Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase.

Citrullination is a post-translational modification of higher organisms that deiminates arginines in proteins and peptides. It occurs in physiological processes but also pathologies such as multiple sclerosis, fibrosis, Alzheimer's disease and rheumatoid arthritis (RA). The reaction is catalyzed by peptidylarginine deiminases (PADs), which are found in vertebrates but not in lower organisms. RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis. Uniquely among microbes, P. gingivalis secretes a PAD, termed PPAD (Porphyromonas peptidylarginine deiminase), which is genetically unrelated to eukaryotic PADs. Here, we studied function of PPAD and its substrate-free, substrate-complex, and substrate-mimic-complex structures. It comprises a flat cylindrical catalytic domain with five-fold α/β-propeller architecture and a C-terminal immunoglobulin-like domain. The PPAD active site is a funnel located on one of the cylinder bases. It accommodates arginines from peptide substrates after major rearrangement of a "Michaelis loop" that closes the cleft. The guanidinium and carboxylate groups of substrates are tightly bound, which explains activity of PPAD against arginines at C-termini but not within peptides. Catalysis is based on a cysteine-histidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes. We provide a working mechanism hypothesis based on 18 structure-derived point mutants

The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal \beta-sandwich domain

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway

Effect of actin C-terminal modification on tropomyosin isoforms binding and thin filament regulation

Tropomyosins, a family of actin-binding regulatory proteins, are present in muscle and non-muscle cells. Multiple tropomyosin (TM) isoforms differ in actin affinity and regulatory properties, but little is known about the molecular bases of these differences. The C-terminus of actin stabilizes contacts between actin subunits in the filament and interacts with myosin and regulatory proteins. The goal of this work was to reveal how structural changes in actin and differences between TM isoforms affect binding between these proteins and affect thin filament regulation. Actin proteolytically truncated by three C-terminal amino acids exhibited 1.2–1.5 fold reduced affinity for non-muscle and smooth muscle tropomyosin isoforms. The truncation increased the cooperativity of myosin S1-induced tropomyosin binding for short tropomyosins (TM5a and TM1b9a), but it was neutral for long isoforms (smTM and TM2). Actin modification affected regulation of actomyosin ATPase activity in the presence of all tropomyosins by shifting the filament into a more active state. We conclude that the integrity of the actin C-terminus is important for actin–tropomyosin interactions, however the increased affinity of tropomyosin binding in the S1-induced state of the filament appears not to be involved in the tropomyosin isoform-dependent mechanism of the actomyosin ATPase activation

Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase.

Citrullination is a post-translational modification of higher organisms that deiminates arginines in proteins and peptides. It occurs in physiological processes but also pathologies such as multiple sclerosis, fibrosis, Alzheimer's disease and rheumatoid arthritis (RA). The reaction is catalyzed by peptidylarginine deiminases (PADs), which are found in vertebrates but not in lower organisms. RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis. Uniquely among microbes, P. gingivalis secretes a PAD, termed PPAD (Porphyromonas peptidylarginine deiminase), which is genetically unrelated to eukaryotic PADs. Here, we studied function of PPAD and its substrate-free, substrate-complex, and substrate-mimic-complex structures. It comprises a flat cylindrical catalytic domain with five-fold α/β-propeller architecture and a C-terminal immunoglobulin-like domain. The PPAD active site is a funnel located on one of the cylinder bases. It accommodates arginines from peptide substrates after major rearrangement of a "Michaelis loop" that closes the cleft. The guanidinium and carboxylate groups of substrates are tightly bound, which explains activity of PPAD against arginines at C-termini but not within peptides. Catalysis is based on a cysteine-histidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes. We provide a working mechanism hypothesis based on 18 structure-derived point mutants

Citrullination and proteolytic processing of chemokines by Porphyromonas gingivalis

The outgrowth of Porphyromonas gingivalis within the inflammatory subgingival plaque is associated with periodontitis characterized by periodontal tissue destruction, loss of alveolar bone, periodontal pocket formation, and eventually, tooth loss. Potential virulence factors of P. gingivalis are peptidylarginine deiminase (PPAD), an enzyme modifying free or peptide-bound arginine to citrulline, and the bacterial proteases referred to as gingipains (Rgp and Kgp). Chemokines attract leukocytes during inflammation. However, posttranslational modification (PTM) of chemokines by proteases or human peptidylarginine deiminases may alter their biological activities. Since chemokine processing may be important in microbial defense mechanisms, we investigated whether PTM of chemokines by P. gingivalis enzymes occurs. Upon incubation of interleukin-8 (IL-8; CXCL8) with PPAD, only minor enzymatic citrullination was detected. In contrast, Rgp rapidly cleaved CXCL8 in vitro. Subsequently, different P. gingivalis strains were incubated with the chemokine CXCL8 or CXCL10 and their PTMs were investigated. No significant CXCL8 citrullination was detected for the tested strains. Interestingly, although considerable differences in the efficiency of CXCL8 degradation were observed with full cultures of various strains, similar rates of chemokine proteolysis were exerted by cell-free culture supernatants. Sequencing of CXCL8 incubated with supernatant or bacteria showed that CXCL8 is processed into its more potent forms consisting of amino acids 6 to 77 and amino acids 9 to 77 (the 6-77 and 9-77 forms, respectively). In contrast, CXCL10 was entirely and rapidly degraded by P. gingivalis, with no transient chemokine forms being observed. In conclusion, this study demonstrates PTM of CXCL8 and CXCL10 by gingipains of P. gingivalis and that strain differences may particularly affect the activity of these bacterial membrane-associated proteases

Peptidyl arginine deiminase from Porphyromonas gingivalis abolishes anaphylatoxin C5a activity

Evasion of killing by the complement system, a crucial part of innate immunity, is a key evolutionary strategy of many human pathogens. A major etiological agent of chronic periodontitis, the Gram-negative bacterium Porphyromonas gingivalis produces a vast arsenal of virulence factors that compromise human defense mechanisms. One of these is peptidylarginine deiminase (PPAD), an enzyme unique to P. gingivalis among bacteria, which converts Arg residues in polypeptide chains into citrulline. Here, we report that PPAD citrullination of a critical C-terminal arginine of the anaphylatoxin C5a disabled the protein function. Treatment of C5a with PPAD in vitro resulted in decreased chemotaxis of human neutrophils and diminished calcium signaling in monocytic cell line U937 transfected with the C5a receptor (C5aR) and loaded with a fluorescent intracellular calcium probe: Fura 2-AM. Moreover, a low degree of citrullination of internal arginine residues by PPAD was also detected using mass spectrometry. Further, after treatment of C5 with outer membrane vesicles (OMVs) naturally shed by P. gingivalis we observed generation of C5a totally citrullinated at the C-terminal Arg74 residue (Arg74Cit). In stark contrast only native C5a was detected after treatment with PPAD-null OMVs. Our study suggests reduced antibacterial and proinflammatory capacity of citrullinated C5a, achieved via lower level of chemotactic potential of the modified molecule, and weaker cell activation. In the context of previous studies, which showed crosstalk between C5aR and toll-like receptors, as well as enhanced arthritis development in mice infected with PPAD expressing P. gingivalis, our findings support a crucial role of PPAD in the virulence of P. gingivalis

Exposure to Ti4Al4V Titanium Alloy Leads to Redox Abnormalities, Oxidative Stress, and Oxidative Damage in Patients Treated for Mandible Fractures

Due to the high biotolerance, favourable mechanical properties, and osseointegration ability, titanium is the basic biomaterial used in maxillofacial surgery. The passive layer of titanium dioxide on the surface of the implant effectively provides anticorrosive properties, but it can be damaged, resulting in the release of titanium ions to the surrounding tissues. The aim of our work was to evaluate the influence of Ti6Al4V titanium alloy on redox balance and oxidative damage in the periosteum surrounding the titanium miniplates and screws as well as in plasma and erythrocytes of patients with mandibular fractures. The study included 31 previously implanted patients (aged 21–29) treated for mandibular fractures and 31 healthy controls. We have demonstrated increased activity/concentration of antioxidants both in the mandibular periosteum and plasma/erythrocytes of patients with titanium mandibular fixations. However, increased concentrations of the products of oxidative protein and lipid modifications were only observed in the periosteum of the study group patients. The correlation between the products of oxidative modification of the mandible and antioxidants in plasma/erythrocytes suggests a relationship between the increase of oxidative damage at the implantation site and central redox disorders in patients with titanium miniplates and screws

The Redox Balance in Erythrocytes, Plasma, and Periosteum of Patients with Titanium Fixation of the Jaw

Titanium miniplates and screws are commonly used for fixation of jaw fractured or osteotomies. Despite the opinion of their biocompatibility, in clinical practice symptoms of chronic inflammation around the fixation develop in some patients, even many years after the application of miniplates and screws. The cause of these complications is still an unanswered question. Taking into account that oxidative stress is one of the toxic action of titanium, we have evaluated the antioxidant barrier as well as oxidative stress in the erythrocytes, plasma and periosteum covering the titanium fixation of the jaw. The study group was composed of 32 patients aged 20–30 with inserted miniplates and screws. The antioxidant defense: catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase-1 (SOD1), uric acid (UA), total antioxidant capacity (TAC), as well as oxidative damage products: advanced oxidation protein products (AOPP), advanced glycation end products (AGE), dityrosine, kynurenine, N-formylkynurenine, tryptophan, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), total oxidant status (TOS), and oxidative status index (OSI) were evaluated. SOD1 activity (↓37%), and tryptophan levels (↓34%) showed a significant decrease while AOPP (↑25%), TOS (↑80%) and OSI (↑101%) were significantly elevated in maxillary periosteum of patients who underwent bimaxillary osteotomies as compared to the control group. SOD-1 (↓55%), TAC (↓58.6%), AGE (↓60%) and N-formylkynurenine (↓34%) was statistically reduced while AOPP (↑38%), MDA (↑29%), 4-HNE (↑114%), TOS (↑99%), and OSI (↑381%) were significantly higher in the mandibular periosteum covering miniplates/screw compared with the control tissues. There were no correlations between antioxidants and oxidative stress markers in the periosteum of all patients and the blood. As exposure to the Ti6Al4V titanium alloy leads to disturbances of redox balance in the periosteum surrounding titanium implants of the maxilla and the mandible so antioxidant supplementation should be recommended to the patients undergoing treatment of dentofacial deformities with the use of titanium implants. The results we obtained may also indicate a need to improve the quality of titanium jaw fixations through increase of TiO2 passivation layer thickness or to develop new, the most highly biodegradable materials for their production

Peptidylarginine deiminase from Porphyromonas gingivalis contributes to infection of gingival fibroblasts and induction of prostaglandin E_{2}-signaling pathway

Porphyromonas gingivalis (Pg) expresses the enzyme peptidylarginine deiminase (PPAD), which has a strong preference for C-terminal arginines. Due to the combined activity of PPAD and Arg-specific gingipains, Pg on the cell surface is highly citrullinated. To investigate the contribution of PPAD to the interaction of Pg with primary human gingival fibroblasts (PHGF) and Pg-induced synthesis of prostaglandin E2 (PGE2), PHGF were infected with wild-type Pg ATCC 33277, an isogenic PPAD-knockout strain (Δppad) or a mutated strain (C351A) expressing an inactive enzyme in which the catalytic cysteine has been mutated to alanine (PPAD(C351A)). Cells were infected in medium containing the mutants alone or in medium supplemented with purified, active PPAD. PHGF infection was assessed by colony-forming assay, microscopic analysis and flow cytometry. Expression of COX-2 and mPGES-1, key factors in the prostaglandin synthesis pathway, was examined by qRT-PCR, while PGE2 synthesis was evaluated by EIA. PHGF were infected more efficiently by wt-Pg than the Δppad strain, which correlated with strong induction of COX-2 and mPGES-1 expression by wt-Pg, but not by the PPAD activity-null mutant strains (ΔPPAD and C351A). The impaired ability of the ΔPPAD strain to adhere to and/or invade PHGF and both ΔPPAD and C351A to stimulate the PGE2-synthesis pathway was fully restored by the addition of purified PPAD. The latter effect was strongly inhibited by aspirin. Collectively, our results implicate PPAD activity, but not PPAD itself, as an important factor for gingival fibroblast infection and activation of PGE2 synthesis, the latter of which may strongly contribute to bone resorption and eventual tooth loss