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The main etiological agent of periodontitis is the anaerobic bacterium Porphyromonas gingivalis. Virulence of this pathogen is controlled by various mechanisms and executed by major virulence factors including the gingipain proteases, peptidylarginine deiminase (PPAD), and RagB, an outer membrane macromolecular transport component. Although the structures and functions of these proteins are well characterized, little is known about their posttranslational maturation. Here, we determined the phosphoproteome of P. gingivalis in which phosphorylated tyrosine residues constitute over 80% of all phosphoresidues. Multiple phosphotyrosines were found in gingipains, PPAD, and RagB. Although mutation of phosphorylated residues in PPAD and RagB had no effect on secretion or activity, site-directed mutagenesis showed that phosphorylation in hemagglutinin/adhesin domains of RgpA and Kgp, and in the catalytic domain of RgpB, had a strong influence on secretion, processing, and enzymatic activity. Moreover, preventing phosphorylation of one gingipain influenced the others, suggesting multiple phosphorylation-dependent pathways of gingipain maturation in P. gingivalis. Various candidate kinases including Ptk1 BY kinase and ubiquitous bacterial kinase 1 (UbK1) may be involved, but their contribution to gingipain processing and activation remains to be confirmed.

The PglZ family of proteins belongs to the alkaline phosphatase superfamily, which consists of metallohydrolases with limited sequence identity but similar metal-coordination architectures in otherwise divergent active sites. Proteins with a well-defined PglZ domain are ubiquitous among prokaryotes as essential components of BREX phage defence systems and two-component systems (TCSs). Whereas other members of the alkaline phosphatase superfamily are well characterized, the activity, structure and biological function of PglZ family proteins remain unclear. We therefore investigated the structure and function of PorX, an orphan response regulator of the Porphyromonas gingivalis TCS containing a putative PglZ effector domain. The crystal structure of PorX revealed a canonical receiver domain, a helical bundle, and an unprecedented PglZ domain, similar to the general organization of the phylogenetically related BREX-PglZ proteins. The PglZ domain of PorX features an active site cleft suitable for large substrates. An extensive search for substrates revealed that PorX is a phosphodiesterase that acts on cyclic and linear oligonucleotides, including signalling molecules such as cyclic oligoadenylates. These results, combined with mutagenesis, biophysical and enzymatic analysis, suggest that PorX coordinates oligonucleotide signalling pathways and indirectly regulates gene expression to control the secretion of virulence factors., National Science Centre, Poland [UMO-2015/19/N/NZ1/00322 to M.M., UMO-2016/23/N/NZ1/01513 to Z.N., UMO-2018/31/B/NZ1/03968 to J.P., UMO-2019/35/B/NZ1/03118 to M.K.]; Spanish Ministry of Science, Innovation and Universities [MCIN/AEI/ 10.13039/501100011033 ERDF ‘A way to make Europe’, BFU2015-70645-R, RTI2018-101015-B-100, PID2021-129038NB-I00, MDM-2014–0435 to M.S., BES-2013–063407 to C.S., PGC2018-101370-B-I00 and PID2021-128751NB-I00 (MICINN/AEI/FEDER/UE) to I.U.]; Generalitat de Catalunya [2014-SGR-99, 2017-SGR-1192 to M.S.]; European Social Fund [09.3.3-LMT-K-712-01-0126 to V.S.]; National Institute of Dental and Craniofacial Research (NIDCR) [DE012505, DE011111 to R.J.L.]; Polish Ministry of Science and Higher Education for the Faculty of Chemistry of Wroclaw University of Science and Technology. Funding for open access charge: National Science Centre, Poland [UMO-2018/31/B/NZ1/03968].

Significance The newly described type IX secretion systems (T9SSs) translocate virulence factors and can mediate specialized gliding motility among bacterial pathogens of the Fibrobacteres–Chlorobi–Bacterioidetes superphylum. We visualized the spatial organization of the T9SS in its native context in the Porphorymonas gingivalis cell by cryoelectron tomography. The T9SS exhibits distinct symmetries across the bacterial cell envelope: a cytoplasmic complex requiring PorL and PorM for assembly exhibits 12-fold symmetry; a periplasmic complex composed of PorM exhibits 18-fold symmetry and attaches to a PorKN ring near the outer membrane; and eight Sov translocons are arranged with 8-fold symmetry at the cell surface. The T9SS is the largest of the known bacterial secretion systems and evidently arranges as multiple, independently functioning translocation motors.

© 2021 Madej et al., Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two β-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggesting that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the “attachment complex” composed of PorU, PorZ, and the integral OM β-barrel proteins PorV and PorQ, which is involved in posttranslational modification and retention of T9SS cargos on the bacterial surface., This study was supported in part by grants from Spanish and Catalan public and private bodies (PID2019-107725RG-I00, 2017SGR3, and Fundació “La Marató de TV3” 201815 to F.X.G.-R.); National Science Centre, Poland grants UMO-2015/19/N/NZ1/ 00322 to M.M., UMO-2012/04/A/NZ1/00051 to J.P., and UMO-2016/23/N/NZ1/01513 to Z.N.; Polish Ministry of Science and Higher Education grant 1306/MOB/IV/2015/0 to M. K.; U.S. National Institutes of Health, NIDCR, grants DE09761 and DE026280 to J.P.; and Medical Research Council grant MR/P012175/2 to M.A.C.

Cargo proteins of the type IX secretion system (T9SS) in human pathogens from phylum Bacteroidetes invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of selected cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ, and found that it consists of two β-propeller moieties followed by a CTD. In this study, we performed structure-based modelling suggesting that PorZ is a carbohydrate-binding protein. We found that recombinant PorZ specifically binds A-LPS. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of the A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS–dependent manner. This interaction on the cell surface is crucial for the function of the attachment complex composed of PorU, PorZ, and the integral OM β-barrel proteins PorV and PorQ, which is involved in post-translational modification and retention of T9SS cargos on the bacterial surface.Author summaryBacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis. Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS– bound PorZ interacts with sortase with significantly greater affinity, facilitating modification of cargo proteins by the cell-surface attachment complex of the T9SS.

Porphyromonas gingivalis, an asaccharolytic member of the Bacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory diseases. P. gingivalis utilizes protease-generated peptides derived from extracellular proteins for growth, but how these peptides enter the cell is not clear. Here, we identify RagAB as the outer-membrane importer for these peptides. X-ray crystal structures show that the transporter forms a dimeric RagA(2)B(2) complex, with the RagB substrate-binding surface-anchored lipoprotein forming a closed lid on the RagA TonB-dependent transporter. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus provide direct evidence for a 'pedal bin' mechanism of nutrient uptake. Together with mutagenesis, peptide-binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic, selective outer-membrane oligopeptide-acquisition machine that is essential for the efficient utilization of proteinaceous nutrients by P. gingivalis.Porphyromonas gingivalis, an oral anaerobe involved in the pathogenesis of periodontitis, relies on extracellular proteases to degrade proteins into peptides for growth, but how these peptides enter the cell is unknown. Here, the authors identify RagAB as the outer-membrane importer for these peptides and solve its structure, elucidating that it works via a 'pedal bin' mechanism of nutrient uptake.

Smokers are more likely than non-smokers to harbour Porphyromonas gingivalis, they are more susceptible to destructive periodontal disease and smokers may, ultimately, benefit from tobacco-specific preventive and treatment strategies. A Mariner transposon insertion library for P. gingivalis ATCC 33277 was exploited to define 256 genes as essential for P. gingivalis survival in a tobacco-rich environment. Genes whose products play roles in protein transport and catabolism, nicotinamide processing, protection against oxidative stress, drug resistance, and transcriptional regulation have all been identified as essential for CSE survival. Many of these tobacco-essential genes are also requisite for epithelial colonization and abscess formation, suggestive of a core stress-related P. gingivalis genome. Single-gene deletions in several of the TnSeq-implicated genes led to significantly reduced P. gingivalis fitness upon competition with the parent strain, under conditions of cigarette smoke extract-induced stress (1,000 ng/ml nicotine equivalents). This study identifies, for the first time, a subset of P. gingivalis genes required for surviving the plethora of insults present in cigarette smoke. Such conditionally essential genes may delineate bacterial persistence strategies and represent novel therapeutic foci for the prevention of P. gingivalis infection and related diseases in smokers and in general.

Porphyromonas gingivalis is a key pathogen in chronic periodontitis and has recently been mechanistically linked to the development of rheumatoid arthritis via the activity of peptidyl arginine deiminase generating citrullinated epitopes in the periodontium. In this project the outer membrane vesicles (OMV) from P. gingivalis W83 wild-type (WT), a W83 knock-out mutant of peptidyl arginine deiminase (ΔPPAD), and a mutant strain expressing PPAD with the active site cysteine mutated to alanine (C351A), have been analyzed using a two-dimensional HFBA-based separation system combined with LC-MS. For optimal and positive identification and validation of citrullinated peptides and proteins, high resolution mass spectrometers and strict MS search criteria were utilized. This may have compromised the total number of identified citrullinations but increased the confidence of the validation. A new two-dimensional separation system proved to increase the strength of validation, and along with the use of an in-house build program, Citrullia, we establish a fast and easy semi-automatic (manual) validation of citrullinated peptides. For the WT OMV we identified 78 citrullinated proteins having a total of 161 citrullination sites. Notably, in keeping with the mechanism of OMV formation, the majority (51 out of 78) of citrullinated proteins were predicted to be exported via the inner membrane and to reside in the periplasm or being translocated to the bacterial surface. Citrullinated surface proteins may contribute to the pathogenesis of rheumatoid arthritis. For the C351A-OMV a single citrullination site was found and no citrullinations were identified for the ΔPPAD-OMV, thus validating the unbiased character of our method of citrullinated peptide identification.

Objectives To establish whether two families from Malopolska and Mazovia provinces in Poland are affected by hereditary gingival fibromatosis type 1, caused by a single-cytosine insertion in exon 21 of the Son-of-Sevenless-1 gene. Material and methods Six subjects with hereditary gingival fibromatosis and five healthy subjects were enrolled in the study. Gingival biopsies were collected during gingivectomy or tooth extraction and used for histopathological evaluation. Total RNA and genomic DNA were purified from cultured gingival fibroblasts followed by cDNA and genomic DNA sequencing and analysis. Results Hereditary gingival fibromatosis was confirmed by periodontal examination, X-ray, and laboratory tests. Histopathological evaluation showed hyperplastic epithelium, numerous collagen bundles, and abundant-to-moderate fibroblasts in subepithelial and connective tissue. Sequencing of exons 19-22 of the Son-of-Sevenless-1 gene did not reveal a single-cytosine insertion nor other mutations. Conclusions Patients from two Polish families under study had not been affected by hereditary gingival fibromatosis type 1, caused by a single-cytosine insertion in exon 21 of the Son-of-Sevenless-1 gene. Further studies of the remaining regions of this gene as well as of other genes are needed to identify disease-related mutations in these patients. This will help to unravel the pathogenic mechanism of gingival overgrowth.

Light-dependent protochlorophyllide oxidoreductase (POR, E.C. 1.3.1.33) is a plant enzyme that directly needs light to conduct a biochemical reaction. In the present paper we confirmed that POR forms large oligomers in solution before binding of substrates. We carried out the research using different techniques: cross-linking, native gel electrophoresis and FRET measurements. Mass spectrometry analysis of the cross-link products provided the first structural data about the organisation of the oligomer of POR. The results indicated that the catalytic motifs of the adjacent subunits become close to each other upon binding of substrates. Moreover, we identified two mutations of POR that disturbed its oligomerisation properties: Δ85–88 and Δ240–270. Additionally, a complete loss of the catalytic activity was observed for the following mutations: Δ189–194, Δ240–270, Δ318–331 and Δ392–393.

Porphyromonas gingivalis uses a type IX secretion system (T9SS) to deliver more than 30 proteins to the bacterial surface using a conserved C-terminal domain (CTD) as an outer membrane translocation signal. On the surface, the CTD is cleaved and an anionic lipopolysaccharide (A-PLS) is attached by PorU sortase. Among T9SS cargo proteins are cysteine proteases, gingipains, which are secreted as inactive zymogens requiring removal of an inhibiting N-terminal prodomain (PD) for activation. Here, we have shown that the gingipain proRgpB isolated from the periplasm of a T9SS-deficient P. gingivalis strain was stable and did not undergo autocatalytic activation. Addition of purified, active RgpA or RgpB, but not Lys-specific Kgp, efficiently cleaved the PD of proRgpB but catalytic activity remained inhibited because of inhibition of the catalytic domain in trans by the PD. In contrast, active RgpB was generated from the zymogen, although at a slow rate, by gingipain-null P. gingivalis lysate or intact bacterial cell suspension. This activation was dependent on the presence of the PorU sortase. Interestingly, maturation of proRgpB with the catalytic cysteine residues mutated to Ala expressed in the ΔRgpA mutant strain was indistinguishable from that in the parental strain. Cumulatively, this suggests that PorU not only has sortase activity but is also engaged in activation of gingipain zymogens on the bacterial cell surface.

Porphyromonas gingivalis, an asaccharolyticBacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory diseases, such as rheumatoid arthritis, cardiovascular disease and Alzheimer’s disease.P. gingivalisutilizes protease-generated peptides derived from extracellular proteins for growth, but how those peptides enter the cell is not clear. Here we identify RagAB as the outer membrane importer for peptides. X-ray crystal structures show that the transporter forms a dimeric RagA2B2complex with the RagB substrate binding surface-anchored lipoprotein forming a closed lid on the TonB-dependent transporter RagA. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus provide direct evidence for a “pedal bin” nutrient uptake mechanism. Together with mutagenesis, peptide binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic OM oligopeptide acquisition machine with considerable substrate selectivity that is essential for the efficient utilisation of proteinaceous nutrients byP. gingivalis.

Porphyromonas gingivalis is a key pathogen in chronic periodontitis and has recently been mechanistically linked to the development of rheumatoid arthritis via the activity of peptidyl arginine deiminase generating citrullinated epitopes in the periodontium. In this project the outer membrane vesicles (OMV) from P. gingivalis W83 wild-type (WT), a W83 knock-out mutant of peptidyl arginine deiminase (ΔPPAD), and a mutant strain expressing PPAD with the active site cysteine mutated to alanine (C351A), have been analyzed using a two-dimensional HFBA-based separation system combined with LC-MS. For optimal and positive identification and validation of citrullinated peptides and proteins, high resolution mass spectrometers and strict MS search criteria were utilized. This may have compromised the total number of identified citrullinations but increased the confidence of the validation. A new two-dimensional separation system proved to increase the strength of validation, and along with the use of an in-house build program, Citrullia, we establish a fast and easy semi-automatic (manual) validation of citrullinated peptides. For the WT OMV we identified 78 citrullinated proteins having a total of 161 citrullination sites. Notably, in keeping with the mechanism of OMV formation, the majority (51 out of 78) of citrullinated proteins were predicted to be exported via the inner membrane and to reside in the periplasm or being translocated to the bacterial surface. Citrullinated surface proteins may contribute to the pathogenesis of rheumatoid arthritis. For the C351A-OMV a single citrullination site was found and no citrullinations were identified for the ΔPPAD-OMV, thus validating the unbiased character of our method of citrullinated peptide identification.

Investigate the content of fibrotic fibrils in gingival tissue and the proliferation of fibroblasts collected from recurrent and non-recurrent hereditary gingival fibromatosis (HGF) and idiopathic gingival fibromatosis (IGF).Gingival biopsies were collected from HGF (n = 3) and IGF (n = 3) donors with recurrent and non-recurrent gingival overgrowths and from a control group (Ctrl, n = 3). Hematoxylin staining was performed to evaluate the histomorphology of gingival tissue. Heidenhain's AZAN trichrome staining served for visualization of fibrotic fibrils in gingiva. Quantitative analysis of the content of fibrotic fibrils in gingival tissue was performed using a polarized light microscope. Proliferation was evaluated at 24 h, 48 h, and 72 h in fibroblast cultures using a cell proliferation ELISA assay based on 5-bromo-2'-deoxyuridine (BrdU).Numerous blood vessels and fibroblasts were observed in recurrent overgrowths, whereas moderate blood vessels and moderate to scanty fibroblasts were detected in non-recurrent overgrowths. Heidenhain's staining revealed numerous collagen fibers in both recurrent and non-recurrent overgrowths. Quantitative analysis in a polarizing microscope showed significant accumulation of fibrotic fibrils exclusively in the overgrowths with the recurrence. In all time-points, increased proliferation of cells from all recurrent overgrowths was observed, but not from overgrowths which do not reoccur.The study revealed that recurrent gingival overgrowths consist of highly fibrotic and dense connective tissue with numerous blood vessels and abundant fibroblasts. We also demonstrated that unlike fibroblasts derived from overgrowths, which did not present recurrence, fibroblasts derived from highly fibrotic and recurrent overgrowths maintain high rate of proliferation in vitro.

Objectives To investigate the processes associated with the excessive production of collagen I in hereditary gingival fibromatosis (HGF). Materials and methods Three HGF subjects and five controls were enrolled in the study. Histomorphological and immunohistological analyses were performed on gingival tissues. The expression of heat-shock protein 47 (HSP47), collagen I, transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) by gingival fibroblasts isolated from HGF and controls was analysed using qRT-PCR, Western blotting and ELISA. Results Considerable accumulation of fibrotic fibrils and increased synthesis of HSP47 were noted in HGF gingival tissues. The synthesis of collagen I, HSP47, TGF-β1, CTGF and TIMP-1 was significantly elevated in HGF gingival fibroblasts compared with controls, while the production of MMP-1 was decreased. Conclusions We report that fibrosis in HGF gingival tissues is associated with increased synthesis of HSP47. This finding was confirmed by an in vitro study, where excessive production of collagen I was associated with increased synthesis of HSP47, TGF-β1 and CTGF by HGF gingival fibroblasts. Moreover, the shift in the TIMP-1/MMP-1 ratio identifies increased synthesis of TIMP-1 as one of the processes associated with collagen I overproduction in HGF fibroblasts.

Background Periodontitis (PD) is the most common chronic inflammatory disease caused by bacterial infection resulting in alveolar bone resorption and tooth loss.1,2 A main causative agent of PD is Porphyromonas gingivalis (Pg).3,4 This periodontopathogen produces peptidylarginine deiminase (PPAD) catalysing conversion of arginine to citrulline.5,6 PD shares common mechanism and risk factors with rheumatoid arthritis (RA). Due to the presence of pathogenic autoantibodies reactive with citrullinated proteins in RA, citrullination of host and bacterial proteins by PPAD was proposed as a mechanistic link between PD and RA.7–9 The aim of this study was to investigate the impact of a novel polymorphism identified in the PPAD gene on bacterial virulence and PD clinical status. Materials and methods Gingival crevicular fluid (GCF) from 20 patients with PD was plated on blood agar and Pg colonies re-cultured to isolate individual strains of the bacterium. From each strain the PPAD gene was cloned, sequenced and analysed. The native PPAD gene in the reference Pg ATCC33277 strain was substituted with the polymorphic gene. The phenotype of clinical isolates harbouring polymorphism, the mutated and native ATCC33277 strains was examined. Further, periodontal clinical parameters were compared amongst patients infected by Pg expressing PPAD with and w/o polymorphism. Results A three amino acid polymorphism (G231N, E232T, N235D) was identified in the vicinity of the PPAD catalytic His residue in Pg isolates obtained from 30% of PD patients. The PPAD activity of clinical strains with polymorphism and the ATCC33277 mutant was 2-fold higher in comparison to the reference strain. Gingival fibroblasts infection with strains carrying polymorphic PPAD caused significantly higher upregulation of cyclooxygenase 2 (COX-2) than infection with native ATCC33277. Probing pocket depth (PPD) and clinical attachment level (CAL) assessment revealed that patients infected with Pg expressing polymorphic PPAD suffered from more severe disease than those carrying ‘classical’ Pg w/o polymorphism. Conclusions A three amino acid polymorphism of PPAD augments the virulence of Pg and severity of periodontitis apparently due to higher PPAD activity. The increased citrullination of bacterial and/or host proteins by Pg with the characterised PPAD genotype can trigger autoimmune response in RA. Acknowledgments National Science Centre, Poland (2012/07/B/NZ6/03524, K.G.), Foundation for Polish Science (TEAM, DPS/424-329/10, J.P.). References 1. Petersen PE, Bourgeois D, Ogawa H, et al.The global burden of oral diseases and risks to oral health. Bull World Health Organ 2003;83:661–69. 2. Eke PI, Dye BA, Wei L, et al.CDC Periodontal Disease Surveillance workgroup and representatives of the American Academy of Periodontology. Prevalence of periodontitis in adults in the United States: 2009 and 2010. J Dent Res 2012;91:914–20. 3. Socransky SS, Haffajee AD, Smith C, et al.Relation of counts of microbial species to clinical status at the sampled site. J Clin Periodontol 1991;18:766–75. 4. Holt SC, Ebersole J, Felton J, et al.Implantation of Bacteroides gingivalis in nonhuman primates initiates progression of periodontitis. Science 1998;239:55–57. 5. McGraw WT, Potempa J, Farley D, et al.Purification, characterisation, and sequence analysis of a potential virulence factor from Porphyromonas gingivalis, peptidylarginine deiminase. Infect Immun 1999;67:3248–56. 6. Rodriguez SB, Stitt BL, Ash DE.Expression of peptidylarginine deiminase from Porphyromonas gingivalis in Escherichia coli: enzyme purification and characterisation. Arch Biochem Biophys 2009;488:14–22. 7. Wegner N, Wait R, Sroka A, et al.Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and α-enolase: implications for autoimmunity in rheumatoid arthritis. Arthritis Rheum 2010;62:2662–72. 8. Lundberg K, Kinloch A, Fisher BA, et al.Antibodies to citrullinated alpha-enolase peptide 1 are specific for rheumatoid arthritis and cross-react with bacterial enolase. Arthritis Rheum 2008;58:3009–19. 9. Quirke AM, Lugli EB, Wegner N, et al.Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis. Ann Rheum Dis 2014;73:263–69.

Skewing of the human oral microbiome causes dysbiosis and preponderance of bacteria such as Porphyromonas gingivalis, the main etiological agent of periodontitis. P. gingivalis secretes proteolytic gingipains (Kgp and RgpA/B) as zymogens inhibited by a pro-domain that is removed during extracellular activation. Unraveling the molecular mechanism of Kgp zymogenicity is essential to design inhibitors blocking its activity. Here, we found that the isolated 209-residue Kgp pro-domain is a boomerang-shaped all-β protein similar to the RgpB pro-domain. Using composite structural information of Kgp and RgpB, we derived a plausible homology model and mechanism of Kgp-regulating zymogenicity. Accordingly, the pro-domain would laterally attach to the catalytic moiety in Kgp and block the active site through an exposed inhibitory loop. This loop features a lysine (Lys129) likely occupying the S1 specificity pocket and exerting latency. Lys129 mutation to glutamate or arginine led to misfolded protein that was degraded in vivo. Mutation to alanine gave milder effects but still strongly diminished proteolytic activity, without affecting the subcellular location of the enzyme. Accordingly, the interactions of Lys129 within the S1 pocket are also essential for correct folding. Uniquely for gingipains, the isolated Kgp pro-domain dimerized through an interface, which partially overlapped with that between the catalytic moiety and the pro-domain within the zymogen, i.e. both complexes are mutually exclusive. Thus, pro-domain dimerization, together with partial rearrangement of the active site upon activation, explains the lack of inhibition of the pro-domain in trans. Our results reveal that the specific latency mechanism of Kgp differs from those of Rgps.

Iñaki de Diego et al., 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., This study was financially supported in part by grants from European, US American, Polish, Spanish, and Catalan agencies (UMO-2012/04/A/NZ1/00051, UMO-2012/05/B/NZ6/00581, UMO-2013/08/W/NZ1/00696, UMO-2011/01/D/NZ1/01169, 2975/7.PR/13/2014/2, NIH NIDCR DE09761; FP7-PEOPLE-2011-ITN-290246 “RAPID”; FP7-HEALTH-2012-306029-2 “TRIGGER”; BFU2012-32862; BIO2013-49320-EXP; MDM-2014-0435; 1306/MOB/IV/2015/0 (“Mobilność Plus” MK) and 2014SGR9). The Department of Structural Biology of IBMB is a “María de Maeztu” Unit of Excellence from the Ministry of Economy and Competitiveness. Funding for data collection was provided in part by ESRF

Porphyromonas gingivalis (P. gingivalis) expres-ses 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, P. gingivalis on the cell surface is highly citrullinated. To investigate the contribution of PPAD to the interaction of P. gingivalis with primary human gingival fibroblasts (PHGF) and P. gingivalis-induced synthesis of prostaglandin E2 (PGE2 ), PHGF were infected with wild-type P. gingivalis ATCC 33277, an isogenic PPAD-knockout strain (∆ppad) 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 cyclo-oxygenase 2 (COX-2) and microsomal PGE synthase-1 (mPGES-1), key factors in the prostaglandin synthesis pathway, was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), while PGE2 synthesis was evaluated by enzyme immunoassay. PHGF were infected more efficiently by wild-type P. gingivalis than by the ∆ppad strain, which correlated with strong induction of COX-2 and mPGES-1 expression by wild-type P. gingivalis, 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.