Structural determinants of inhibition of Porphyromonas gingivalis gingipain K by KYT-36, a potent, selective, and bioavailable peptidase inhibitor

Abstract Porphyromonas gingivalis is a member of the dysbiotic oral microbiome and a “keystone pathogen” that causes severe periodontal disease, which is among the most prevalent infectious diseases. Part of the virulence factors secreted by P. gingivalis are the essential cysteine peptidases gingipain K (Kgp) and R (RgpA and RgpB), which account for 85% of the extracellular proteolytic activity of the pathogen and are thus prime targets for inhibition. We report the high-resolution (1.20 Å) complex structure of Kgp with KYT-36, a peptide-derived, potent, bioavailable and highly selective inhibitor, which is widely used for studies in vitro, in cells and in vivo. Sub-nanomolar inhibition of Kgp is achieved by tight binding to the active-site cleft, which is covered for its sub-sites S3 through S1’ under establishment of nine hydrophobic interactions, 14 hydrogen bonds and one salt bridge. In addition, an inhibitor carbonyl carbon that mimics the scissile carbonyl of substrates is pyramidalized and just 2.02 Å away from the catalytic nucleophile of Kgp, C477Sγ. Thus, the crystal structure emulates a reaction intermediate of the first nucleophilic attack during catalysis of cysteine peptidases. The present study sets the pace for the development of tailored next-generation drugs to tackle P. gingivalis

Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

Gingipain cysteine proteases are considered key virulence factors of Porphyromonas gingivalis. They significantly influence antibacterial and homeostatic functions of macrophages, neutrophils, the complement system, and cytokine networks. Recent data indicate the role of P. gingivalis in T cell differentiation;however, the involvement of gingipains in this process remains elusive. Therefore, the aim of this study was to investigate the contribution of danger signals triggered by the gingipains on the generation of Th17 cells, which play a key role in protection against bacterial diseases but may cause chronic inflammation and bone resorption. To this end we compared the effects of the wild-type strain of P. gingivalis (W83) with its isogenic mutant devoid of gingipain activity (Delta K Delta RAB), and bacterial cells pretreated with a highly-specific inhibitor of gingipains activity (KYTs). Antigen presenting cells (APCs), both professional (dendritic cells), and non-professional (gingival keratinocytes), exposed to viable bacteria expressed high amounts of cytokines (IL-6, IL-21, IL-23). These cytokines are reported to either stimulate or balance the Th17-dependent immune response. Surprisingly, cells infected with P. gingivalis devoid of gingipain activity showed increased levels of all tested cytokines compared to bacteria with fully active enzymes. The effect was dependent on both the reduction of cytokine proteolysis and the lack of cross-talk with other bacterial virulence factors, including LPS and fimbriae that induce de novo synthesis of cytokines. The profile of lymphocyte T differentiation from naive T cells showed enhanced generation of Th17 in response to bacteria with inactive gingipains. Moreover, we found that gingipain-dependent induction of Th17 cells was highly specific, since other T cell-subsets remained unchanged. Finally, inhibition of IL-6 signaling in dendritic cells led to a significant depletion of the Th17 population. Cumulatively, this study revealed a previously undisclosed role of gingipain activity in the process of Th17 differentiation reliant on blocking signaling through IL-6. Since inactivation of gingipains accelerates the skewing of T cells toward Th17 cells, which are detrimental in periodontitis, IL-6 signaling may serve as an attractive target for treatment of the disease

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

Antibodies against Porphyromonas gingivalis in serum and saliva and their association with rheumatoid arthritis and periodontitis. Data from two rheumatoid arthritis cohorts in Sweden

BackgroundPeriodontitis and oral pathogenic bacteria can contribute to the development of rheumatoid arthritis (RA). A connection between serum antibodies to Porphyromonas gingivalis (P. gingivalis) and RA has been established, but data on saliva antibodies to P. gingivalis in RA are lacking. We evaluated antibodies to P. gingivalis in serum and saliva in two Swedish RA studies as well as their association with RA, periodontitis, antibodies to citrullinated proteins (ACPA), and RA disease activity.MethodsThe SARA (secretory antibodies in RA) study includes 196 patients with RA and 101 healthy controls. The Karlskrona RA study includes 132 patients with RA ≥ 61 years of age, who underwent dental examination. Serum Immunoglobulin G (IgG) and Immunoglobulin A (IgA) antibodies and saliva IgA antibodies to the P. gingivalis–specific Arg-specific gingipain B (RgpB) were measured in patients with RA and controls.ResultsThe level of saliva IgA anti-RgpB antibodies was significantly higher among patients with RA than among healthy controls in multivariate analysis adjusted for age, gender, smoking, and IgG ACPA (p = 0.022). Saliva IgA anti-RgpB antibodies were associated with RA disease activity in multivariate analysis (p = 0.036). Anti-RgpB antibodies were not associated with periodontitis or serum IgG ACPA.ConclusionPatients with RA had higher levels of saliva IgA anti-RgpB antibodies than healthy controls. Saliva IgA anti-RgpB antibodies may be associated with RA disease activity but were not associated with periodontitis or serum IgG ACPA. Our results indicate a local production of IgA anti-RgpB in the salivary glands that is not accompanied by systemic antibody production

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

Mucus detachment by host metalloprotease meprin \beta requires shedding of its inactive pro-form, which is abrogated by the pathogenic protease RgpB

The host metalloprotease meprin β is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease

Identification and characterization of aptameric inhibitors of human neutrophil elastase

Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation and tissue remodelling. 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 systematic evolution of ligands by exponential enrichment (SELEX) to develop single-stranded DNA 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