Strategies for the inhibition of gingipains for the potential treatment of periodontitis and associated systemic diseases

Gingipains are the major virulence factors of Porphyromonas gingivalis, the main periodontopathogen. It is expected that inhibition of gingipain activity in vivo could prevent or slow down the progression of adult periodontitis. To date, several classes of gingipain inhibitors have been recognized. These include gingipain N-terminal prodomains, synthetic compounds, inhibitors from natural sources, antibiotics, antiseptics, antibodies, and bacteria. Several synthetic compounds are potent gingipain inhibitors but inhibit a broad spectrum of host proteases and have undesirable side effects. Synthetic compounds with high specificity for gingipains have unknown toxicity effects, making natural inhibitors more promising as therapeutic gingipain blockers. Cranberry and rice extracts interfere with gingipain activity and prevent the growth and biofilm formation of periodontopathogens. Although the ideal gingipain inhibitor has yet to be discovered, gingipain inhibition represents a novel approach to treat and prevent periodontitis. Gingipain inhibitors may also help treat systemic disorders that are associated with periodontitis, including cardiovascular disease, rheumatoid arthritis, aspiration pneumonia, pre-term birth, and low birth weight

Complement Activation as a Helping Hand for Inflammophilic Pathogens and Cancer

The complement system, an evolutionarily ancient component of innate immunity, is capable of protecting hosts from invading pathogens, either directly, by lysis of target cells, or indirectly, by mobilization of host immune mechanisms. However, this potentially cytotoxic cascade must be tightly regulated, since improperly controlled complement can damage healthy cells and tissues. The practical importance of this axis is highlighted when impairment of complement regulators or bacterial mechanisms of complement evasion result in pathogenic conditions. Recognition of complement as a “double-edged sword” is widely acknowledged, but another, currently underappreciated aspect of complement function has emerged as an important player in homeostatic balance—the dual outcome of complement-mediated inflammation. In most cases, the proinflammatory properties of complement are beneficial to the host. However, certain pathogens have developed the ability to utilize local inflammation as a source of nutrients and as a way to establish a niche for further colonization. Such a strategy can be illustrated in the example of periodontitis. Interestingly, certain tumors also seem to benefit from complement activation products, which promote a proangiogenic and immunosuppressive microenvironment

Genes contributing to Porphyromonas gingivalis fitness in abscess and epithelial cell colonization environments

Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis, and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism

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

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 ESRFPeer Reviewe

Bacterial and human peptidylarginine deiminases: targets for inhibiting the autoimmune response in rheumatoid arthritis?

Peptidylarginine deiminases (PADs) convert arginine within a peptide (peptidylarginine) into peptidylcitrulline. Citrullination by human PADs is important in normal physiology and inflammation. Porphyromonas gingivalis, a major pathogen in periodontitis, is the only prokaryote described to possess PAD. P. gingivalis infection may generate citrullinated peptides, which trigger anti-citrullinated peptide antibodies. In susceptible individuals, host protein citrullination by human PADs in the joint probably perpetuates antibody formation, paving the way for the development of chronic arthritis. Blockades of bacterial and human PADs may act as powerful novel therapies by inhibiting the generation of the antigens that trigger and sustain autoimmunity in rheumatoid arthritis

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

A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme karilysin can be truncated to Ser-Trp-Phe-Pro

Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin