SARS-CoV-2 structural proteins trigger periodontal fibrosis

In a recent study published on bioRxiv* preprint server, researchers examined the pathological impacts of structural components of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on human periodontal cells and tissues to understand the association between coronavirus disease 2019 (COVID -19) and deterioration of oral health.

Background

Recent studies have shown that the symptoms of the long-running coronavirus disease (COVID) manifest beyond the pulmonary system, with lingering complications also occurring in the cardiac, renal and neurological systems. In addition to nasal swabs, saliva samples have been used to test for SARS-CoV-2, and the oral cavity is believed to be a viral reservoir. COVID-19 could pose a serious risk to periodontal health since periodontal tissues are vulnerable to infectious diseases. However, the pathology of the deterioration of periodontal health due to COVID-19 remains unclear.

The spike protein region of SARS-CoV-2 is the most studied structural part of the virus since most mutations that give rise to variants with increased transmissibility and immune evasion occur in the receptor binding domain of peak protein. The spike protein has also been the main target of vaccines and monoclonal antibody therapies. However, SARS-CoV-2 also consists of three other structural components – envelope, membrane and nucleocapsid proteins – whose pathological roles have not been thoroughly explored.

About the study

In the present study, researchers used cultured human periodontal ligament fibroblasts (HPLF) and human gingival epithelial cells (HGEPp) to study the pathology of periodontal fibrosis related to COVID-19.

Immunofluorescence analysis was used to detect the expression of angiotensin-converting enzyme-2 (ACE-2) and transmembrane serine protease 2 (TMPRSS2) receptors in cells of the gingival epithelium and periodontal ligament, which was confirmed by Western blot analysis. The potential of SARS-CoV-2 to infect HPLFs was explored by treating cells with a SARS-CoV-2 spike protein conjugated to a polyhistidine tag (His Tag). Anti-His Tag (APC) allophycocyanin-conjugated antibodies were then used to localize the spike protein by immunofluorescence analysis.

To determine whether COVID-19 caused fibrosis in periodontal tissues, HPLFs were either infected with lentiviruses carrying plasmids for SARS-CoV-2 envelope, membrane, and nucleocapsid proteins or treated with recombinant SARS-CoV-2 peak proteins. Acute and long-lasting infections were stimulated in periodontal fibroblasts, and cell proliferation was assessed by immunostaining for anti-Bromodeoxyuridine (BrdU) antibodies. Additionally, Western blot was used to determine the production of collagen I and metalloproteinase-1 (MMP1) in the extracellular matrix to assess the tissue integrity of the periodontal ligament.

Periodontal ligament fibroblasts treated with the structural components of SARS-CoV-2 were subjected to proteomic analysis to determine the molecular mechanisms regulating COVID-19 pathology. Additionally, the Seahorse Mito stress test was performed to understand the effects of SARS-CoV-2 infections on mitochondrial fatty acid pathway function. The results were validated using etomoxir, which inhibits the mitochondrial β-oxidation pathway.

Results

The results revealed that ACE-2 and TMPRSS2 were expressed at high levels in periodontal tissues, and that exposure to structural components of SARS-CoV-2 such as membrane and envelope proteins increased periodontal fibroblast hyperproliferation, as well as apoptosis and senescence.

Molecular mechanisms mediating periodontal tissue fibrosis consisted primarily of downregulation of β-oxidation in mitochondria by membrane and envelope proteins of SARS-CoV-2. Other studies have found associations between downregulated mitochondrial β-oxidation and fibrosis in the lungs and kidneys. The results also highlighted the role of structural components of SARS-CoV-2 other than the spike protein in disease pathology. The authors discussed various studies that have identified alternative mechanisms by which envelope proteins increased the pathogenicity of SARS-CoV-2, including increased pH in the Golgi apparatus and formation of cation channels. .

Researchers also performed deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) terminal labeling (TUNEL) to understand the involvement of structural components of SARS-CoV-2 in increasing apoptosis and senescence, and the results showed indicated that spike or nucleocapsid proteins did not contribute to increased apoptosis and senescence, and only envelope and membrane proteins did. Membrane and envelope proteins also upregulated collagen I production and decreased MMP1 enzyme production in the extracellular matrix, contributing to periodontal fibrosis.

conclusion

Overall, the study provided new insights into the impact of SARS-CoV-2 infections on periodontal health. The results revealed that SARS-CoV-2 membrane and envelope proteins alter fatty acid degradation pathways in mitochondria that are essential for maintaining energy homeostasis. Dysregulation of the mitochondrial β-oxidation pathway leads to hyperproliferation of periodontal fibroblasts, causing fibrosis, and increased apoptosis and senescence. These results could contribute to a better understanding of the long covid symptoms manifest in other organ systems and provide targets for therapy.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.