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Cell death is a natural consequence of infection. However, although the induction of cell death was solely thought to benefit the pathogen, compelling data now show that the activation of cell death pathways serves as a nuanced antimicrobial strategy that couples pathogen elimination with the generation of inflammatory cytokines and the priming of innate and adaptive cellular immunity. Following cell death, the phagocytic uptake of the infected dead cell by antigen‐presenting cells and the subsequent lysosomal fusion of the apoptotic body containing the pathogen serve as an important antimicrobial mechanism that furthers the development of downstream adaptive immune responses. Despite the complexity of regulated cell death pathways, pathogens are highly adept at evading them. Here, we provide an overview of the remarkable diversity of cell death and efferocytic pathways and discuss illustrative examples of virulence strategies employed by pathogens, including oral pathogens, to counter their activation and persist within the host. [ABSTRACT FROM AUTHOR]

Research in recent decades has brought significant advancements in understanding of the molecular basis of the etiology of autoimmune diseases, including rheumatoid arthritis, a common systemic disease in which an inappropriate or inadequate immune response to environmental challenges leads to joint destruction. Recent studies have indicated that the classical viewpoint of the immunological processes underpinning the pathobiology of rheumatoid arthritis is restricted and needs to be expanded to include a more holistic and interdisciplinary approach incorporating bacteria-induced inflammatory reactions as an important pathway in rheumatoid arthritis etiology. Here, we discuss in detail data showing the clinical and molecular association of rheumatoid arthritis development with periodontal diseases. We also describe the unique role of periopathogens, which have been proposed to be crucial in the initiation and progression of this autoimmune pathological disorder. [ABSTRACT FROM AUTHOR]

RagA and RagB proteins are major components of the outer membrane of the oral pathogen Porphyromonas gingivalis and, while recently suggested to represent a novel peptide uptake system, their full function is still under investigation. Herein, we (a) discuss the evidence that the rag locus contributes to P. gingivalis virulence; (b) provide insight to Rag protein potential biological function in macromolecular transport and other aspects of bacterial physiology; (c) address the host response to Rag proteins which are immunodominant and immunomodulatory; and (d) review the potential of Rag‐focused therapeutic strategies for the control of periodontal diseases. [ABSTRACT FROM AUTHOR]

Gingipains are extracellular cysteine proteases of the oral pathogen Porphyromonas gingivalis and are its most potent virulence factors. They can degrade a great variety of host proteins, thereby helping the bacterium to evade the host immune response, deregulate signaling pathways, trigger anoikis and, finally, cause tissue destruction. Host cell-surface proteins targeted by gingipains are the main focus of this review and span three groups of substrates: immune-regulatory proteins, signaling pathways regulators and adhesion molecules. The analysis of published data revealed that gingipains predominantly inactivate their substrates by cleaving them at one or more sites, or through complete degradation. Sometimes, gingipains were even found to initially shed their membrane substrates, but this was mostly just the first step in the degradation of cell-surface proteins. [ABSTRACT FROM AUTHOR]

Pathogens have developed sophisticated strategies to evade the immune response, among which manipulation of host cellular epigenetic mechanisms plays a prominent role. In the last decade, modulation of histone acetylation in host cells has emerged as an efficient strategy of bacterial immune evasion. Virulence factors and metabolic products of pathogenic microorganisms alter expression and activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs) to suppress transcription of host defense genes through epigenetic changes in histone acetylation marks. This new avenue of pathogen-host interactions is particularly important in light of introduction of HDAC inhibitors into clinical practice. Considerable effort is currently being applied to better understand the effects of HDAC inhibitors on the quality of immune responses to pathogens and to characterize the therapeutic potential of these compounds in microbial infections. In this review, we will discuss the recently discovered mechanisms utilized by bacteria to facilitate their survival within infected hosts through subversion of the host acetylation system and the effects of acetylation modulators, including HDAC inhibitors and bromodomain-containing BET protein inhibitors, on innate immune responses against microbial pathogens. Integration of these two lines of experimental evidence provides critical information on the perspectives of epigenetic therapies targeting protein acetylation in infectious diseases. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA), an autoimmune disease that affects ∼1% of the human population, is driven by autoantibodies that target modified self-epitopes, whereas ∼11% of the global adult population are affected by severe chronic periodontitis, a disease in which the commensal microflora on the tooth surface is replaced by a dysbiotic consortium of bacteria that promote the chronic inflammatory destruction of periodontal tissue. Despite differences in aetiology, RA and periodontitis are similar in terms of pathogenesis; both diseases involve chronic inflammation fuelled by pro-inflammatory cytokines, connective tissue breakdown and bone erosion. The two diseases also share risk factors such as smoking and ageing, and have strong epidemiological, serological and clinical associations. In light of the ground-breaking discovery that Porphyromonas gingivalis, a pivotal periodontal pathogen, is the only human pathogen known to express peptidylarginine deiminase, an enzyme that generates citrullinated epitopes that are recognized by anti-citrullinated protein antibodies, a new paradigm is emerging. In this Review, the clinical and experimental evidence supporting this paradigm is discussed and the potential mechanisms involved in linking periodontitis to RA are presented. [ABSTRACT FROM AUTHOR]

Recent reports focusing on virulence factors of periodontal pathogens implicated proteinases as major determinants of remarkable pathogenicity of these species, with special emphasis on their capacity to modulate complement activity. In particular, bacteriamediated cleavage of C5 and subsequent release of C5a seems to be an important phenomenon in the manipulation of the local inflammatory response in periodontitis. In this study, we present mirolysin, a novel metalloproteinase secreted by Tannerella forsythia, a well-recognized pathogen strongly associated with periodontitis. Mirolysin exhibited a strong effect on all complement pathways. It inhibited the classical and lectin complement pathways due to efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4, whereas inhibition of the alternative pathway was caused by degradation of C5. This specificity toward complement largely resembled the activity of a previously characterized metalloproteinase of T. forsythia, karilysin. Interestingly, mirolysin released the biologically active C5a peptide in human plasma and induced migration of neutrophils. Importantly, we demonstrated that combination of mirolysin with karilysin, as well as a cysteine proteinase of another periodontal pathogen, Prevotella intermedia, resulted in a strong synergistic effect on complement. Furthermore, mutant strains of T. forsythia, devoid of either mirolysin or karilysin, showed diminished survival in human serum, providing further evidence for the synergistic inactivation of complement by these metalloproteinases. Taken together, our findings on interactions of mirolysin with complement significantly add to the understanding of immune evasion strategies of T. forsythia and expand the knowledge on molecular mechanisms driving pathogenic events in the infected periodontium. [ABSTRACT FROM AUTHOR]

The skin constitutes a formidable barrier against commensal and pathogenic bacteria, which permanently and transiently colonise the skin, respectively. Commensal and pathogenic species inhabiting skin both express proteases. Whereas proteases secreted by commensals contribute to homeostatic bacterial coexistence on skin, proteases from pathogenic bacteria are used as virulence factors, helping them colonise skin with breached integrity of the epithelial layer. From these initial sites of colonisation, pathogens can disseminate into deeper layers of skin, possibly leading to the spread of infection. Secreted bacterial proteases probably play an important role in this process and in the deterrence of innate defence mechanisms. For example, Staphylococcus aureus proteases are essential for changing the bacterial phenotype from adhesive to invasive by degrading adhesins on the bacterial cell surface. Secreted staphylococcal proteases mediate pathogen penetration by degrading collagen and elastin, essential components of connective tissue in the dermis. The activation of the contact system and kinin generation by Streptococcus pyogenes and S. aureus proteases contributes to an inflammatory reaction manifested by oedema, redness and pain. Kinin-enhanced vascular leakage might help bacteria escape into the circulation thereby causing possible systemic dissemination of the infection. The inflammatory reaction can also be fueled by the activation of protease-activated receptors on keratinocytes. Concomitantly, bacterial proteases are involved in degrading antimicrobial peptides, disarming the complement system and neutrophils and preventing the infiltration of the infected sites with immune cells by inactivation of chemoattractants. Together, this provides protection for colonising and/or invading pathogens from attack by antibacterial forces of the skin. [ABSTRACT FROM AUTHOR]

The human immune system has evolved a variety of mechanisms for the primary task of neutralizing and eliminating microbial intruders. As the first line of defense, the complement system is responsible for rapid recognition and opsonization of bacteria, presentation to phagocytes and bacterial cell killing by direct lysis. All successful human pathogens have mechanisms of circumventing the antibacterial activity of the complement system and escaping this stage of the immune response. One of the ways in which pathogens achieve this is the deployment of proteases. Based on the increasing number of recent publications in this area, it appears that proteolytic inactivation of the antibacterial activities of the complement system is a common strategy of avoiding targeting by this arm of host innate immune defense. In this review, we focus on those bacteria that deploy proteases capable of degrading complement system components into non-functional fragments, thus impairing complement-dependent antibacterial activity and facilitating pathogen survival inside the host. [ABSTRACT FROM AUTHOR]

Tannerella forsythia is a poorly studied pathogen despite being one of the main causes of periodontitis, which is an inflammatory disease of the supporting structures of the teeth. We found that despite being recognized by all complement pathways, T. forsythia is resistant to killing by human complement, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with karilysin, a metalloproteinase of T. forsythia, resulted in a decrease in bactericidal activity of the serum. T. forsythia strains expressing karilysin at higher levels were more resistant than low-expressing strains. Furthermore, the low-expressing strain was significantly more opsonized with activated complement factor 3 and membrane attack complex from serum compared with the other strains. The high-expressing strain was more resistant to killing in human blood. The protective effect of karilysin against serum bactericidal activity was attributable to its ability to inhibit complement at several stages. The classical and lectin complement pathways were inhibited because of the efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4 by karilysin, whereas inhibition of the terminal pathway was caused by degradation of C5. Interestingly, karilysin was able to release biologically active C5a peptide in human plasma and induce migration of neutrophils. Importantly, we detected the karilysin gene in >90% of gingival crevicular fluid samples containing T. forsythia obtained from patients with periodontitis. Taken together, the newly characterized karilysin appears to be an important virulence factor of T. forsythia and might have several important implications for immune evasion. [ABSTRACT FROM AUTHOR]

The article focuses on the concepts of the two innate immune components which are the toll-like receptors (TLRs) and the complement systems. It defines innate immunity as a phylogenetically ancient system of host defense and an inherited resistance to infection. It discusses concepts on how the systems relates with periodontal bacteria. It further presents a brief information on the interaction of TLRs and complement system with periodontal bacteria.

Periodontitis is an inflammatory disease of the supporting structures of the teeth caused by, among other pathogens, Prevotella intermedia. Many strains of P. intermedia are resistant to killing by the human complement system, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with recombinant cysteine protease of P. intermedia (interpain A) resulted in a drastic decrease in bactericidal activity of the serum. Furthermore, a clinical strain 59 expressing interpain A was more serum-resistant than another clinical strain 57, which did not express interpain A, as determined by Western blotting. Moreover, in the presence of the cysteine protease inhibitor E64, the killing of strain 59 by human serum was enhanced. Importantly, we found that the majority of P. intermedia strains isolated from chronic and aggressive periodontitis carry and express the interpain A gene. The protective effect of interpain A against serum bactericidal activity was found to be attributable to its ability to inhibit all three complement pathways through the efficient degradation of the a-chain of C3—the major complement factor common to all three pathways. P. intermedia has been known to co-aggregate with P. gingivalis, which produce gingipains to efficiently degrade complement factors. Here, interpain A was found to have a synergistic effect with gingipains on complement degradation. In addition, interpain A was able to activate the C1 complex in serum, causing deposition of C1q on inert and bacterial surfaces, which may be important at initial stages of infection when local inflammatory reaction may be beneficial for a pathogen. Taken together, the newly characterized interpain A proteinase appears to be an important virulence factor of P. intermedia. [ABSTRACT FROM AUTHOR]

Abstract: Physiologically the only acceptable fate for almost all damaged or unwanted cells is their apoptotic death, followed by engulfment of the corpses by healthy neighbors or professional phagocytes. Efficient clearance of cells that have succumbed to apoptosis is crucial for normal tissue homeostasis, and for the modulation of immune responses. The disposal of apoptotic cells is finely regulated by a highly redundant system of receptors, bridging molecules and ‘eat me’ signals. The complexity of the system is reflected by the term: ‘engulfment synapse’, used to describe the interaction between a phagocytic cell and its target. In healthy humans, dying neutrophils are the most abundant and important targets for such recognition and engulfment. In inflammation the scope and importance of this complicated task is further increased. Paradoxically, despite growing evidence highlighting the priority of neutrophils clearance, the recognition of these cells by phagocytes is not as well understood as the recognition of other apoptotic cell types. New findings indicate that the interaction of phosphatidylserine (PS) on apoptotic neutrophils with its receptor on macrophages is not as critical for the specific clearance of neutrophil corpses it was previously believed. In this review we focus on recent findings regarding alternative, PS-independent “eat me” signals expressed on neutrophils during cell death and activation. Based on our own research, we emphasize the clearance of dying neutrophils, especially at the focus of bacterial infection; and the associated inflammatory reaction, which occurs in a highly proteolytic milieu containing both host and bacteria-derived proteinases. In these environments, eat-me signals expressed by neutrophils are drastically modified; arguing against the phospholipid-based detection of apoptotic cells, but supporting the importance of proteinaceous ligand(s) for the recognition of neutrophils by macrophages. In this context we discuss the effect of the gingipain R (Rgp) proteinases from Porphyromonas gingivalis on neutrophils interactions with macrophages. Since the recognition of apoptotic neutrophils is an important fundamental process, serving multiple functions in the regulation of immunity and homeostasis, we hypothesize that many pathogenic bacteria may have developed similar strategies to confuse macrophage-neutrophil interaction as a common pathogenic strategy. [Copyright &y& Elsevier]

Pseudomonas aeruginosa secretes several proteases considered as important virulence factors. In this report we present data indicating that two key proinflammatory cytokines, interleukin-6 (IL-6) and IL-8, are substrates for pseudolysin (elastase) and aeruginolysin (alkaline protease). While IL-6 was totally digested by both proteases, a long form of IL-8 (IL-8-77) was first rapidly processed into a 72-residue form with enhanced chemokine activity, then very slowly degraded. Interestingly, aeruginolysin bearing two additional residues at the N-terminus (Leu-Lys-aeruginolysin) in the absence of calcium degraded both IL-6 and IL-8-72 far more efficiently than the shorter form of the enzyme. [ABSTRACT FROM AUTHOR]

The genes encoding secreted, broad-spectrum activity cysteine proteases of Staphylococcus spp. (staphopains) and Streptococcus pyogenes (streptopain, SpeB) are genetically linked to genes encoding cytoplasmic inhibitors. While staphopain inhibitors have lipocalin-like folds, streptopain is inhibited by a protein bearing the scaffold of the enzyme profragment. Bioinformatic analysis of other prokaryotic genomes has revealed that two more species may utilize this same genetic arrangement to control streptopain-like proteases with lipocalin-like inhibitors, while three other species may employ a C-terminally located domain that resembles the profragment. This apparently represents a novel system that bacteria use to control the intracellular activity of their proteases. [ABSTRACT FROM AUTHOR]

Staphostatins are the endogenous, highly specific inhibitors of staphopains, the major secreted cysteine proteases from Staphylococcus aureus. We have previously shown that staphostatins A and B are competitive, active site-directed inhibitors that span the active site clefts of their target proteases in the same orientation as substrates. We now report the crystal structure of staphostatin B in complex with wild-type staphopain B at 1.9 Å resolution. In the complex structure, the catalytic residues are found in exactly the positions that would be expected for uncomplexed papain-type proteases. There is robust, continuous density for the staphostatin B binding loop and no indication for cleavage of the peptide bond that comes closest to the active site cysteine of staphopain B. The carbonyl carbon atom C of this peptide bond is 4.1 Å away from the active site cysteine sulfur Sγ atom. The carbonyl oxygen atom O of this peptide bond points away from the putative oxyanion hole and lies almost on a line from the Sγ atom to the C atom. The arrangement is strikingly similar to the ‘ion-molecule’ arrangement for the complex of papain-type enzymes with their substrates but differs significantly from the arrangement conventionally assumed for the Michaelis complex of papain-type enzymes with their substrates and also from the arrangement that is crystallographically observed for complexes of standard mechanism inhibitors and their target serine proteases. [ABSTRACT FROM AUTHOR]

A previously identified gene sprE of Enterococcus faecalis strain OG1 was shown to encode an extracellular serine protease that appears to belong to the glutamyl endopeptidase I staphylococcal group. A single form of SprE with a molecular mass of 25 kDa and a pH optimum between 7.0 and 7.5 was isolated from culture supernatant of wild-type E. faecalis strain OG1RF (TX4002); this form was apparently generated by cleavage of the Ser-1-Leu-1 and Arg230-Leu231 peptide bonds of the secreted zymogen. In contrast, the culture supernatant of the gelatinase-null mutant, TX5264, with a nonpolar deletion of gelE which encodes the E. faecalis gelatinase, was found to contain several forms of SprE proteolytically processed on both the N and C termini; in addition to a full-length zymogen and a truncated zymogen, three mature forms of the SprE proteinase, Leu¹-Ala237, Ser-1-Glu227, and Leu¹-Glu227, were identified. As with the V8 proteinase of Staphylococcus aureus, the closest homologue of SprE, all of the active forms cleaved specifically Glu-Xaa peptide bonds but with substantially different efficiencies, while none was able to hydrolyze peptide bonds with Asp in the P1 position. The most active of all these enzyme forms against several substrates, including human fibrinogen and s-chain insulin, was the Ser-1-Glu227 (-1S-SprE) isolated from TX5264; -1S-SprE, in contrast to other forms of SprE, was unstable at 37°C, apparently due to autodegradation. In conclusion, our results demonstrate that sprE encodes a highly specific serine-type glutamyl endopeptidase, the maturation of which is dependent on the presence of gelatinase. In the absence of gelatinase activity, the aberrant processing of pro-SprE results in the appearance of a ‘superactive’ form of the enzyme, -1S-SprE. [ABSTRACT FROM AUTHOR]

Kinins are released from kininogens through the activation of the Hageman factor-prekallikrein system or by tissue kallikrein. These peptides exert various biological activities, such as vascular permeability increase, smooth muscle contraction, pain sensation and induction of hypotension. In many instances kinins are thought to be involved in the pathophysiology of various diseases. Recent studies have revealed that microbial and human cell proteinases activate Hageman factor and/or prekallikrein, or directly release kinin from kininogens. This review discusses the activation of the kinin-release system by mast-cell tryptase and microbial proteinases, including gingipains, which are cysteine proteinases from Porphyromonas gingivalis , the major pathogen of periodontal disease. Each enzyme is evaluated in the context of its association to allergy and infectious diseases, respectively. Furthermore, a novel system of kinin generation directly from kininogens by the concerted action of two proteinases is described. An interesting example of this system with implications to bacterial pathogenicity is the release of kinins from kininogens by neutrophil elastase and a synergistic action of cysteine proteinases from Staphylococcus aureus . This alternative production of kinins by proteinases present in diseased sites indicates a significant contribution of proteinases other than kallikreins in kinin generation. Therefore kinin receptor antagonists and proteinase inhibitors may be useful as therapeutic agents. [ABSTRACT FROM AUTHOR]

Discusses the role of bacterial proteinases in matrix destruction and modulation of host responses. Evasion of host defense mechanisms; Mechanism of gingipain inference with host defense system; Enhancement of pathogen dissemination from site of infection to systemic circulation.

Examines the role of cysteine proteinases (gingipains R) in the pathogenesis of Porphyromonas gingivalis-mediated periodontal disease. Association of proteinase/hemagglutinin with the bacterial surface; Location of the functional subdomains within the hemagglutinin domain; Biological functions of the arginine- and lysine-specific cysteine proteinases; Clinical hallmarks of periodontitis.

Presents a study which examined the role of individual components of the complexes in hemagglutination (HA) and the binding activities of proteinase-adhesin complexes from Porphyromonas gingivalis. How P. gingivalis obtains nutrients and evade host defense mechanisms; Proposal from the International Union of Biochemistry; Results of study.

Epigenetic mechanisms, namely DNA and histone modifications, are critical regulators of immunity and inflammation which have emerged as potential targets for immunomodulating therapies. The prevalence and significant morbidity of periodontitis, in combination with accumulating evidence that genetic, environmental and lifestyle factors cannot fully explain the susceptibility of individuals to disease development, have driven interest in epigenetic regulation as an important factor in periodontitis pathogenesis. Aberrant promoter methylation profiles of genes involved in inflammatory activation, including TLR2, PTGS2, IFNG, IL6, IL8, and TNF, have been observed in the gingival tissue, peripheral blood or buccal mucosa from patients with periodontitis, correlating with changes in expression and disease severity. The expression of enzymes that regulate histone acetylation, in particular histone deacetylases (HDACs), is also dysregulated in periodontitis-affected gingival tissue. Infection of gingival epithelial cells, gingival fibroblasts and periodontal ligament cells with the oral pathogens Porphyromonas gingivalis or Treponema denticola induces alterations in expression and activity of chromatin-modifying enzymes, as well as site-specific and global changes in DNA methylation profiles and in histone acetylation and methylation marks. These epigenetic changes are associated with excessive production of inflammatory cytokines, chemokines, and matrix-degrading enzymes that can be suppressed by small molecule inhibitors of HDACs (HDACi) or DNA methyltransferases. HDACi and inhibitors of bromodomain-containing BET proteins ameliorate inflammation, osteoclastogenesis, and alveolar bone resorption in animal models of periodontitis, suggesting their clinical potential as host modulation therapeutic agents. However, broader application of epigenomic methods will be required to create a comprehensive map of epigenetic changes in periodontitis. The integration of functional studies with global analyses of the epigenetic landscape will provide critical information on the therapeutic and diagnostic potential of epigenetics in periodontal disease. [ABSTRACT FROM AUTHOR]

Several studies have shown a clear association between periodontal disease and increased risk of cardiovascular disease. Porphyromonas gingivalis (Pg), a key oral pathogen, and its cell surface‐expressed gingipains, induce oedema in a zebrafish larvae infection model although the mechanism of these vascular effects is unknown. Here, we aimed to determine whether Pg‐induced vascular damage is mediated by gingipains. In vitro, human endothelial cells from different vascular beds were invaded by wild‐type (W83) but not gingipain‐deficient (ΔK/R‐ab) Pg. W83 infection resulted in increased endothelial permeability as well as decreased cell surface abundance of endothelial adhesion molecules PECAM‐1 and VE‐cadherin compared to infection with ΔK/R‐ab. In agreement, when transgenic zebrafish larvae expressing fluorescently labelled PECAM‐1 or VE‐cadherin were systemically infected with W83 or ΔK/R‐ab, a significant reduction in adhesion molecule fluorescence was observed specifically in endothelium proximal to W83 bacteria through a gingipain‐dependent mechanism. Furthermore, this was associated with increased vascular permeability in vivo when assessed by dextran leakage microangiography. These data are the first to show that Pg directly mediates vascular damage in vivo by degrading PECAM‐1 and VE‐cadherin. Our data provide a molecular mechanism by which Pg might contribute to cardiovascular disease. [ABSTRACT FROM AUTHOR]

Periodontitis is a widespread chronic inflammatory disease caused by a changed dysbiotic oral microbiome. Although multiple species and risk factors are associated with periodontitis, Porphyromonas gingivalis has been identified as a keystone pathogen. The immune-modulatory function of P. gingivalis is well characterized, but the mechanism by which this bacterium secretes peptidyl arginine deiminase (PPAD), a protein/peptide citrullinating enzyme, thus contributing to the infinite feed-forward loop of inflammation, is not fully understood. To determine the functional role of citrullination in periodontitis, neutrophils were stimulated by P. gingivalis bearing wild-type PPAD and by a PPAD mutant strain lacking an active enzyme. Flow cytometry showed that PPAD contributed to prolonged neutrophil survival upon bacterial stimulation, accompanied by the secretion of aberrant IL-6 and TNF-α. To further assess the complex mechanism by which citrullination sustains a chronic inflammatory state, the ROS production and phagocytic activity of neutrophils were evaluated. Flow cytometry and colony formation assays showed that PPAD obstructs the resolution of inflammation by promoting neutrophil survival and the release of pro-inflammatory cytokines, while enhancing the resilience of the bacteria to phagocytosis. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis is an oral pathogen that promotes dysbiosis by quenching the bactericidal activity of the host immune system while maintaining chronic inflammation, leading to periodontitis. This involves the secretion of virulence factors such as P. gingivalis peptidyl arginine deiminase (PPAD), which converts the C‐terminal Arg residues of bacterial and host‐derived proteins and peptides into citrulline. We have previously shown that PPAD activity and major fimbriae (containing FimA) are necessary for P. gingivalis to activate Toll‐like receptor 2 (TLR2). TLR2 is an important component of the innate immune system and plays a predominant role in the recognition of P. gingivalis by host cells. Here, we extend those findings to show that P. gingivalis strains deficient for PPAD and fimbriae induced almost identical transcriptional profiles in infected primary human gingival fibroblasts (PHGFs), but these differed substantially from the transcriptome elicited by the wild‐type ATCC 33277 strain. Apparently, PPAD‐modified fimbriae trigger the host cell response to P. gingivalis, as confirmed by showing that the proinflammatory host cell response mediated by TLR2 is dependent on PPAD activity and the presence of fimbriae, with type I fimbriae as the most potent TLR2 activators. We also found that PPAD‐modified accessory fimbrial subunits (FimC, FimD, and FimE) alone or in combination are TLR2 ligands in a reporter cell line. Although FimA polymerization to form the fimbrial shaft was not required for TLR2 activation, the secretion and proteolytic maturation of FimA were necessary for signaling by accessory Fim proteins. This was supported by showing that the proinflammatory activation of PHGFs is dependent on PPAD and accessory fimbrial subunits. We conclude that accessory fimbrial subunits are modified by PPAD and stimulate the response to P. gingivalis infection in a TLR2‐dependent manner. [ABSTRACT FROM AUTHOR]

Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation, and tissue remodeling. Aberrant proteolytic activity of HNE contributes to organ destruction in various chronic inflammatory diseases including emphysema, asthma, and cystic fibrosis. Therefore, elastase inhibitors could alleviate the progression of these disorders. Here, we used the systematic evolution of ligands by exponential enrichment to develop ssDNA aptamers that specifically target HNE. We determined the specificity of the designed inhibitors and their inhibitory efficacy against HNE using biochemical and in vitro methods, including an assay of neutrophil activity. Our aptamers inhibit the elastinolytic activity of HNE with nanomolar potency and are highly specific for HNE and do not target other tested human proteases. As such, this study provides lead compounds suitable for the evaluation of their tissue-protective potential in animal models. [ABSTRACT FROM AUTHOR]

The Msp protein complex and the serine protease dentilisin are the best‐characterized virulence factors in Treponema denticola, the major etiological agent of chronic periodontitis. In addition to these outer sheath factors, the cysteine protease dentipain contributes to pathogenicity, but its secretion, processing, cellular localization, and role in T. denticola virulence are not fully understood. In this study, we found that full‐sized dentipain (74‐kDa) and the 52‐kDa truncated form of the enzyme are located, respectively, in the outer sheath derived from T. denticola dentilisin‐ and the Msp‐deficient mutants. Furthermore, dentipain was barely detected in the wild‐type strain. These results suggest that dentilisin and Msp, the major outer sheath proteins, are involved in the secretion and maturation of dentipain. Inactivation of the dentipain gene slowed the growth of T. denticola, and the effect was more profound in serum‐free medium than in serum‐containing medium. Several genes, including those encoding transporters and methyl‐accepting chemotaxis proteins, were differentially expressed in the dentipain‐deficient mutant. Furthermore, the mutant strain was more hydrophobic than the wild‐type strain. Finally, the mutant showed less autoaggregation activity and adhesion to IgG in a serum‐free medium than the wild‐type strain. These findings suggest that dentipain contributes to the virulence of T. denticola by facilitating adhesion and acquisition of nutrients essential for colonization and proliferation in the gingival crevice under serum‐rich conditions. [ABSTRACT FROM AUTHOR]

Metalloproteases derived from microbial pathogens are important virulence factors contributing to evasion of antimicrobial mechanisms of the innate immune system. Karilysin is a metalloprotease recently discovered in the periodonto-pathogen Tanneralla forsythia and currently no monoclonal antibodies exist against karilysin, which is a gap in the molecular toolbox for structure-function studies of karilysin. In this study we have used phage display for fast selection of single domain antibodies (VHs) towards the karilysin catalytic domain (Kly18) using a human domain library based on a VH framework. Following five panning rounds, phage clones were sequenced, and three unique sequences were identified (termed Kly18-VHI-III). Initial screens identified Kly18-VHII-phage as capable of inhibiting Kly18 proteolytic activity. The free Kly18-VHII was expressed in the periplasmic space of BL21 E. coli using the pET22b (+) vector and purified by IMAC and the inhibition capacity of purified Kly18-VHII was confirmed. The data presented in this study provides input to the molecular toolbox for the study of karilysin and Kly18-VHII could serve as a lead molecule for development of a karilysin-specific inhibitor. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that respond to pathogen-associated molecular patterns (PAMPs). The recognition of specific microbial ligands by TLRs triggers an innate immune response and also promotes adaptive immunity, which is necessary for the efficient elimination of invading pathogens. Successful pathogens have therefore evolved strategies to subvert and/or manipulate TLR signaling. Both the impairment and uncontrolled activation of TLR signaling can harm the host, causing tissue destruction and allowing pathogens to proliferate, thus favoring disease progression. In this context, microbial proteases are key virulence factors that modify components of the TLR signaling pathway. In this review, we discuss the role of bacterial and viral proteases in the manipulation of TLR signaling, highlighting the importance of these enzymes during the development of infectious diseases. [ABSTRACT FROM AUTHOR]

Gingival fibromatosis is a rare and heterogeneous group of disorders that develop as slowly progressive, local or diffuse enlargements within marginal and attached gingiva or interdental papilla. In severe cases, the excess tissue may cover the crowns of the teeth, thus causing functional, esthetic, and periodontal problems, such as bone loss and bleeding, due to the presence of pseudopockets and plaque accumulation. It affects both genders equally. Hereditary, drug-induced, and idiopathic gingival overgrowth have been reported. Hereditary gingival fibromatosis can occur as an isolated condition or as part of a genetic syndrome. The pathologic manifestation of gingival fibromatosis comprises excessive accumulation of extracellular matrix proteins, of which collagen type I is the most prominent example. Mutation in the Son-of-Sevenless-1 gene has been suggested as one possible etiological cause of isolated (non-syndromic) hereditary gingival fibromatosis, but mutations in other genes are also likely to be involved, given the heterogeneity of this condition. The most attractive concept of mechanism for drug-induced gingival overgrowth is epithelial-to-mesenchymal transition, a process in which interactions between gingival cells and the extracellular matrix are weakened as epithelial cells transdifferentiate into fibrogenic fibroblast-like cells. The diagnosis is mainly made on the basis of the patient's history and clinical features, and on histopathological evaluation of affected gingiva. Early diagnosis is important, mostly to exclude oral malignancy. Differential diagnosis comprises all pathologies in the mouth with excessive gingival overgrowth. Hereditary gingival fibromatosis may present as an autosomal-dominant or less commonly autosomal-recessive mode of inheritance. If a systemic disease or syndrome is suspected, the patient is directed to a geneticist for additional clinical examination and specialized diagnostic tests. Treatments vary according to the type of overgrowth and the extent of disease progression, thus, scaling of teeth is sufficient in mild cases, while in severe cases surgical intervention is required. Prognosis is precarious and the risk of recurrence exists. [ABSTRACT FROM AUTHOR]

Background: Prevotella intermedia is a Gram-negative black-pigmenting oral anaerobe associated with periodontitis in humans, and has a haem requirement for growth, survival and virulence. It produces an iron porphyrincontaining pigment comprising monomeric iron (III) protoporphyrin IX (Fe(III)PPIX.OH; haematin). The bacterium expresses a 90-kDa cysteine protease termed interpain A (InpA) which both oxidizes and subsequently degrades haemoglobin, releasing haem. However, it is not known whether the enzyme may play a role in degrading other haem-carrying plasma proteins present in the gingival sulcus or periodontal pocket from which to derive haem. This study evaluated the ability of InpA to degrade apo- and haem-complexed albumin. Results: Albumin breakdown was examined over a range of pH and in the presence of reducing agent; conditions which prevail in sub- and supra-gingival plaque. InpA digested haemalbumin more efficiently than apoalbumin, especially under reducing conditions at pH 7.5. Under these conditions InpA was able to substantially degrade the albumin component of whole human plasma. Conclusions: The data point to InpA as an efficient "albuminase" with the ability to degrade the minor fraction of haem-bound albumin in plasma. InpA may thus contribute significantly to haem acquisition by P. intermedia under conditions of low redox potential and higher pH in the inflamed gingival crevice and diseased periodontal pocket where haem availability is tightly controlled by the host. [ABSTRACT FROM AUTHOR]

Highlights: [•] Optimal protocol for freeze-drying that allows safe and effective preservation of HCoV-NL63 infectious material was developed. [•] Lyophilized virus preparations can be stored either at ambient temperature or at +4°C. In the latter case samples may be stored for at least two months. [•] HCoV-NL63 virions are exquisitely stable in liquid media and can be stored also without preservatives at ambient temperature for up to 14 days. [ABSTRACT FROM AUTHOR]

The Porphyromonas gingivalis type IX secretion system (T9SS) promotes periodontal disease by secreting gingipains and other virulence factors. By in situ cryoelectron tomography, we report that the P. gingivalis T9SS consists of 18 PorM dimers arranged as a large, caged ring in the periplasm. Near the outer membrane, PorM dimers interact with a PorKN ring complex of ∼52 nm in diameter. PorMKN translocation complexes of a given T9SS adopt distinct conformations energized by the proton motive force, suggestive of different activation states. At the inner membrane, PorM associates with a cytoplasmic complex that exhibits 12-fold symmetry and requires both PorM and PorL for assembly. Activated motors deliver substrates across the outer membrane via one of eight Sov translocons arranged in a ring. The T9SSs are unique among known secretion systems in bacteria and eukaryotes in their assembly as supramolecular machines composed of apparently independently functioning translocation motors and export pores. [ABSTRACT FROM AUTHOR]

Abstract: Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease. [Copyright &y& Elsevier]

Abstract: Human coronavirus NL63 was identified in 2004 in the Netherlands. Due to the high prevalence and world-wide distribution of this pathogen, it is essential to develop a sensitive and specific detection assay suitable for use in a routine diagnostic laboratory. Techniques based on PCR or real-time PCR are laborious and expensive. Detailed analysis of the HCoV-NL63 genome permitted the identification of a conserved nucleic acid sequential motif, which was sufficient for the design of a loop-mediated isothermal amplification (LAMP) assay. Evaluation of the method showed that the test is specific to HCoV-NL63 and that it does not cross-react with other respiratory viruses. The detection limit was found to be 1 copy of RNA template per reaction in cell culture supernatants and clinical specimens. [Copyright &y& Elsevier]

Abstract: In metazoan organisms protein inhibitors of peptidases are important factors essential for regulation of proteolytic activity. In vertebrates genes encoding peptidase inhibitors constitute up to 1% of genes reflecting a need for tight and specific control of proteolysis especially in extracellular body fluids. In stark contrast unicellular organisms, both prokaryotic and eukaryotic consistently contain only few, if any, genes coding for putative peptidase inhibitors. This may seem perplexing in the light of the fact that these organisms produce large numbers of proteases of different catalytic classes with the genes constituting up to 6% of the total gene count with the average being about 3%. Apparently, however, a unicellular life-style is fully compatible with other mechanisms of regulation of proteolysis and does not require protein inhibitors to control their intracellular and extracellular proteolytic activity. So in prokaryotes occurrence of genes encoding different types of peptidase inhibitors is infrequent and often scattered among phylogenetically distinct orders or even phyla of microbiota. Genes encoding proteins homologous to alpha-2-macroglobulin (family I39), serine carboxypeptidase Y inhibitor (family I51), alpha-1-peptidase inhibitor (family I4) and ecotin (family I11) are the most frequently represented in Bacteria. Although several of these gene products were shown to possess inhibitory activity, with an exception of ecotin and staphostatins, the biological function of microbial inhibitors is unclear. In this review we present distribution of protein inhibitors from different families among prokaryotes, describe their mode of action and hypothesize on their role in microbial physiology and interactions with hosts and environment. [ABSTRACT FROM AUTHOR]

The article discusses the role of gingipains, a cysteine protease produced by porphyromonas gingivalis, in the development of periodontal infection. It states that gingipains mediate the adherence of P. gingivalis to different sites within the oral cavity by acting as nonfimbrial adhesions or enabling the assembly of fimbriae. Meanwhile, gingipains attach themselves to the extracellular matrix proteins and trigger apoptosis by attacking adhesion molecules used by the host cells.

Porphyromonas gingivalis HmuY is a putative heme-binding lipoprotein associated with the outer membrane. It is part of an operon together with a gene encoding an outer-membrane hemin utilization receptor (HmuR) and four uncharacterized genes. A similar operon organization was found in Bacteroides fragilis and B. thetaiotaomicron, with the former containing an additional HmuY homologue encoded upstream of the hmuR-like gene. In P. gingivalis cultured under heme-limited conditions, a ∼1-kb hmuY transcript was produced at high levels along with some ∼3.5 and ∼9-kb transcripts. Compared with the parental strain, mutants deficient in hmuY or hmuR or hmuY– hmuR gene function grew more slowly and bound lower amounts of hemin and hemoglobin. Significantly, they grew more slowly or were unable to grow when human serum was used as the sole iron/heme source. Analysis of the hmu promoter showed that it is regulated by iron. The HmuY protein normally occurs as a homodimer, but in the presence of hemin it may form tetramers. These results show that HmuY may be the first reported member of a new class of proteins in Porphyromonas and Bacteroides species involved in heme utilization, a function being exerted in conjunction with HmuR, an outer-membrane heme transporter. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus infection triggers polyclonal B-cell activation. It was sought to further characterize the hypergammaglobulinemia seen in Staphylococcus aureus infection, focusing on the significance of protease-specific B-cell responses. Sera from mice infected with Staphylococcus aureus wild-type strain 8325-4 and two of its isogenic mutants devoid of protease expression were analyzed for the occurrence of polyclonal B-cell activation and the presence of specific antibodies against a set of exoproteases and superantigens. Furthermore, the functional properties of anti-V8-protease antibodies were analyzed in vitro. Polyclonal activation was manifested by increased levels of total serum IgG and IgM in all infected animals and by antibodies to staphylococcal toxins and aureolysin whether these antigens were present in the inoculate or not. Importantly, Staphylococcus aureus mutant lacking the V8-protease did not trigger a response against this enzyme. In contrast, strains expressing the V8-protease elicited V8-protease antibodies, proving the antigen-specific nature of this response. In vitro tests revealed that these antibodies had the capacity to inhibit the V8 protease activity in a dose-dependent manner. It was concluded that exposure to Staphylococcus aureus, in addition to a massive polyclonal B-cell response, gives rise to production of exoprotease-specific antibodies displaying functional properties. [ABSTRACT FROM AUTHOR]

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase found in human plasma, presumably as an inactive zymogen. The current dogma is that proteolytic activation by thrombin/thrombomodulin generates the active enzyme (TAFIa), which down-regulates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin. In this study, we have shown that the zymogen exhibits continuous and stable carboxypeptidase activity against large peptide substrates, and we suggest that the activity down-regulates fibrinolysis in vivo. [ABSTRACT FROM AUTHOR]

Heme utilization in Porphyromonas gingivalis requires the participation of an outer membrane hemin/hemoglobin receptor, HmuR, the lysine-specific gingipain proteinase (Kgp) and arginine-specific gingipain proteinase (Rgp). In this study, the expression of hmuR , kgp and rgpA genes in response to growth with different heme sources was examined by reverse transcription-polymerase chain reaction and enzyme-linked immunoassay. Coordinate regulation of hmuR , kgp and rgpA gene expression was evaluated through utilization of P. gingivalis hmuR and kgp mutants or by selective inactivation of proteinases with Kgp- and Rgp-specific inhibitors. We observed that expression of the kgp and rgpA genes was not tightly regulated by heme, but rather by the growth phase. In contrast, expression of the hmuR gene was negatively regulated by heme, while growth of P. gingivalis with human serum resulted in increased hmuR expression. A P. gingivalis kgp isogenic mutant demonstrated significantly increased hmuR gene expression, and inactivation of Kgp and Rgp activity by specific inhibitors up-regulated hmuR gene transcription. Moreover, inactivation of Kgp up-regulated rgpA transcription. Finally, a P. gingivalis hmuR mutant exhibited repressed kgp gene expression and lysine-specific proteinase activity. Collectively, these results indicate that kgp , rgpA and hmuR gene transcription is coordinately regulated and may facilitate greater efficiency of heme utilization in P. gingivalis . [ABSTRACT FROM AUTHOR]

Staphylococcus aureus has several extracellular proteases with proposed roles in virulence. SspA (serine protease), SspB (cysteine protease) and Aur (metalloprotease) have been characterized previously and SspA and SspB were found to be cotranscribed. The coding region for the cysteine protease ScpA has been identified and characterized. It is in a probable bi-cistronic operon with scpA located immediately upstream of a coding region for a 108 aa protein that is a specific inhibitor of ScpA. Using primer extension analysis promoters have been mapped and it was found that σ[sup A] is the only sigma factor involved in the transcription of scpA, sspABC and aur. The transcription of all the genes occurs maximally at post-exponential phase, being positively regulated by agr (accessory gene regulator) and negatively regulated by sara (staphylococcal accessory regulator). Furthermore σ[sup B] represses transcription from the aur and scp operons similarly to the previously shown effect on ssp [Horsburgh, M., Aish, J., White, I., Shaw, L., Lithgow, J. & Foster, S. (2002). J Bacteriol 184, 5457-5467]. Using mutations in each protease gene the proteolytic cascade of activation has been analysed. Aur, SspA, SspB and ScpA are all produced as zymogens, activated by proteolytic cleavage. Although the metalloprotease, Aur, does catalyse activation of the SspA zymogen, it is not the sole agent capable of conducting this process. Site-directed mutagenesis revealed that Aur is not capable of undergoing auto-proteolysis to achieve activation. The cysteine protease, ScpA, appears to reside outside this cascade of activation, as mature ScpA was observed in the aur, sspA and sspB mutant strains. Using a mouse abscess model, it has been shown that insertional inactivation of sspA or sspB results in significant attenuation of virulence, whilst mutations in aur or scpA do not. It is likely the attenuation observed in the sspA strain is due to polarity on the sspB gene. [ABSTRACT FROM AUTHOR]