Search

Accurate, robust, and rapid diagnostics is the basis for all well-functioning healthcare. There is a large need in point-of-care biosensors to facilitate diagnosis and reduce the need for cumbersome laboratory equipment. Proteases are key virulence factors in periodontitis. Periodontal disease is very common and characterized by inflammation and infection in the tooth-supporting structures and is linked to many systemic diseases such as cardiovascular disease, diabetes, and Alzheimer's disease. Proteases present in periodontal disease, gingipains, are highly responsible for the disease onset and progression and are therefore a promising biomarker. Here we show a novel nanopore-based biosensor strategy for protease activity monitoring. Solid-state nanopores were modified with a proteolytic substrate, restricting the ionic current through the apertures of the nanopores. Protease can digest the proteolytic substrate thus enlarge the aperture and the ionic current. Trypsin was used as an initial model protease to investigate the performance of the sensor. We show that the solid-state nanoporebiosensor can detect trypsin with a limit of detection (LOD) of 0.005 ng/mL (0.2 pM). The detection system developed for the model enzyme was then applied to the detection of gingipains. The LOD for detection of gingipains was 1 ng/mL (0.02 nM), with a 27% recovery of the signal at 0.1 mu g/mL, indicating that the sensitivity and dynamic range are relevant for the clinical diagnosis of periodontitis. The generic detection of protease activity and high sensitivity make this a promising sensor technology for both diagnosis of periodontal disease and monitoring of other disease-related proteases., Funding agency:Wallenberg Academy Fellow Program KAW 2020.0190 KAW 2016.0231

Neuronal lesions in Parkinson’s disease (PD) are commonly associated with α-synuclein (α-Syn)-induced cell damage that are present both in the central and peripheral nervous systems of patients, with the enteric nervous system also being especially vulnerable. Here, we bring together evidence that the development and presence of PD depends on specific sets of interlinking factors that include neuroinflammation, systemic inflammation, α-Syn-induced cell damage, vascular dysfunction, iron dysregulation, and gut and periodontal dysbiosis. We argue that there is significant evidence that bacterial inflammagens fuel this systemic inflammation, and might be central to the development of PD. We also discuss the processes whereby bacterial inflammagens may be involved in causing nucleation of proteins, including of α-Syn. Lastly, we review evidence that iron chelation, pre-and probiotics, as well as antibiotics and faecal transplant treatment might be valuable treatments in PD. A most important consideration, however, is that these therapeutic options need to be validated and tested in randomized controlled clinical trials. However, targeting underlying mechanisms of PD, including gut dysbiosis and iron toxicity, have potentially opened up possibilities of a wide variety of novel treatments, which may relieve the characteristic motor and nonmotor deficits of PD, and may even slow the progression and/or accompanying gut-related conditions of the disease.

Periodontitis is an inflammatory oral disease that affects a large part of the adult population, causing significant costs and suffering. The key pathogen, Porphyromonas gingivalis, secretes gingipains, which are highly destructive proteases and the most important virulence factors in the pathogenesis of the disease. Currently, periodontitis is diagnosed mainly by mechanical manual probing and radiography, often when the disease has already progressed significantly. The possibilities of detecting gingipain activity in gingival fluid could enable early-stage diagnosis and facilitate treatment. Here, we describe a sensitive nanoparticle-based nanoplasmonic biosensor for the detection of the proteolytic activity of gingipains. Gold nanoparticles (AuNPs) were self-assembled as a submonolayer in multiwell plates and further modified with casein or IgG. The proteolytic degradation of the protein coating was tracked by monitoring the shift in the localized surface plasmon resonance (LSPR) peak position. The sensor performance was investigated using model systems with trypsin and purified gingipains (subtypes Kgp and RgpB) and further validated using supernatants from cultures of P. gingivalis. Proteolytic degradation by proteases in buffer results in a concentration- and time-dependent blueshift of the LSPR band of about 1-2 nm when using casein as a substrate. In bacterial supernatants, the degradation of the protein coating resulted in unspecific binding of proteins present in the complex sample matrix to the nanoparticles, which instead triggered a redshift of about 2 nm of the LSPR band. A significant LSPR shift was seen only in samples with gingipain activity. The sensor showed a limit of detection < 0.1 mu g/mL (4.3 nM), which is well below gingipain concentrations detected in severe chronic periodontitis cases (similar to 50 mu g/mL). This work shows the possibility of developing cost-effective nanoparticle-based biosensors for rapid detection of protease activit

Background and objective: Several studies have shown an association between periodontitis and cardiovascular disease (CVD). Atherosclerosis is the major cause of CVD and a key event in the development of atherosclerosis is accumulation of lipoproteins within the arterial wall. Bacteria are the primary etiologic agents in periodontitis and Porphyromonas gingivalis (P. gingivalis) is the major pathogen in the disease. Several studies support the role of modified LDL in atherogenesis, however, the pathogenic stimuli that induce the changes and the mechanisms by which this occur are unknown. This study aims at identifying alterations in plasma lipoproteins induced by the periodontopathic bacteria P. gingivalis in vitro.Material and Methods: Plasma lipoproteins were isolated from whole blood treated with wild type and gingipain-mutants (lacking either the Rgp- or Kgp gingipains) of P. gingivalis by density/gradient-ultracentrifugation and studied using two-dimensional gel electrophoresis followed by matrix-assisted laser desorption ionization mass spectrometry. P. gingivalis-induced lipid peroxidation and antioxidant levels were measured by thiobarbituric acid reactive substances (TBARs) and antioxidant assay kits, respectively, and lumiaggregometry was used for reactive oxygen species (ROS) and aggregation.Results: P. gingivalis exerted substantial proteolytic effects on the lipoproteins. The Rgp gingipains were responsible for producing two apoE, as well as two apoB-100, fragments in LDL, and the Kgp gingipain produced an un-identified fragment in HDL. P. gingivalis and its different gingipain variants induced ROS, and consumed antioxidants. Both the Rgp and Kgp gingipains were involved in inducing lipid peroxidation.Conclusions: P. gingivalis has the potential to change the expression of lipoproteins in blood which may represent a crucial link between periodontitis and cardiovascular disease.

Background and objective: Several studies have shown an association between periodontitis and cardiovascular disease (CVD). Atherosclerosis is the major cause of CVD and a key event in the development of atherosclerosis is accumulation of lipoproteins within the arterial wall. Bacteria are the primary etiologic agents in periodontitis and Porphyromonas gingivalis (P. gingivalis) is the major pathogen in the disease. Several studies support the role of modified LDL in atherogenesis, however, the pathogenic stimuli that induce the changes and the mechanisms by which this occur are unknown. This study aims at identifying alterations in plasma lipoproteins induced by the periodontopathic bacteria P. gingivalis in vitro.Material and Methods: Plasma lipoproteins were isolated from whole blood treated with wild type and gingipain-mutants (lacking either the Rgp- or Kgp gingipains) of P. gingivalis by density/gradient-ultracentrifugation and studied using two-dimensional gel electrophoresis followed by matrix-assisted laser desorption ionization mass spectrometry. P. gingivalis-induced lipid peroxidation and antioxidant levels were measured by thiobarbituric acid reactive substances (TBARs) and antioxidant assay kits, respectively, and lumiaggregometry was used for reactive oxygen species (ROS) and aggregation.Results: P. gingivalis exerted substantial proteolytic effects on the lipoproteins. The Rgp gingipains were responsible for producing two apoE, as well as two apoB-100, fragments in LDL, and the Kgp gingipain produced an un-identified fragment in HDL. P. gingivalis and its different gingipain variants induced ROS, and consumed antioxidants. Both the Rgp and Kgp gingipains were involved in inducing lipid peroxidation.Conclusions: P. gingivalis has the potential to change the expression of lipoproteins in blood which may represent a crucial link between periodontitis and cardiovascular disease.

BACKGROUND AND OBJECTIVE: Several studies have shown an association between periodontitis and cardiovascular disease (CVD). Atherosclerosis is the major cause of CVD, and a key event in the development of atherosclerosis is accumulation of lipoproteins within the arterial wall. Bacteria are the primary etiologic agents in periodontitis and Porphyromonas gingivalis is the major pathogen in the disease. Several studies support a role of modified low-density lipoprotein (LDL) in atherogenesis; however, the pathogenic stimuli that induce the changes and the mechanisms by which this occur are unknown. This study aims to identify alterations in plasma lipoproteins induced by the periodontopathic species of bacterium, P. gingivalis, in vitro. MATERIAL AND METHODS: Plasma lipoproteins were isolated from whole blood treated with wild-type and gingipain-mutant (lacking either the Rgp- or Kgp gingipains) P. gingivalis by density/gradient-ultracentrifugation and were studied using 2-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry. Porphyromonas gingivalis-induced lipid peroxidation and antioxidant levels were measured by thiobarbituric acid-reactive substances and antioxidant assay kits, respectively, and lumiaggregometry was used for measurement of reactive oxygen species (ROS) and aggregation. RESULTS: Porphyromonas gingivalis exerted substantial proteolytic effects on the lipoproteins. The Rgp gingipains were responsible for producing 2 apoE fragments, as well as 2 apoB-100 fragments, in LDL, and the Kgp gingipain produced an unidentified fragment in high-density lipoproteins. Porphyromonas gingivalis and its different gingipain variants induced ROS and consumed antioxidants. Both the Rgp and Kgp gingipains were involved in inducing lipid peroxidation. CONCLUSIONS: Porphyromonas gingivalis has the potential to change the expression of lipoproteins in blood, which may represent a crucial link between periodontitis and

The pathogenicity of the periodontal biofilm is highly dependent on a few key species, of which Porphyromonas gingivalis is considered to be one of the most important pathogens. P. gingivalis expresses a broad range of virulence factors, of these cysteine proteases (gingipains) are of special importance both for the bacterial survival/proliferation and for the pathological outcome. Several cell types, for example, epithelial cells, endothelial cells, dendritic cells, osteoblasts, and fibroblasts, reside in the periodontium and are part of the innate host response, as well as platelets, neutrophils, lymphocytes, and monocytes/macrophages. These cells recognize and respond to P. gingivalis and its components through pattern recognition receptors (PRRs), for example, Toll-like receptors and protease-activated receptors. Ligation of PRRs induces downstream-signaling pathways modifying the activity of transcription factors that regulates the expression of genes linked to inflammation. This is followed by the release of inflammatory mediators, for example, cytokines and reactive oxygen species. Periodontal disease is today considered to play a significant role in various systemic conditions such as cardiovascular disease (CVD). The mechanisms by which P. gingivalis and its virulence factors interact with host immune cells and contribute to the pathogenesis of periodontitis and CVD are far from completely understood., Periodontitis - A Useful Reference is a comprehensive book compiled by a team of experts with the objective of providing an overview of the basic pathology of "periodontitis" and its implication on oral health and general systemic health. Periodontitis has become a global health burden in recent days. It is noteworthy that oral health is being considered as the mirror of general health and the study of oral-systemic health connections has advanced among scientists, clinicians, and the public as well. We wish the array of chapters that highlights the importance and impact of periodontal health could be a useful guide for the community of public, students, and clinicians.

The pathogenicity of the periodontal biofilm is highly dependent on a few key species, of which Porphyromonas gingivalis is considered to be one of the most important pathogens. P. gingivalis expresses a broad range of virulence factors, of these cysteine proteases (gingipains) are of special importance both for the bacterial survival/proliferation and for the pathological outcome. Several cell types, for example, epithelial cells, endothelial cells, dendritic cells, osteoblasts, and fibroblasts, reside in the periodontium and are part of the innate host response, as well as platelets, neutrophils, lymphocytes, and monocytes/macrophages. These cells recognize and respond to P. gingivalis and its components through pattern recognition receptors (PRRs), for example, Toll-like receptors and protease-activated receptors. Ligation of PRRs induces downstream-signaling pathways modifying the activity of transcription factors that regulates the expression of genes linked to inflammation. This is followed by the release of inflammatory mediators, for example, cytokines and reactive oxygen species. Periodontal disease is today considered to play a significant role in various systemic conditions such as cardiovascular disease (CVD). The mechanisms by which P. gingivalis and its virulence factors interact with host immune cells and contribute to the pathogenesis of periodontitis and CVD are far from completely understood., Periodontitis - A Useful Reference is a comprehensive book compiled by a team of experts with the objective of providing an overview of the basic pathology of "periodontitis" and its implication on oral health and general systemic health. Periodontitis has become a global health burden in recent days. It is noteworthy that oral health is being considered as the mirror of general health and the study of oral-systemic health connections has advanced among scientists, clinicians, and the public as well. We wish the array of chapters that highlights the importance and impact of periodontal health could be a useful guide for the community of public, students, and clinicians.

Periodontitis is one of the most common adult infections. Duing bacteremia in healthy individuals or patients with chronic periodontitis, a number of oral bacteria such as Porphyromonas gingivalis encounter inflammatory cells in the blood eg. platelets, neutrophils and monocytes. Although several studies have suggested an association between periodontitis and cardiovascular diseases, the infection and inflammatory mechanisms are poorly understood. Hence, the aim of this thesis was to elucidate the mechanisms that are involved in P. gingivalis interaction with blood leukocytes, in order to further understand the molecular pathogenesis that renders periodontitis as a risk factor for several systemic conditions. We have demonstrated that P. gingivalis induces ROS production in neutrophils, THP1 cells and in whole blood, through activation of pattern recognition receptors, such as toll-like receptors, nuclear oligomerizing domains and protease- activated receptors. Besides, we have also shown that monocytes secrete IL-1β and CXCL8 in response to P. gingivalis. Both these cytokines prime neutrophils, endothelial cells and other vascular cells in an autocrine and paracrine manner. P. gingivalis has a plethora of virulence factors of which gingipains are very unique. In addition to activating inflammatory signalling pathways in cells, gingipains also regulate CXCL8 and IL-1β, thereby curtailing the host defence strategies. We demonstrated that oxidized LDL, but not native LDL, induces IL-1β release and CD36 expression on THP1 cells. Furthermore, LDL mildly modifies P. gingivalis-induced inflammatory responses as well as CD36 expression in THP1 cells. We also observed that P. gingivalis is eliminated mainly by phagocytosis in neutrophils. In summary, these studies clarify the mechanisms of interaction between P. gingivalis and leukocytes, which can increase the understanding of the pathogenesis of periodontitis and associated systemic disorders.

Porphyromonas gingivalis utilises the Bacteroidetes-specific Type IX Secretion System (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo post-translational modification with anionic lipopolysaccharide (A-LPS) and are attached to the OM. Comparative analyses of several Bacteroidetes genomes identified PG1058 as a candidate for a novel component of the T9SS. Inactivation of pg1058 in P. gingivalis resulted in slowed growth, loss of colonial pigmentation and surface-associated gingipain activity, a phenotype common to T9SS mutants. This study revealed normal cell architecture and no difference in OM solute permeability between the wild-type and pg1058- mutant. Thus the mutant phenotype was unlikely due to structural effects on cell division or perturbation of OM integrity causing aberrant assembly and function of the T9SS. T9SS substrates accumulated within the pg1058- mutant periplasm indicated perturbed T9SS function and the Kgp gingipain was shown to be absent from the cell surface, whilst A-LPS was present. This indicated that PG1058 is crucial for export of T9SS substrates but not for the export and surface-association of A-LPS. PG1058 was localised as an OM-associated periplasmic protein. Several proteins crucial to the T9SS and modification processes have been identified in the OM including PorT, PorV and PorU the CTD peptidase, that is itself a substrate of the T9SS. Increased abundance of PorT and PorV was seen in the pg1058- mutant, along with several other T9SS components. PorU accumulated in the periplasm with other T9SS substrates. The localisation and abundance of PG1058 in porV-, porT- and porU- mutants was not grossly altered. Bioinformatic analyses revealed that numerous Bacteroidetes possess multiple PG1058 homologues which cor

Platelets are considered to have important functions in inflammatory processes and as actors in the innate immunity. Several studies have shown associations between cardiovascular disease and periodontitis, where the oral anaerobic pathogen Porphyromonas gingivalis has a prominent role in modulating the immune response. Porphyromonas gingivalis has been found in atherosclerotic plaques, indicating spreading of the pathogen via the circulation, with an ability to interact with and activate platelets via e.g. Toll-like receptors (TLR) and protease-activated receptors. We aimed to evaluate how the cysteine proteases, gingipains, of P.gingivalis affect platelets in terms of activation and chemokine secretion, and to further investigate the mechanisms of platelet-bacteria interaction. This study shows that primary features of platelet activation, i.e. changes in intracellular free calcium and aggregation, are affected by P.gingivalis and that arg-gingipains are of great importance for the ability of the bacterium to activate platelets. The P.gingivalis induced a release of the chemokine RANTES, however, to a much lower extent compared with the TLR2/1-agonist Pam(3)CSK(4), which evoked a time-dependent release of the chemokine. Interestingly, the TLR2/1-evoked response was abolished by a following addition of viable P.gingivalis wild-types and gingipain mutants, showing that both Rgp and Kgp cleave the secreted chemokine. We also demonstrate that Pam(3)CSK(4)-stimulated platelets release migration inhibitory factor and plasminogen activator inhibitor-1, and that also these responses were antagonized by P.gingivalis. These results supports immune-modulatory activities of P.gingivalis and further clarify platelets as active players in innate immunity and in sensing bacterial infections, and as target cells in inflammatory reactions induced by P.gingivalis infection., Funding Agency:Foundation of Olle Engkvist

Background: Porphyromonas gingivalis is an important bacterial etiological agent involved in periodontitis. The bacterium expresses two kinds of cysteine proteases called gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). This study evaluated the interaction between P. gingivalis and THP-1 cells, a widely used monocytic cell line, in vitro with a focus on CXCL8 at the gene and protein levels and its fate thereafter in cell culture supernatants. THP-1 cells were stimulated with viable and heat-killed wild-type strains ATCC 33277 or W50 or viable isogenic gingipain mutants of W50, E8 (Rgp mutant) or K1A (Kgp mutant), for 24 hours. Results: ELISA and qPCR results show an elevated CXCL8 expression and secretion in THP-1 cells in response to P. gingivalis, where the heat-killed ATCC33277 and W50 induced higher levels of CXCL8 in comparison to their viable counterparts. Furthermore, the Kgp-deficient mutant K1A caused a higher CXCL8 response compared to the Rgp-deficient E8. Chromogenic quantification of lipopolysaccharide (LPS) in supernatant showed no significant differences between viable and heat killed bacteria except that W50 shed highest levels of LPS. The wild-type strains secreted relatively more Rgp during the co-culture with THP-1 cells. The CXCL8 degradation assay of filter-sterilized supernatant from heat-killed W50 treated cells showed that Rgp was most efficient at CXCL8 hydrolysis. Of all tested P. gingivalis strains, adhesion and internalization in THP-1 cells was least conspicuous by Rgp-deficient P. gingivalis (E8), as demonstrated by confocal imaging. Conclusions: W50 and its Kgp mutant K1A exhibit a higher immunogenic and proteolytic function in comparison to the Rgp mutant E8. Since K1A differs from E8 in the expression of Rgp, it is rational to conclude that Rgp contributes to immunomodulation in a more dynamic manner in comparison to Kgp. Also, W50 is a more virulent strain when compared to the laboratory strain ATCC33277., Funding Agencies:Foundation of Olle EngkvistKnowledge Foundation