SUMMARY Porphyromonas gingivalis is a low-abundance oral anaerobic bacterium implicated in periodontitis, a polymicrobial inflammatory disease, and the associated systemic conditions. However, the mechanism by which P. gingivalis contributes to inflammation and disease has remained elusive. Here we show that P. gingivalis, at very low colonization levels, triggers changes to the amount and composition of the oral commensal microbiota leading to inflammatory periodontal bone loss. The commensal microbiota and the complement pathway were both required for P. gingivalis-induced bone loss as germ-free mice or conventionally raised C3a and C5a receptor deficient mice did not develop bone loss after inoculation with P. gingivalis. These findings demonstrate that a single, low-abundance species can disrupt host-microbial homeostasis to cause inflammatory disease. The identification and targeting of similar low-abundance pathogens with community-wide impact may be important for treating inflammatory diseases of polymicrobial etiology.

Aging is linked to increased susceptibility to chronic inflammatory diseases several of which, including periodontitis, involve neutrophil-mediated tissue injury. Here, we found that aging-associated periodontitis was accompanied by diminished expression of Del-1 (EDIL3), an endogenous inhibitor of LFA-1 integrin-dependent neutrophil adhesion, and by a reciprocal increase in IL-17 expression. Consistently, IL-17 inhibited gingival endothelial cell expression of Del-1, thereby promoting LFA-1-dependent neutrophil recruitment. Young Del-1-deficient mice developed spontaneous periodontitis featuring excessive neutrophil infiltration and IL-17 expression; disease was prevented in Del-1–LFA-1 and Del-1–IL-17 receptor double-deficient mice. Locally administered Del-1 inhibited IL-17 production, neutrophil accumulation, and bone loss. Therefore, Del-1 suppresses LFA-1-dependent neutrophil recruitment and IL-17-triggered inflammatory pathology and may thus be a promising therapeutic for inflammatory diseases.

SUMMARY Certain low-abundance bacterial species, such as the periodontitis-associated oral bacterium Porphyromonas gingivalis can subvert host immunity to remodel a normally symbiotic microbiota into a dysbiotic, disease-provoking state. However, such pathogens also exploit inflammation to thrive in dysbiotic conditions. How these bacteria evade immunity while maintaining inflammation is unclear. As previously reported, P. gingivalis remodels the oral microbiota into a dysbiotic state by exploiting complement. Now we show that in neutrophils P. gingivalis disarms a host-protective TLR2-MyD88 pathway via proteasomal degradation of MyD88, whereas it activates an alternate TLR2-Mal-PI3K pathway. This alternate TLR2-Mal-PI3K pathway blocks phagocytosis, provides ‘bystander’ protection to otherwise susceptible bacteria, and promotes dysbiotic inflammation in vivo. This mechanism to disengage bacterial clearance from inflammation required an intimate crosstalk between TLR2 and the complement receptor C5aR, and can contribute to the persistence of microbial communities that drive dysbiotic diseases.

SummaryMice lacking the transcription factor T-bet in the innate immune system develop microbiota-dependent colitis. Here, we show that interleukin-17A (IL-17A)-producing IL-7Rα+ innate lymphoid cells (ILCs) were potent promoters of disease in Tbx21−/−Rag2−/− ulcerative colitis (TRUC) mice. TNF-α produced by CD103−CD11b+ dendritic cells synergized with IL-23 to drive IL-17A production by ILCs, demonstrating a previously unrecognized layer of cellular crosstalk between dendritic cells and ILCs. We have identified Helicobacter typhlonius as a key disease trigger driving excess TNF-α production and promoting colitis in TRUC mice. Crucially, T-bet also suppressed the expression of IL-7R, a key molecule involved in controlling intestinal ILC homeostasis. The importance of IL-7R signaling in TRUC disease was highlighted by the dramatic reduction in intestinal ILCs and attenuated colitis following IL-7R blockade. Taken together, these data demonstrate the mechanism by which T-bet regulates the complex interplay between mucosal dendritic cells, ILCs, and the intestinal microbiota.

SummaryThe Arg-gingipains (RgpsA and B) of Porphyromonas gingivalis are a family of extracellular cysteine proteases and are important virulence determinants of this periodontal bacterium. A monoclonal antibody, MAb1B5, which recognizes an epitope on glycosylated monomeric RgpAs also cross-reacts with a cellsurface polysaccharide of P. gingivalis W50 suggesting that the maturation pathway of the Arg-gingipains may be linked to the biosynthesis of a surface carbohydrate. We report the purification and structural characterization of the cross-reacting anionic polysaccharide (APS), which is distinct from both the lipopolysaccharide and serotype capsule polysaccharide of P. gingivalis W50. The structure of APS was determined by 1D and 2D NMR spectroscopy and methylation analysis, which showed it to be a phosphorylated branched mannan. The backbone is built up of α α α α -1,6-linked mannose residues and the sidechains contain α α α α -1,2-linked mannose oligosaccharides of different lengths (one to two sugar residues) attached to the backbone via 1,2-linkage. One of the side-chains in the repeating unit contains Man α α α α 1-2Man α α α α 1-phosphate linked via phosphorus to a backbone mannose at position 2. De-O -phosphorylation of APS abolished cross-reactivity suggesting that Man α α α α 1-2Man α α α α 1-phosphate fragment forms part of the epitope recognized by MAb1B5. This phosphorylated branched mannan represents a novel polysaccharide that is immunologically related to the post-translational additions of Arg-gingipains.

We previously described a cell surface anionic polysaccharide (APS) in Porphyromonas gingivalis that is required for cell integrity and serum resistance. APS is a phosphorylated branched mannan that shares a common epitope with posttranslational additions to some of the Arg-gingipains. This study aimed to determine the mechanism of anchoring of APS to the surface of P. gingivalis. APS was purified on concanavalin A affinity columns to minimize the loss of the anchoring system that occurred during chemical extraction.1 H nuclear magnetic resonance spectroscopy of the lectin-purified APS confirmed the previous structure but also revealed additional signals that suggested the presence of a lipid A. This was confirmed by fatty acid analysis of the APS and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the lipid A released by treatment with sodium acetate buffer (pH 4.5). Hence, P. gingivalis synthesizes two distinct lipopolysaccharide (LPS) macromolecules containing different glycan repeating units: O-LPS (with O-antigen tetrasaccharide repeating units) and A-LPS (with APS repeating units). Nonphosphorylated penta-acylated and nonphosphorylated tetra-acylated species were detected in lipid A from P. gingivalis total LPS and in lipid A from A-LPS. These lipid A species were unique to lipid A derived from A-LPS. Biological assays demonstrated a reduced proinflammatory activity of A-LPS compared to that of total LPS. Inactivation of a putative O-antigen ligase (waaL) at PG1051, which is required for the final step of LPS biosynthesis, abolished the linkage of both the O antigen and APS to the lipid A core of O-LPS and A-LPS, respectively, suggesting that WaaL in P. gingivalis has dual specificity for both O-antigen and APS repeating units.The gram-negative anaerobic bacterium Porphyromonas gingivalis is an important etiological agent in periodontal disease and produces several virulence factors. Among them are the cysteine proteases Arg-gingipain (Rgp) and Lys-gingipain (Kgp), which are capable of causing the degradation of several host proteins and lipopolysaccharide (LPS), which may exacerbate the inflammatory response in periodontal tissues of the infected host (2, 9) These factors are also important antigens in patients with periodontal disease and may account for a significant proportion of the immune response directed against P. gingivalis (27, 38). A monoclonal antibody (MAb), 1B5, raised against one of the five isoforms of Arg-gingipains (Rgps), RgpA cat , also cross-reacts with two other Rgps, namely, mtRgpA cat and mt-RgpB, and also cross-reacts with an anionic cell surface polysaccharide (APS) (10, 30). Chemical deglycosylation of RgpA cat and mt-RgpA cat with anhydrous trifluoromethane sulfonic acid abolishes their cross-reactivity to MAb 1B5, indicating that this antibody recognizes a carbohydrate epitope that is also present in APS (10, 30).We established that APS was distinct from LPS and capsular polysaccharide (PS) (K antigen) in P. gingivalis (30). LPS purified by a pr...

Summary The oral and intestinal host tissues both carry a heavy microbial burden. Although commensal bacteria contribute to healthy intestinal tissue structure and function, their contribution to oral health is poorly understood. A crucial component of periodontal health is the recruitment of neutrophils to periodontal tissue. To elucidate this process, gingival tissues of specific‐pathogen‐free and germ‐free wild‐type mice and CXCR2KO and MyD88KO mice were examined for quantitative analysis of neutrophils and CXCR2 chemoattractants (CXCL1, CXCL2). We show that the recruitment ofneutrophils to the gingival tissue does not require commensal bacterial colonization but is entirely dependent on CXCR2 expression. Strikingly, however, commensal bacteria selectively upregulate the expression of CXCL2, but not CXCL1, in a MyD88‐dependent way that correlates with increased neutrophil recruitment as compared with germ‐free conditions. This is the first evidence that the selective use of chemokine receptor ligands contributes to neutrophil homing to healthy periodontal tissue.

Capsular polysaccharides of gram-negative bacteria play an important role in maintaining the structural integrity of the cell in hostile environments and, because of their diversity within a given species, can act as useful taxonomic aids. In order to characterize the genetic locus for capsule biosynthesis in the oral gramnegative bacterium Porphyromonas gingivalis, we analyzed the genome of P. gingivalis W83 which revealed two candidate loci at PG0106-PG0120 and PG1135-PG1142 with sufficient coding capacity and appropriate gene functions based on comparisons with capsule-coding loci in other bacteria. Insertion and deletion mutants were prepared at PG0106-PG0120 in P. gingivalis W50-a K1 serotype. Deletion of PG0109-PG0118 and PG0116-PG0120 both yielded mutants which no longer reacted with antisera to K1 serotypes. Restriction fragment length polymorphism analysis of the locus in strains representing all six K-antigen serotypes and K ؊ strains demonstrated significant variation between serotypes and limited conservation within serotypes. In contrast, PG1135-PG1142 was highly conserved in this collection of strains. Sequence analysis of the capsule locus in strain 381 (K ؊ strain) demonstrated synteny with the W83 locus but also significant differences including replacement of PG0109-PG0110 with three unique open reading frames, deletion of PG0112-PG0114, and an internal termination codon within PG0106, each of which could contribute to the absence of capsule expression in this strain. Analysis of the Arg-gingipains in the capsule mutants of strain W50 revealed no significant changes to the glycan modifications of these enzymes, which indicates that the glycosylation apparatus in P. gingivalis is independent of the capsule biosynthetic machinery.