In human monocyte‐derived macrophages, the MCPIP gene (monocyte chemoattractant protein‐induced protein) is strongly activated by interleukin‐1β (IL‐1β). Using bioinformatics, a PIN domain was identified, spanning amino acids 130‐280; such domains are known to possess structural features of RNases. Recently, RNase properties of MCPIP were confirmed on transcripts coding for interleukins IL‐6 and IL‐12p40. Here we present evidence that siRNA‐mediated inhibition of the MCPIP gene expression increases the level of the IL‐1β transcript in cells stimulated with LPS, whereas overexpression of MCPIP exerts opposite effects. Cells with an increased level of wild‐type MCPIP showed lower levels of IL‐1β mRNA. However, this was not observed when mutant forms of MCPIP, either entirely lacking the PIN domain or with point mutations in this domain, were used. The results of experiments with actinomycin D indicate that lower levels of IL‐1β mRNA are due to shortening of the IL‐1β transcript half‐life, and are not related to the presence of AU‐rich elements in the 3′ UTR. The interaction of the MCPIP with transcripts of both IL‐1β and MCPIP observed in an RNA immunoprecipitation assay suggests that this novel RNase may be involved in the regulation of expression of several genes.

Rheumatoid arthritis and periodontitis are two prevalent chronic inflammatory diseases in humans and are associated with each other both clinically and epidemiologically. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic autoimmune response to citrullinated epitopes. Here we showed that infection with viable periodontal pathogen Porphyromonas gingivalis strain W83 exacerbated collagen-induced arthritis (CIA) in a mouse model, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique P. gingivalis peptidylarginine deiminase (PPAD), which converts arginine residues in proteins to citrulline. Infection with wild type P. gingivalis was responsible for significantly increased levels of autoantibodies to collagen type II and citrullinated epitopes as a PPAD-null mutant did not elicit similar host response. High level of citrullinated proteins was also detected at the site of infection with wild-type P. gingivalis. Together, these results suggest bacterial PAD as the mechanistic link between P. gingivalis periodontal infection and rheumatoid arthritis.

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.

A novel gene ZC3H12A, encoding MCP‐1‐induced protein 1 (MCPIP), was recently identified in human peripheral blood monocytes treated with monocyte chemotactic protein 1 (MCP‐1) and in human monocyte‐derived macrophages stimulated with interleukin (IL)‐1β. These experiments revealed that the gene undergoes rapid and potent transcription induction upon stimulation with proinflammatory molecules, such as MCP‐1, IL‐1β, tumour necrosis factor α and lipopolysaccharide. Here we show that the induction of ZC3H12A by IL‐1β is predominantly NF‐κB‐dependent because inhibition of this signalling pathway results in the impairment of ZC3H12A transcription activation. Our results indicate the presence of an IL‐1β‐responding region within the second intron of the ZC3H12A gene, which contains four functional NF‐κB‐binding sites. Therefore, we propose that this transcription enhancer transduces a ZC3H12A transcription‐inducing signal after IL‐1β stimulation. Recent reports suggest that MCPIP acts as a negative regulator of inflammatory processes because it is engaged in the degradation of transcripts coding for certain proinflammatory cytokines. Our observations provide evidence for a novel negative feedback loop in the activation of NF‐κB and point to potential significance of MCPIP in the treatment of various pathological states, such as diabetes or cancer that involve disturbances in the functioning of the NF‐κB system.

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.

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Background: Serpins are uncommon in bacteria; little is known about their function. Results: Serpin from T. forsythia (miropin) inhibits a broad array of proteases with divergent specificities. Conclusion: Miropin may allow T. forsythia to dwell in a highly proteolytic environment. Significance: Miropin is the first pathogen-derived serpin with the unusual ability to efficiently inhibit different proteases at several active sites.

Comparative genomics of virulent Tannerella forsythia ATCC 43037 and a close health-associated relative, Tannerella BU063, revealed, in the latter, the absence of an entire array of genes encoding putative secretory proteases that possess a nearly identical C-terminal domain (CTD) that ends with a -Lys-Leu-Ile-Lys-Lys motif. This observation suggests that these proteins, referred to as KLIKK proteases, may function as virulence factors. Re-sequencing of the loci of the KLIKK proteases found only six genes grouped in two clusters. All six genes were expressed by T. forsythia in routine culture conditions, although at different levels. More importantly, a transcript of each gene was detected in gingival crevicular fluid (GCF) from periodontitis sites infected with T. forsythia indicating that the proteases are expressed in vivo. In each protein, a protease domain was flanked by a unique N-terminal profragment and a C-terminal extension ending with the CTD. Partially purified recombinant proteases showed variable levels of proteolytic activity in zymography gels and toward protein substrates, including collagen, gelatin, elastin, and casein. Taken together, these results indicate that the pathogenic strain of T. forsythia secretes active proteases capable of degrading an array of host proteins, which likely represents an important pathogenic feature of this bacterium.