Inhibition of SARS-CoV-2 infection by Porphyromonas gingivalis and the oral microbiome.

The COVID-19 pandemic persists despite the availability of vaccines, and it is, therefore, crucial to develop new therapeutic and preventive approaches. In this study, we investigated the potential role of oral microbiome in SARS-CoV-2 infection. Using an in vitro SARS-CoV-2 pseudovirus infection assay, we found a potent inhibitory effect exerted by Porphyromonas gingivalis on SARS-CoV-2 infection mediated by known P. gingivalis compounds such as phosphoglycerol dihydroceramide (PGDHC) and gingipains as well as by unknown bacterial factors. We found that the gingipain-mediated inhibition of infection is likely due to cytotoxicity, whereas PGDHC inhibited virus infection by an unknown mechanism. Unidentified factors present in P. gingivalis supernatant inhibited SARS-CoV-2 likely via the fusion step of the virus life cycle. We addressed the role of other oral bacteria and found certain periodontal pathogens capable of inhibiting SARS-CoV-2 pseudovirus infection by inducing cytotoxicity on target cells. In the human oral cavity, we observed that the modulatory activity of oral microbial communities varied among individuals, in that some saliva-based cultures were capable of inhibiting while others were enhancing infection. These findings contribute to our understanding of the complex relationship between the oral microbiome and viral infections, offering potential avenues for innovative therapeutic strategies in combating COVID-19. IMPORTANCE: The oral microbiome is important in health and disease, and in this study, we addressed the potential role of the oral microbiome in COVID-19 infection. Our in vitro studies suggest that certain bacteria of the oral microbiome such as P. gingivalis produce compounds that could potentially inhibit SARS-CoV-2 infection. These findings elucidating the interactions between the oral microbiome and SARS-CoV-2 infection will be important in our understanding of COVID-19 pathogenesis and the development of innovative therapeutic and preventive strategies against COVID-19 infection.

Unveiling the molecular mechanisms of the type IX secretion system's response regulator: Structural and functional insights.

The type IX secretion system (T9SS) is a nanomachinery utilized by bacterial pathogens to facilitate infection. The system is regulated by a signaling cascade serving as its activation switch. A pivotal member in this cascade, the response regulator protein PorX, represents a promising drug target to prevent the secretion of virulence factors. Here, we provide a comprehensive characterization of PorX both in vitro and in vivo. First, our structural studies revealed PorX harbors a unique enzymatic effector domain, which, surprisingly, shares structural similarities with the alkaline phosphatase superfamily, involved in nucleotide and lipid signaling pathways. Importantly, such pathways have not been associated with the T9SS until now. Enzymatic characterization of PorX's effector domain revealed a zinc-dependent phosphodiesterase activity, with active site dimensions suitable to accommodate a large substrate. Unlike typical response regulators that dimerize via their receiver domain upon phosphorylation, we found that zinc can also induce conformational changes and promote PorX's dimerization via an unexpected interface. These findings suggest that PorX can serve as a cellular zinc sensor, broadening our understanding of its regulatory mechanisms. Despite the strict conservation of PorX in T9SS-utilizing bacteria, we demonstrate that PorX is essential for virulence factors secretion in Porphyromonas gingivalis and affects metabolic enzymes secretion in the nonpathogenic Flavobacterium johnsoniae, but not for the secretion of gliding adhesins. Overall, this study advances our structural and functional understanding of PorX, highlighting its potential as a druggable target for intervention strategies aimed at disrupting the T9SS and mitigating virulence in pathogenic species.

Carbamylation Is Instrumental in End-Stage Kidney Disease Coagulopathies: The Impact on von Willebrand Factor and Platelet Functionality.

BACKGROUND: Chronic kidney disease (CKD) is a progressive, irreversible, and incurable condition characterized by high morbidity and mortality, affecting approximately one-tenth of the global population. Rise of urea-derived cyanate levels in CKD patients, severalfold higher in comparison to those found in healthy individuals, leads to an increased rate of carbamylation of lysine residues of proteins and peptides. This posttranslational modification plays an important role in the progression of kidney failure but also in the onset of CKD-related complications, including previously reported coagulopathies. In this study, we have explored the impact of carbamylation on the functionality of von Willebrand factor (vWF), a pivotal player in hemostasis, and its implications for platelet adhesion. MATERIALS AND METHODS: We have explored carbamylated vWF's interactions with its partner proteins via ELISA. Mass spectrometry was employed to identify modified lysine residues. Blood platelets isolated from healthy donors were carbamylated, and their activation, binding to endothelium and thromboxane release were evaluated using flow cytometry, adhesion assays and ELISA, respectively. RESULTS: Using mass spectrometry we detected the vWF's lysine residue smost susceptible to carbamylation. This modification has in turn affected vWF's interactions with its key binding partners: decreased binding to collagen types I/III but increased the affinity to factor FVIII, while its binding to fibrinogen remained unchanged. Carbamylation of vWF impeded vWF-blood platelet binding, but carbamylation of platelets led to their increased thrombin-dependent activation as observed by enhanced phosphatidylserine exposure, improved their binding to vascular endothelium, at the same time decreasing the production of the prothrombotic mediator, thromboxane A2. CONCLUSION: Our findings highlight the multifaceted impact of carbamylation on vWF and platelets, disturbing the delicate balance of coagulation cascade. These alterations could contribute to the complex hemostatic imbalance in ESKD, underscoring the need for further research to fully understand these mechanisms and their clinical implications.

Antiviral activity of temporin-1CEb analogues against gingival infection with herpes simplex virus type 1.

Introduction: Oral herpes infections caused by herpes simplex virus type 1 (HSV-1) are one of the most common in the human population. Recently, they have been classified as an increasing problem in immunocompromised patients and those suffering from chronic inflammation of the oral mucosa and gums. Treatment mainly involves nucleoside analogues, such as acyclovir and its derivatives, which reduce virus replication and shedding. As drug-resistant strains of herpes emerge rapidly, there is a need for the development of novel anti-herpes agents. The aim of the study was to design an antiviral peptide, based on natural compounds, non-toxic to the host, and efficient against drug-resistant HSV-1. Here, we designed a lysine-rich derivative of amphibian temporin-1CEb conjugated to peptides penetrating the host cell membrane and examined their activity against HSV-1 infection of oral mucosa. Methods: We assessed the antiviral efficiency of the tested compound in simple 2D cell models (VeroE6 and TIGKs cells) and a 3D organotypic model of human gingiva (OTG) using titration assay, qPCR, and confocal imaging. To identify the molecular mechanism of antiviral activity, we applied the Azure A metachromatic test, and attachment assays techniques. Toxicity of the conjugates was examined using XTT and LDH assays. Results: Our results showed that temporin-1CEb analogues significantly reduce viral replication in oral mucosa. The mechanism of peptide analogues is based on the interaction with heparan sulfate, leading to the reduce attachment of HSV-1 to the cell membrane. Moreover, temporin-1CEb conjugates effectively penetrate the gingival tissue being effective against acyclovir-resistant strains. Collectively, we showed that temporin-1CEb can be regarded as a novel, naturally derived antiviral compound for HSV-1 treatment.

Structural and functional insights into the C-terminal signal domain of the Bacteroidetes type-IX secretion system.

Gram-negative bacteria from the Bacteroidota phylum possess a type-IX secretion system (T9SS) for protein secretion, which requires cargoes to have a C-terminal domain (CTD). Structurally analysed CTDs are from Porphyromonas gingivalis proteins RgpB, HBP35, PorU and PorZ, which share a compact immunoglobulin-like antiparallel 3+4 ß-sandwich (ß1-ß7). This architecture is essential as a P. gingivalis strain with a single-point mutant of RgpB disrupting the interaction of the CTD with its preceding domain prevented secretion of the protein. Next, we identified the C-terminus ('motif C-t.') and the loop connecting strands ß3 and ß4 ('motif Lß3ß4') as conserved. We generated two strains with insertion and replacement mutants of PorU, as well as three strains with ablation and point mutants of RgpB, which revealed both motifs to be relevant for T9SS function. Furthermore, we determined the crystal structure of the CTD of mirolase, a cargo of the Tannerella forsythia T9SS, which shares the same general topology as in Porphyromonas CTDs. However, motif Lß3ß4 was not conserved. Consistently, P. gingivalis could not properly secrete a chimaeric protein with the CTD of peptidylarginine deiminase replaced with this foreign CTD. Thus, the incompatibility of the CTDs between these species prevents potential interference between their T9SSs.

Exhaled Nitric Oxide Reflects the Immune Reactions of the Airways in Early Rheumatoid Arthritis.

Patients with rheumatoid arthritis (RA) have altered levels of exhaled nitric oxide (NO) compared with healthy controls. Here, we investigated whether the clinical features of and immunological factors in RA pathogenesis could be linked to the NO lung dynamics in early disease. A total of 44 patients with early RA and anti-citrullinated peptide antibodies (ACPAs), specified as cyclic citrullinated peptide 2 (CCP2), were included. Their exhaled NO levels were measured, and the alveolar concentration, the airway compartment diffusing capacity and the airway wall concentration of NO were estimated using the Högman-Meriläinen algorithm. The disease activity was measured using the Disease Activity Score for 28 joints. Serum samples were analysed for anti-CCP2, rheumatoid factor, free secretory component, secretory component containing ACPAs, antibodies against Porphyromonas gingivalis (Rgp) and total levels of IgA, IgA1 and IgA2. Significant negative correlations were found between the airway wall concentration of NO and the number of swollen joints (Rho -0.48, p = 0.004), between the airway wall concentration of NO and IgA rheumatoid factor (Rho -0.41, p = 0.017), between the alveolar concentration and free secretory component (Rho -0.35, p = 0.023) and between the alveolar concentration and C-reactive protein (Rho -0.36, p = 0.016), but none were found for anti-CCP2, IgM rheumatoid factor or the anti-Rgp levels. In conclusion, altered NO levels, particularly its production in the airway walls, may have a role in the pathogenesis of ACPA-positive RA.

Unveiling the Molecular Mechanisms of the Type-IX Secretion System's Response Regulator: Structural and Functional Insights.

The Type-IX secretion system (T9SS) is a nanomachinery utilized by bacterial pathogens to facilitate infection. The system is regulated by a signaling cascade serving as its activation switch. A pivotal member in this cascade, the response regulator protein PorX, represents a promising drug target to prevent the secretion of virulence factors. Here, we provide a comprehensive characterization of PorX both in vitro and in vivo . First, our structural studies revealed PorX harbours a unique enzymatic effector domain, which, surprisingly, shares structural similarities with the alkaline phosphatase superfamily, involved in nucleotide and lipid signaling pathways. Importantly, such pathways have not been associated with the T9SS until now. Enzymatic characterization of PorX's effector domain revealed a zinc-dependent phosphodiesterase activity, with active site dimensions suitable to accommodate a large substrate. Unlike typical response regulators that dimerize via their receiver domain upon phosphorylation, we found that zinc can also induce conformational changes and promote PorX's dimerization via an unexpected interface. These findings suggest that PorX can serve as a cellular zinc sensor, broadening our understanding of its regulatory mechanisms. Despite the strict conservation of PorX in T9SS-utilizing bacteria, we demonstrate that PorX is essential for virulence factors secretion in Porphyromonas gingivalis and affects metabolic enzymes secretion in the non-pathogenic Flavobacterium johnsoniae , but not for the secretion of gliding adhesins. Overall, this study advances our structural and functional understanding of PorX, highlighting its potential as a druggable target for intervention strategies aimed at disrupting the T9SS and mitigating virulence in pathogenic species.

Identification of a new genetic variant (G231N, E232T, N235D) of peptidylarginine deiminase from P. gingivalis in advanced periodontitis.

Chronic periodontitis (CP), an inflammatory disease of periodontal tissues driven by a dysbiotic subgingival bacterial biofilm, is also associated with several systemic diseases, including rheumatoid arthritis (RA). Porphyromonas gingivalis, one of the bacterial species implicated in CP as a keystone pathogen produces peptidyl arginine deiminase (PPAD) that citrullinates C-terminal arginine residues in proteins and peptides. Autoimmunity to citrullinated epitopes is crucial in RA, hence PPAD activity is considered a possible mechanistic link between CP and RA. Here we determined the PPAD enzymatic activity produced by clinical isolates of P. gingivalis, sequenced the ppad gene, and correlated the results with clinical determinants of CP in patients from whom the bacteria were isolated. The analysis revealed variations in PPAD activity and genetic diversity of the ppad gene in clinical P. gingivalis isolates. Interestingly, the severity of CP was correlated with a higher level of PPAD activity that was associated with the presence of a triple mutation (G231N, E232T, N235D) in PPAD in comparison to W83 and ATCC 33277 type strains. The relation between mutations and enhanced activity was verified by directed mutagenesis which showed that all three amino acid residue substitutions must be introduced into PPAD expressed by the type strains to obtain the super-active enzyme. Cumulatively, these results may lead to the development of novel prognostic tools to assess the progress of CP in the context of associated RA by analyzing the ppad genotype in CP patients infected with P. gingivalis.

Organotypic model of the gingiva for studying bacterial and viral pathogens implicated in periodontitis.

Background: Three-dimensional (3D) tissue models bridge the gap between conventional two-dimensional cell cultures and animal models. The aim of this study was to develop an organotypic 3D gingival (OTG) model to provide a tool to investigate bacterial and viral pathogens in periodontitis. Methods: The OTG model composed of gingival fibroblasts (GFs) and telomerase-immortalized gingival keratinocytes (TIGKs) was constructed and applied to study infections by Porphyromonas gingivalis and herpes simplex virus 1 (HSV-1). Immunohistochemical staining, confocal microscopy, qPCR, titration techniques, and colony-forming unit counts were applied to interrogate epithelial markers expression, monitor P. gingivalis and HSV-1 presence, and evaluate the immune response along with the efficiency of antimicrobial drugs. Results: The OTG model resembled the morphology of the human gingiva. During infection, both pathogens penetrated deep into the tissue and persisted for a few days with P. gingivalis also forming a biofilm on the cell surface. The infection triggered the expression of inflammatory mediators in cells and both pathogens were efficiently eliminated by specific antimicrobials. Conclusions: Presented OTG model constitutes a simple and convenient tool to study the interaction between bacterial and viral pathogens within the gingival tissue, including penetration, persistence and biofilm formation. It is also suitable to examine the efficiency of antimicrobial drugs.

Inhibition of SARS-CoV-2 infection by Porphyromonas gingivalis and the oral microbiome.

The COVID-19 pandemic persists despite the availability of vaccines, and it is therefore crucial to develop new therapeutic and preventive approaches. In this study, we investigated the potential role of the oral microbiome in SARS-CoV-2 infection. Using an in vitro SARS-CoV-2 pseudovirus infection assay, we found a potent inhibitory effect exerted by Porphyromonas gingivalis on SARS-CoV-2 infection mediated by known P. gingivalis compounds such as phosphoglycerol dihydroceramide (PGDHC) and gingipains as well as by unknown bacterial factors. We found that the gingipain-mediated inhibition of infection is likely due to cytotoxicity, while PGDHC inhibited virus infection by an unknown mechanism. Unidentified factors present in P. gingivalis supernatant inhibited SARS-CoV-2 likely via the fusion step of the virus life cycle. We addressed the role of other oral bacteria and found certain periodontal pathogens capable of inhibiting SARS-CoV-2 pseudovirus infection by inducing cytotoxicity on target cells. In the human oral cavity, we observed the modulatory activity of oral microbial communities varied among individuals in that some saliva-based cultures were capable of inhibiting while others were enhancing infection. These findings contribute to our understanding of the complex relationship between the oral microbiome and viral infections, offering potential avenues for innovative therapeutic strategies in combating COVID-19.

Neutrophil functions in patients with neutropenia due to glycogen storage disease type 1b treated with empagliflozin.

ABSTRACT: Neutropenia and neutrophil dysfunction in glycogen storage disease type 1b (GSD1b) are caused by the accumulation of 1,5-anhydroglucitol-6-phosphate in granulocytes. The antidiabetic drug empagliflozin reduces the concentration of 1,5-anhydroglucitol (1,5-AG), thus restoring neutrophil counts and functions, leading to promising results in previous case reports. Here, we present a comprehensive analysis of neutrophil function in 7 patients with GSD1b and 11 healthy donors, aiming to evaluate the immediate (after 3 months) and long-term (after 12 months) efficacy of empagliflozin compared with the reference treatment with granulocyte-colony stimulating factor (G-CSF). We found that most patients receiving G-CSF remained neutropenic with dysfunctional granulocytes, whereas treatment with empagliflozin increased neutrophil counts and improved functionality by inhibiting apoptosis, restoring phagocytosis and the chemotactic response, normalizing the oxidative burst, and stabilizing cellular and plasma levels of defensins and lactotransferrin. These improvements correlated with the decrease in serum 1,5-AG levels. However, neither G-CSF nor empagliflozin overcame deficiencies in the production of cathelicidin/LL-37 and neutrophil extracellular traps. Given the general improvement promoted by empagliflozin treatment, patients were less susceptible to severe infections. G-CSF injections were therefore discontinued in 6 patients (and the dose was reduced in the seventh) without adverse effects. Our systematic analysis, the most extensive reported thus far, has demonstrated the superior efficacy of empagliflozin compared with G-CSF, restoring the neutrophil population and normal immune functions. This trial was registered as EudraCT 2021-000580-78.

Screening and characterization of aptamers recognizing the periodontal pathogen Tannerella forsythia.

Periodontal disease is one of the most common forms of inflammation. It is currently diagnosed by observing symptoms such as gingival bleeding and attachment loss. However, the detection of biomarkers that precede such symptoms would allow earlier diagnosis and prevention. Aptamers are short oligonucleotides or peptides that fold into three-dimensional conformations conferring the ability to bind molecular targets with high affinity and specificity. Here we report the selection of aptamers that bind specifically to the bacterium Tannerella forsythia, a pathogen frequently associated with periodontal disease. Two aptamers with the highest affinity were examined in more detail, revealing that their binding is probably dependent on mirolysin, a surface-associated protease secreted by the T. forsythia type-9 secretion system. The aptamers showed minimal cross-reactivity to other periodontopathogens and are therefore promising leads for the development of new tools to study the composition of the periodontitis-associated dysbiotic bacteriome as well as inexpensive new diagnostic assays.

Gingival fibroblast activation by Porphyromonas gingivalis is driven by TLR2 and is independent of the LPS-TLR4 axis.

Gingival fibroblasts (GFs) are abundant structural cells of the periodontium that contribute to the host's innate immunity by producing cytokines and chemokines in response to oral pathogens, such as Porphyromonas gingivalis. Isolated lipopolysaccharide (Pg-LPS) is commonly used to study GF responses to P. gingivalis; however, this approach produced conflicting observations regarding its proinflammatory potential and the engagement of specific Toll-like receptors (TLRs). In this work, we demonstrate that commercially available Pg-LPS preparations are weak activators of GF innate immune responses compared with live P. gingivalis or other relevant virulence factors, such as P. gingivalis fimbriae or LPS from Escherichia coli. GF's nonresponsiveness to Pg-LPS can be only partly attributed to the low expression of TLR4 and its accessory molecules, CD14 and LY36, and is likely caused by the unique structure and composition of the Pg-LPS lipid A. Finally, we combined gene silencing and neutralizing antibody studies to demonstrate that GF response to infection with live P. gingivalis relies predominantly on TLR2. In contrast, the LPS-TLR4 signaling plays a negligible role in inflammatory cytokine production by GFs exposed to this oral pathogen, confirming that Pg-LPS stimulation is not an optimal model for studies of GF responses to P. gingivalis.