral fluid, and in a neutral environment, vice versa. Moreover research showsthat the intensity of dissolution of enamel hydroxyapatite directly depends on the level of acidity. Under normal conditions, the pH of the oral cavity fluctuates around 6.8–7.0. At these values, saliva is oversaturated with free calcium and phosphorus ions. The process is shifted towards remineralization – saturation of the enamel with calcium. At pH 6.0, saliva begins to work in the opposite direction. Calcium begins to migrate from the bound form in the enamel to the soluble form – the process of demineralization begins.

With regular cleaning, the pellicle acts as a protective layer, facilitating remineralization, and suppresses the growth of microflora due to salivary immunoglobulins. Things get much worse if the enamel surface is not cleaned. There are two possible options. Self-cleaning occurs when eating hard food in the form of carrots, apples, hard celery stalks and the like. This is quite consistent natural diet of wild primateswho ate everything, but consumed large amounts of hard plant foods.

The second option is individual oral hygiene using a soft toothbrush, interdental brushes, floss and a single-tuft brush. This is much more effective than chewing cane and Jerusalem artichoke, although it does not completely eliminate the need for solid food. The most important thing is to mechanically remove plaque from all surfaces using brushes, otherwise the pellicle will quickly turn from a protective shell into an attacker. Cleaning helps to destroy the film, i.e. connections between microorganisms, and prevents them from uniting into an attacking army. By the way, today some pastes (for example, Curaprox, President) add enzymes that reduce the activity of the microbial film. And toothpastes containing fluoride help strengthen teeth overall and resist the acids that plaque bacteria produce.

It’s also worth mentioning the acidity of the oral cavity. After any meal, it increases, and bacteria are comfortable multiplying in such an environment. We can very simply help ourselves and drink some water or simply rinse our mouths with water. Then the acidity returns to a more normal environment, it becomes inconvenient for microorganisms, and they are less violent in our oral cavity.

In general, the concept of hygiene has changed recently, and your (and our) tasks do not include cleaning plaque to sterile cleanliness. It used to be that we had to clean out all the plaque. And now it is believed that microbes are a normal environment in the oral cavity. With the exception of one nuance, when they gather directly into the film, they form a quorum, and so on. And the concept of hygiene now is the destruction of connections between microorganisms.

Plaque

Stained section of two-day dental plaque

To get the picture above, the test subject was taped with an epoxy resin plate in his mouth and forced to walk like that for two days. The dark part is the pellicle. But the rest is bacteria and fungi. Let's look at this process in order.

Attachment of Streptococcus sanguis to surfaces. A – saliva-coated enamel sample, B – uncoated sample. Doesn't stick without saliva

IN first studies Glass plates were used to form dental plaque. Bacteria did not stick well to them. Then the scientists changed their tactics. They began to water the plates with donor saliva, and began to force experimental subjects to wear plates of various materials in their mouths in order to evaluate the effectiveness of the adhesion of various microorganisms and the methods of their attachment. The results were somewhat contradictory. In all cases, the first to start sticking Streptococcus sanguiswhich no one particularly considered pathogenic and responsible for the development of caries. More aggressive and angry Streptococcus mutans did not want to dominate in the samples of artificial dental plaque, although it was precisely this plaque that was sown in large quantities in the areas of development of enamel caries.

Alpha glycans. A special type of polymer made from glucose fragments that allows bacteria to attach to each other under certain conditions

The paradox was resolved by Gibbons' group, which managed to create as close to enamel as possible synthetic hydroxyapatite in sticks. The material most effectively imitated even the electrostatic parameters of natural enamel, which turned out to be important for the process of deposition of microorganisms. In their results Streptococcus sanguis also prevailed over Streptococcus mutansbut Gibbons was able to explain It's a two-step process:

1. First, harmless substances begin to stick to the pellicle of saliva proteins. Streptococcus sanguisas in the illustration above.
2. At the second stage Streptococcus mutans begin to attach to the film formed by sanguis. They use α-1,3-glycans. These are insoluble compounds that are often found in the cells of bacteria, plants, fungi and insects. Bacteria often use them to attach themselves to surfaces and communicate with each other.

Thus, low pathogenic

Streptococcus sanguis

And

Streptococcus oralis

are not aggressive towards enamel themselves. The problem is that they work in tandem with mutans to form the basis of a colony we call plaque. Vickerman and colleagues

proved

an interesting relationship between the number of successfully attached microorganisms and the presence of sucrose in the medium. To put it simply, streptococci eat sucrose and build alpha-glycans insoluble in saliva from its glucose residue in the form of a ball of threads.

Therefore, sweet food greatly simplifies the deployment of a base and colonization for the first invasion group – Streptococcus sanguis. He will build threads from your cookies and soda and create a springboard for the following species. At first they will float freely and stick simply due to electrostatic interaction and van der Waals forces, and then will be “consciously” recordedsynthesizing polymer threads. Bacteria cooperate, helping each other. In addition to glycans, streptococci also synthesize many different adhesin proteins (streptococcal antigens of types I and II), which allow even stronger contact with the substrate and with each other.

Some pairs of microorganisms and the types of adhesins they help hold each other together

Bacteria in dental plaque do not stick haphazardly. They clearly specialize in interacting with their own and other species. Coadhesion between bacteria is a common phenomenon and has been demonstrated for more than 700 strains representing 14 genera. In the table above, I listed only a small part of the highly specific types of interspecific adhesion.

For example, the PlaA adhesin Prevotella loescheii recognizes linear cell wall polysaccharides S. oralis, S. sanguis and other streptococci, and Porphyromonas gingivalis, Treponema denticola and Bacteroides forsythus are fixed on surface proteins of both streptococci and actinomycetes.

Colony of Porphyromonas gulaea close relative of Porphyromonas gingivalis

At this stage of development, dental plaque no longer becomes just a passively adhered neutral mass, but a rather complex and aggressive colony of microorganisms that live and develop as a single whole. Let's look at the example of a bacteroid Porphyromonas gingivalis. At the very beginning of colony development, the environment is rather aerobic. Anaerobes feel extremely uncomfortable and are suppressed in every possible way by aerobic competitors. As plaque accumulates and connections between bacteria form, the colony becomes increasingly isolated from oxygen.

The formation of such plaque in the periodontal sulcus leads to the fact that aerobic microorganisms begin to be replaced by anaerobic ones. And here Porphyromonas gingivalis is unfolding in full. To more effectively capture territory, it synthesizes specific digestive enzymes-endopeptidases – gingipains. In addition to helping them break down gum and periodontal tissue, these enzymes also suppress the host's immune responsereducing the synthesis of inflammatory interleukins.

Moreover, there are a number of studies that talk about a connection between the gingipains of these microorganisms and the development of Alzheimer's disease. An illustration from a scientific paper shows a strong correlation between the concentration of gingipains in the brain and diagnosed Alzheimer's syndrome.

Among other effects, this group of proteins helps bacteroids destroy and digest periodontal structural proteins – metalloproteids of the intercellular matrix, collagens and fibronectins of various types. As a result, they do not just passively stick somewhere in the gum area, but actively dig deep, simultaneously suppressing the immune response so that macrophages do not come to kill them immediately after colonization begins.

For almost a hundred years, it was believed that bacteria in the oral cavity exist in a state of passively floating plankton. Modern research has shown that planktonic forms are a transport state. You encounter such options only when a microorganism arrives with a sip of water, a piece of food or dust. The rest of the time they live and coexist as a single structure. The key element in the formation of such a colony is quorum sense. In fact, with the discovery of such a mechanism, it became clear that microorganisms often interact with each other no less complex and complex than our macroorganism, which also consists of many individual cells. Yes, dental plaque colonies are far from human in complexity and specialization, but they are no longer just a lump of bacteria of different types.

Quorum sensing and communication allow bacteria to regulate gene expression, changing their phenotype depending on external unfavorable factors. Until the research of Kenneth Neilson and John Hastings in 1979, microbiologists considered microbial cells to be independently acting individuals. For example, Pseudomonas aeruginosa can safely reproduce in small quantities without causing harm to humans. But as soon as the immune system weakens, pseudomonads dramatically transform, accelerate their metabolism and quickly overcome the immune barrier, forming persistent biofilms. Thanks to quorum sensing, microorganisms successfully exchange information about the need to begin synthesizing protection against antibiotics, slow down or speed up division, or even begin sporulation in order to survive extreme environmental conditions.

What should we do about it?

You need to brush your teeth. Everything here is quite simple, transparent and understandable. If you brush your teeth poorly, instead of a thin protective pellicle you get an aggressive colony that quickly eats up the enamel and periodontal tissue.

But with the development of molecular biology and the possibility of synthesizing peptide molecules with desired properties, we have new tools—inhibitors of bacterial signaling networks. In fact, we can turn on the jammer and suppress their communication with each other. Or force them to transform into an unfavorable state, where they will quickly be finished off by competitors that are non-pathogenic to humans. Instead of heavy artillery in the form of antiseptics and antibiotics, we are starting to manage the microbiome as a whole.

Suppression of education biofilms on implants with quorum sensing inhibitors

The source for such drugs is usually the bacteria themselves, which not only work together in colonies, but also actively compete. Thus, in one of the studies they successfully demonstrated the suppression of biofilm formation S. mutans strain ATCC 25175. The source was quite unusual, I always wondered how scientists select them – from dolphin intestine allocated Lactiplantibacillus curvatus EIR/DG-1, grown on a nutrient medium, then filtered and used the remaining regulatory molecules to destroy the biofilm S. mutans. The Lactiplantibacillus plantarum EIR/IF-1 strain from children's feces turned out to be even more effective.

In general, it can be recommended to continue to brush your teeth well and see a dentist in order to wait for the new generation of regulatory preventive drugs with the maximum number of healthy teeth.

The coming years promise to be interesting in this area.