Mirolysin-specific mRNA was amplified by RT-PCR and separated on agarose gel

Mirolysin-specific mRNA was amplified by RT-PCR and separated on agarose gel. and increased bacterial counts, which, in turn, causes complement-dependent inflammation of periodontium and consequently bone loss (9, 10). In contrast to the previous hypothesis implicating crucial contribution of red complex species in the pathogenesis of periodontal disease, the new model of polymicrobial synergy and dysbiosis proposes a concept of low-abundant keystone pathogens, capable of breaking down periodontal homeostasis and changing normal benign oral biofilm into a dysbiotic one (9). This novel concept was introduced based on the findings involving the most-intensively studied prototype periodontal pathogen (11). In order to disrupt host homeostasis and induce dysbiosis, this bacterium engages two receptors; complement receptor C5aR C activated by deficient in its C5a-releasing proteinases, gingipains, did not induce dysbiosis in a mouse periodontitis model (11). As we demonstrated before, has not one but three proteinases that all can generate biologically active C5a (15). revealed the existence of an entire array of genes encoding putative secretory proteinases with similarity to karilysin, all possessing a nearly identical C-terminal domain that ends with a -Lys-Leu-Ile-Lys-Lys motif. These proteins, referred to as KLIKK proteinases, may function as virulence factors (17). In the current study we characterize the role of one of these, a novel metalloproteinase of resistant to serum bactericidal activity. Materials and Methods Ethics statement The local ethical review committee in Lund has approved collection of sera from healthy human volunteers. Ethical committee of Jena University approved collection of periodontal plaques and gingival crevicular fluid (GCF). Written informed consent was obtained from patients and volunteers and the investigation was performed according to principles of the Declaration of Helsinki. Sera and proteins Normal human serum (NHS) was obtained from eight healthy volunteers. Heat-inactivated NHS was made by incubating NHS for 30 min at 56C. Sera deficient from various complement components as well (R)-BAY1238097 as matching NHS were obtained from Quidel. Purified complement proteins C3, (R)-BAY1238097 C4 and C5 were purchased from Complement Technology. Mirolysin, cloned from the ATCC 43037 genome, as well as its inactive mutant MirE341A (the catalytic glutamic acid was replaced by alanine), were expressed as glutathione S-transferase (GST)-tagged recombinant proteins in and purified by affinity chromatography on Glutathione (GSH)-Sepharose 4 Fast Flow (GE Healthcare). The GST tag was removed from recombinant proteins bound to GSH-Sepharose by cleavage with PreScission Proteinase (Amersham). Tag-free mirolysin and inactive mutant MirE341A were subsequently purified by size exclusion chromatography using Superdex 75 HiLoad 16/60 (Pharmacia Biotech) column. The metalloproteinase karilysin forms: Kly48, high molecular mass karilysin (Kly38) and (R)-BAY1238097 low molecular mass karilysin (Kly18) were purified as described (16). Interpain A (InpA) was expressed and purified as in (18). Antibodies The following antibodies (Abs) against human antigens were used throughout this study: polyclonal (pAb) rabbit anti-C1q, C4c, C3d antibodies (all from Dako), goat TLK2 anti-MBL (R&D), goat anti-C5 (Quidel); monoclonal (mAb) mouse anti-ficolin-2 (19) or anti-ficolin-3 (20), mouse anti-C9 neoantigen Abs (HyCult). Secondary pAb conjugated with horseradish peroxidase (HRP) against rabbit, goat or mouse were from Dako. Bacterial strains and their culture strain ATCC 43037 was grown on hemin N-acetylomuramic acid vitamin K (HNK) agar plates at 37C in an anaerobic chamber (Concept 400, Biotrace) with an atmosphere of 90% N2, 5% CO2 and 5% H2. The purity and correct identity of the cultures was confirmed by Gram-staining and 16S rDNA sequencing. mutant strains lacking mirolysin (gene (start codon followed by a 221 bp DNA sequence encoding CAT. The second DNA fragment consisted of 449 bp of the CAT gene, followed by 551 bp of a 3UTR, terminated with a KpnI restriction site. The two DNA fragments were ligated after EcoRI digestion, and cloned into the SacI and KpnI site of pUC19. The correct orientation of the DNA fragments in the plasmid was confirmed by sequencing. Deletional inactivation of kly (BFO_2683; formerly known as T0367) gene encoding karilysin metalloproteinase in T. forsythia In order to obtain a plasmid for (genomic DNA. The upstream 972 bp fragment was amplified with primers 5-TGTGAATTCGAGCGAAGCGATGAATCTCCTC-3 and 5-GATCCCGGGCTGTAGTCGTCAAATGGGACG-3, containing sequences for EcoRI and SmaI, respectively. The 1235 bp long downstream fragment was amplified with primers 5-GTAGTCGACGATTAAGAAGTGATGCCCTTCG-3 (containing a SalI site) and 5-GCTCGCCATAGAAATAACAAGCTTAGA-3 (containing a HindIII site). An erythromycin resistance cassette (cells were obtained by a modified procedure as described in (22). Briefly, cells from 5-days old plate were collected to a 50 ml conical tube and washed twice with 20 ml of electroporation buffer (1 mM MgCl2, 10% glycerol) and resuspended in 1 ml of the same buffer. One microgram of plasmid DNA was electroporated to 100 l bacterial slurry in.