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.

Extended Data Fig. 2 RagAB binds a wide range of oligopeptides EDFig2.tiff a-c, LC-MS/MS analysis of peptides bound to RagAB W83 KRAB, showing length distribution (a), total charge (b) and pI (c). d-f, Analysis of peptides bound to RagAB W83 wild-type, showing length distribution (d), total charge (e) and pI (f). For charge calculations, the pH was assumed to be 7.0 and contributions of any His residues were ignored. g, Amino acid frequency of RagAB-bound peptides (KRAB and wild-type combined; black) vs. the amino acid composition in the P. gingivalis proteome (gray), showing a substantial enrichment of Ala, Glu, Lys, Thr and Val. By contrast, aromatics (Phe, Trp) and bulky hydrophobics (Leu) appear to be underrepresented. The peptides bound to RagAB from W83 KRAB vary in length from 7 to 29 residues, with a broad maximum of around 13 residues that fits well with the peptide density observed in the structures. Assuming equal abundance of each detected peptide, there is a slight preference for neutral to slightly acidic peptides, and the pI distribution has a bimodal shape, with maxima for acidic and slightly basic peptides. Analysis of the smaller RagAB-bound peptide set from wild-type W83 (d-f) yields a slightly wider size range from 5-36 residues, but overall there are no dramatic differences in the collective length, net charge and pI of the RagAB-bound peptide populations from W83 KRAB and wild-type strains. Extended Data Fig. 3 Molecular dynamics simulations of RagAB show lid opening EDFig3.tiff a, C α-rmsd values of RagB lids in apo-RagAB (red) and peptide-bound RagAB (green) with reference to the starting crystal structure in the closed conformation. The C α-rmsd values of the RagB lids in RagA 2 B 2 are shown in blue with reference to the OO EM state. Each point indicates an average of 50 ns simulation trajectory. b, Comparison of the RagA 2 B 2 open conformation from EM (magenta) Data description i.e.

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.

Confident identification of citrullinated peptides. Abbreviations: (OMV) outer membrane vesicles, (WT) P. gingivalis W83 wild-type, (ΔPPAD) W83 knockout mutant of peptidyl arginine deiminase, (C351A) W83 expressing PPAD with the active site cysteine mutated to alanine, (2D) two-dimensional HFBA-based, (LC-MS) liquid chromatography mass spectrometry, (PAD) humane peptidyl arginine deiminase, (PPAD) bacterial P. gingivalis peptidyl arginine deiminase, (RA) rheumatoid arthritis, (PGE2) prostaglandin E2, (ACPA) anti-citrullinated protein antibodies, (RgpA/B) Argspecific gingipains, (MS) mass spectrometry, (HFBA) heptafluorobutyric acid, (FA) formic acid, (HPLC) high performance liquid chromatography, (AAA) amino acid analysis, (mgf) Mascot Generic File format, (mgx) Mascot Generic eXtended, (RT) retention time, (TIC) total ion count, (T9SS) type IX secretion system,

SUMMARY Porphyromonas gingivalis (Pg) expresses 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, Pg on the cell surface is highly citrullinated. To investigate the contribution of PPAD to the interaction of Pg with primary human gingival fibroblasts (PHGF) and Pg-induced synthesis of prostaglandin E2 (PGE2), PHGF were infected with wild-type Pg ATCC 33277, an isogenic PPAD-knockout strain (Δppad) or a mutated strain (C351A) expressing an inactive enzyme in which the catalytic cysteine has been mutated to alanine (PPADC351A). 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 COX-2 and mPGES-1, key factors in the prostaglandin synthesis pathway, was examined by qRT-PCR, while PGE2 synthesis was evaluated by EIA. PHGF were infected more efficiently by wt-Pg than the Δppad strain, which correlated with strong induction of COX-2 and mPGES-1 expression by wt-Pg, 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.

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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.

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.