How Sars-Cov-2 orchestrates the virulence of Prevotella spp

Note from the director of publication: Bio Moon's work is aimed at researching and explaining to demonstrate that collaborative research and science allow interested people to develop and share their knowledge. Assumptions have been made and research carried out. Bio Moon has tried on many occasions to contact researchers in this field to make his work available which is summarized hereafter

OPINION: During January, I wanted to ensure that my daughter could go to the nursery, and therefore, stimulated by my scientific instincts, I tried to understand this virus. I looked for and established epidemiological correlations. One difference kept coming up all the time between the airways of an adult and a child: the community of bacteria called Prevotella spp. With age, the bacteria that make up the microbiome are less and less varied. Their biodiversity collapse all the more rapidly as a result of health problems, poor oral hygiene, poor diet, overly systematic use of antibiotics, and so on. This is why the elderly have a rather poor microbiome.

(3)  https://erj.ersjournals.com/content/50/5/1700832

I shared the research path on Prevotella with the Indian bioinformatician Sandeep Charkraborty without coming at it from the same direction. He managed to make himself heard by the scientific community: abnormal amounts of Prevotella proteins were found in the lungs of patients with Covid-19. (  11  ). The role of this bacteria therefore no longer seems to be questioned.

The question is how the interaction between the virus and the Prevotella bacteria in our respiratory tract could explains the severe forms of Covid-19. This article is the simplified version of a more technical version intended for the scientific community (1).

I / Prevotella spp in the respiratory tract.

It does not seem useful to demonstrate the significance of the profiles of patients who are victims of severe forms of Covid-19. My point is not whether or not Prevotella spp prevail, but on the relationship between Prevotella community and the microbiome; and therefore on the relative abundance of the Prevotella spp community in the respiratory tract. However, children have a very diverse microbiota in which Prevotella spp is a strong minority. Moreover, the quantity of Prevotella does not change that much: it is the diversity of the microbiota which decreases with age, comorbidities, chronic diseases, diet, that induces a greater relative abundance of the Prevotella community. spp (  6  ).

We can see from the graph above that if the amount of Prevotella spp does not change significantly with age in a healthy patient; however with age, it becomes, one of the main representative of the microbiome in the respiratory tracts due to the disappearance of many taxa ( 7 ). There is extensive literature on the role of Prevotella spp in many inflammatory pathologies affecting the mucous membranes of the epithelium of the respiratory tract: gingivitis, peridontitis, sinusitis, angina, pulmonary fibrosis, pneumonia, portal vein thrombosis, pulmonary and cerebral abscesses , myocarditis ( 9 ).

1.2 Prevotella my love.

Indeed, if I remain stuck on Prevotella, it is for several important reasons:

- It is an anaerobic Gram-negative bacteria which by definition requires anoxic conditions to develop. To this end, it will be one of the main generators of the creation of bacterial biofilm on the epithelial mucous membranes, in particular when inflammation affects these mucous membranes. I believe it is these biofilms that promote the timely pathogenicity of the microbiome and the low immunogenicity of our tissues.

-The Prevotella Spp community has a very strong and permanent interaction with the immune system. It also has a dual role in the inflammatory reaction. It will be able to inhibit inflammation, thereby modulating the interactions of various pathogens with epithelial cells and the rest of the microbiome. But it can also be a factor of hyper inflammation under certain conditions, which can even cause the famous cytokine storms ( 5 ).

- It is the most discriminating bacteria and the most correlated with the profiles of seriously ill patients, as the epidemiological correlations demonstrate. The other bacteroidetes, firmicutes, Veillonella can also play a role in inflammatory diseases but in a more targeted way and in addition, they are aerobic and gram positive bacteria for many of them.

https://www.openaccessjournals.com/articles/the-airway-microbiome-in-cystic-fibrosis-challenges-for-therapy.pdf

1.3 Supporting clinical data.

At the time of this paper, the bioinformatician Chakraborty, who reached the same conclusions (though through a different path) on a probable role played by the Prevotella bacteria, has managed to make himself heard. With a team of researchers, they analyzed the sputum of patients with Covid-19 and looked for proteins specifically encoded by Prevotella ( 11 ). The results speak for themselves: a particularly high level of gene expression of Prevotella can be found in patients with Covid-19.

https://academic.oup.com/bioinformatics/article/36/13/4065/5831291#.XrOdUxboJ9E.twitter

The role of Prevotella therefore no longer requires demonstration: this bacteria plays a role in the pathogenicity induced by SARS-Cov-2. On the other hand, I think that the interpretation on the role played by Prevotella is not the right one: This increased presence of protein is not explained by the direct cell / Prevotella interaction but by a more complex action involving Prevotella, the Sars-Cov-2 and respiratory tract epithelium cells.

II / SARS-Cov-2 activates Prevotella spp in the respiratory tract.

2.1 The enzyme S-adenosylmethionine (SAM).

The S-adenosyl-methionine (SAM) connects these three organisms. It is an enzyme that plays an essential metabolic role. An enzyme is this mason who generates the link between the architect and your house. The problem is that the body does not itself produce the tools for the mason, the contributions are necessarily exogenous, and most likely food.

The SAM enzyme is also a precursor for the synthesis of proteins that allows bacteria to communicate with each other: autoinducers. Yes indeed, our bacteria communicate with each other to adapt to our body. It is through this communication that infection with SARS-Cov-2 will induce generalized inflammation caused by competition between the Prevotella community and our epithelial cells for the SAM enzyme.

Depending on the density, the diversity of the microbiome and the relative abundance of Prevotella, SARS-Cov-2 will induce more or less inflammation. In some person, the detection threshold of these signal molecules, the autoinducers, will be quickly reached, and will generate cytokine storms.

2.2 The non-structuring proteins np10 and np16 of SARS-Cov-2

In our cells, as for bacteria and for RNA viruses such as SARS-Cov-2, there are molecules that make the link between the masons, called enzymes, and the architects that are the genes, made up of DNA. These molecules are like “work leaders”, we call them RNAs. In order to perform their task, they have to be capped by a methyl group.

In order to ensure its translation and therefore its replication, SARS-Cov-2 has two proteins cap, nsp1O and nsp14.  Their role is precisely to cap the viral RNA, using the SAM enzyme (the mason of our organism).

There haven't been so many mutations between SARS-Cov-2 and other coronaviruses. But many relate to our nsp10 and nsp14 proteins. And these mutations can influence the binding between the works supervisor, the RNA, and the mason, the SAM enzyme. It seems to speed up the site significantly. With the Sars-Cov-2, the works foreman is much faster topped by this mason which is the SAM enzyme. And indeed we can think that the kinetics of styling are much faster than that of other corona viruses: a publication dated April 09, 2020 ( 14) claims that SARS-Cov-2 replication would be much faster. I think it is the kinetics of viral RNA capping that explains this increased rate of replication. And I think it is this speed of replication that explains the role played by Prevotella. https://www.biorxiv.org/content/10.1101/2020.04.26.061705v1.full

Indeed, this replication speed will induce a significant consumption of the mason of our organism, the SAM enzyme. I think that the speed of replication induces a punctual and localized deficiency in SAM. This enzyme, in addition to its central role in many metabolic pathways and in gene expression, is also a substrate for bacterial metabolism. It is easy to understand that infection with SARS-Cov-2 will require an increased consumption of the SAM enzyme, which can quickly represent a limiting factor. However, the SAM enzyme will be abundantly recruited by epithelial cells infected with Sars-Cov-2, but it will also be called upon by bacteria in the microbiome. The importance of SAM and the disturbances caused by its metabolism in all living organisms are already well known.

2.3 Prevotella quorum sensing.

As with eukaryotic organisms, there will be autoregulation in bacteria which will promote the different uses of the enzyme in SAM according to cellular needs and the environment. For Prevotella spp and many other bacteria, there is an additional pathway in the metabolism of the SAM enzyme compared to eukaryotes: the production of communication molecules, the autoinducers.

Depending on the environment and energy needs, the bacteria will either use the SAM enzyme for its functional and metabolic needs, or when the environment is favorable, that is to say when the population density is sufficient and / or a source of stress affects the bacteria, the latter will coordinate their action and therefore the production of autoinducers. And autoinducer production has the potential to activate virulence and resistance genes in the bacteria. The greater the population density, the greater the concentration of autoinducer, the greater the production will also be, up to a threshold, the quorum sensing.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1698510/

Schematic representation of the detection of bacterial quorum. At low population densities, the basal level production of self-inducing molecules results in rapid dilution of self-inducing signals in the environment. At high population densities, an increase in the number of bacteria results in an accumulation of self-inducers above a threshold concentration, leading to the activation of the regulatory proteins of the response, which in turn trigger the cascade of quorum detection.

This is where the specificity of Prevotella compared to other bacteria of the microbiome will intervene: Prevotella is a strict anaerobic bacterium (it does not like oxygen) and therefore to proliferate on the epithelial mucous membranes, it will have to constitute biofilms, kind of microscopic greenhouses, which will isolate it from the oxygen in the respiratory tract. It is the local competition between Prevotella spp and the epithelial cells of the respiratory tract for the SAM enzyme that explains the expression of virulence genes in Prevotella whose proliferation will cause the serious damage observed. I have decisive evidence to show that this is the mechanism that induces virulence in Prevotella: smokers.

In the context of this pandemic, one would have expected that smokers would be at least as affected, if not more, than others. However, it is not the case. It is the SAM enzyme that explains the relative immunity of smokers: researchers have worked on the link between cigarette smoke and the SAM enzyme ( 15 ). They found that cigarette smoke causes the SAM enzyme to increase in cells by activating another metabolic pathway for the synthesis of the SAM enzyme. It can therefore be said that cigarette smoke inhibits competition for SAM, which explains the relative immunity of smokers.

This becomes all the more obvious if we consider the importance of anoxia on the metabolism of SAM at the level of epithelial cells: it has been shown by many researchers that hypoxia, and therefore all the more so anoxia, caused exocytosis, an exit of molecules, SAM enzymes at the level of cells of the pulmonary epithelium ( 16 ).

The effects of low, moderate and severe oxidative stress on the levels of the metabolites involved have been studied in lung epithelial cells. The results indicate that lung epithelial cells release high levels of SAM, possibly as a response. Hypoxia therefore increases the level of SAM secreted in the mucous membranes of epithelial cells, even when the cells are deficient in this enzyme which is a limiting factor. The Prevotella community thus takes advantage of the anoxia created by the biofilm to use the SAM originating from the cells of the epithelium.

The stress induced in Prevotella following the viral infection will cause a competition for SAM, which will take place in a very localized manner, in the sinuous, cramped and inflamed areas where Prevotella is rife, in the sinuses, throat, lungs .

We will note that in smokers, epithelial cells produce more SAM and that is why no stress is induced in the Prevotella community, even when, like in smokers, it occupies many areas of our airways.

2.4 Co-infection rather than over-infection.

The main reason that this coronavirus stands out from the rest is the kinetics of its replication, which is much faster. It is this kinetics that explains the spontaneous and localized competition which causes the wrath of Prevotella. It is the autoinducers produced by Prevotella that will transmit the message of expression of virulence and resistance genes to all bacteria throughout the body. Because the autoinducers circulate throughout the whole organism and they are universal: the message produced by Prevotella can be perceived and translated by numerous pathogenic bacteria in numerous tissues. This is how, in all tissues in contact with an unbalanced environment of the microbiome, bacterial toxins creating lesions and inhibiting the immune system will be diffused by opportunistic germs. It is even the presence of these dysbiosis in the cells of the epithelium of the respiratory tract that promotes infection of the cells by SARS-Cov-2.

Indeed, even in the absence of viral infection, these pathogenic bacteria produce autoinducers. And these autoinducers, when they reach a certain level in the mucous membranes, can alter epithelial cells and promote the crossing of barriers by pathogens.

https://tel.archives-ouvertes.fr/tel-01807764/document

SARS-Cov-2 thus goes to areas of the inflamed respiratory tract, and in hypoxia, preferentially penetrates epithelial cells. It will thus rapidly mobilize a large part of the SAM for its replication even when the conditions of hypoxia favor its exocytosis. I believe that SAM exocytosis is interrupted by infection of epithelial cells and this causes expression of the virulence and resistance genes of Prevotella. I do not believe it is the Sars-Cov-2 that inhibits the immune response. I think it is Prevotella that is known for its powerful impact on the immune system.

Studies show that autoinducers affect signal transduction and innate immune responses of cells. Prevotella is also known to cause destructive cytokine storms, especially in the lungs and intestine ( 18 ). It is this storm of cytokines that causes respiratory distress in patients and sometimes death. It also causes thrombosis ( 19 ) and intestinal disorders.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5506432/

This model would therefore explain absolutely all the specificities of this pandemic  :

- The elderly will be the first to be affected: their microbiome is not very diversified, and their respiratory tracts will be more hypoxic. Prevotella will be relatively more abundant than in a younger one.
- Healthy carriers are infected with the virus but this did not induce the threshold of detection of Prevotella quorum and the virus alone induces only limited inflammation.
- Obese people, diabetics and patients with inflammatory diseases suffer from chronic dysbiosis, reduced biodiversity and therefore an increased presence of Prevotella in the respiratory tract.
- The false negatives have cells containing the virus but whose replication is blocked in particular by the interactions between Prevotella and the epithelial cells and thus the virus no longer circulates.

III / Therapeutic perspectives.

3.1 Drug outlook

I am worried that neither antivirals nor vaccines will work for this disease. This would be due to the kinetics of the disease and the intrinsic inertia of our adaptive immunity and the potential action of antivirals. It does not take long for the virus to induce the immune cascade for which bacteria in the microbiome are responsible. Antivirals will necessarily be administered after infection, therefore after the interference induced in the bacteria of our microbiome. The problem is the same with vaccines: vaccines use the adaptive immune response that recruits lymphocytes to neutralize and destroy pathogens. Again, the latency of the adaptive immune response will not prevent the virus from initiating the immune cascade caused by Prevotella.It could well be that all your investment for a vaccine is as effective as for the vaccine against influenza , the efficiency of which is still questionable.

For the initial stage, it seems to me that there are three axes of attack. The first seems to me the most delicate because it targets the metabolism of methionine which is of extraordinary complexity. This is why this approach seems complicated to me because according to the profiles, the intervention on this metabolism could be beneficial as well as fatal. This being the case, many serious avenues taking this route have been proposed with more than satisfactory results ( 23 ). In the same vein, quroum quetching, a technique that consists of blurring the communication between bacteria, seems to me more than promising even if we are at the beginnings of exploration

The second axis of research, and my preferred one for this initial stage, would be to intervene on the biofilms. In fact, it is the conditions induced by the biofilm which promotes a high level of SAM in the mucous membranes and therefore an increased proliferation of Prevotella. There are many molecules of all kinds to destroy biofilms.

For the stationary stage, I will hypothesize that a combination of antibiotics specific to anaerobic bacteria and with antiparasitic properties would be effective. I use the conditional because the problem is that antibiotics may cause additional dysbiosis in response to viral induced dysbiosis. And that, in the long run, could increase the death toll from the pandemic through chronic inflammatory disease. Also, I would leave it to the doctors to discuss the most effective and least harmful antibiotics, reconciling the death of anaerobic bacteria without further impacting the diversity of the microbiome. In any case, it seems that supplementation with probiotics and prebiotics is essential.

It also seems that it is high time for our societies to take an interest in one of the most effective administrations of probiotics because in this pandemic, it would be the appropriate solution if we managed to induce an inter-bacterial fight to fight against the role played. by commensal bacteria that are opportunely pathogenic in a large number of diseases .

At this stage, the imbalance of the immune system looms . I also think that immunomodulators and other mild anti-allergens and without too many side effects could have a role to play. Plants like harpagophytum, an anti-TNF-alpha plant, may help prevent severe forms. Unlike immunomodulators used in certain autoimmune diseases, their cost is affordable. I would also not hesitate to prescribe aspirin or a herbal anticoagulant because I think the blood clotting starts sooner than we think.

At the final stage, I think the administration of metrodinazol + spiramycin as an injection is inevitable. And this not after having intubated the patients as is currently done, but as soon as they are taken care of. The Prevotella community as well as the many opportunistic germs that accompany its proliferation induce collective multi-resistance to antibiotics. Prevotella does not normally show exceptional resistance. But its collaboration with other bacteria via autoinducers gives it increased resistance. Obviously at this stage, a powerful anticoagulant, heparin, should be administered as soon as possible.

Conclusion  : We probably rushed off and got into a wave of panic. This Sars-Cov-2 would actually only cause a flu at most. It's its interaction with our microbiome that makes it deadly . Faced with a viral infection, we adopted a viral approach: PCR, molecular biology, genetics. And we forgot the fundamental sciences, the purely logical, epidemiological, environmental approach. As a consequence, very good questions arise on our approach of medicine, perhaps too often focused on details, when it is necessary to treat the environment, too quick to confuse cause and consequences, to treat the symptoms rather than to seek the causes. The role of Prevotella in this pandemic raises serious problems: industrial food and denaturalized fresh products, antibiotics and their unsuitable use causing dysbiosis in the long term, the contempt of our big clutches for plants, prebiotics and probiotics, for the preventive approach to which they prefer the invasive and / or medicinal method.

This study also allows us to ask questions about all our certainties of our medicinal approach. In my research, I have updated (updated) that the impact on our health of the communication between bacteria, viruses, microscopic fungi is considerable. Even the flu virus uses its interactions with our microbiome to infect us more efficiently. Antivirals and antibiotics shouldn't be mutually exclusive, that's what this crisis is telling us. Our microbiome is our first immune barrier. Whether it is a viral or bacterial infection, it will be involved in the disease.

We must therefore learn to have a more global approach that takes into account hygiene, diet, the preventive power of herbal medicine and probiotics. This pandemic has broken down the dogmatic barrier between viral infection and bacterial infection. Both are inseparable.