Inhibition of Sphingosine-1-Phosphate Receptor 2 by JTE013 Enhanced Alveolar Bone Regeneration by Promoting Angiogenesis.

Sphingosine-1-phosphate receptor 2 (S1PR2) is a G protein-coupled receptor that regulates various immune responses. Herein, we report the effects of a S1PR2 antagonist (JTE013) on bone regeneration. Murine bone marrow stromal cells (BMSCs) were treated with dimethylsulfoxide (DMSO) or JTE013 with or without infection by an oral bacterial pathogen Aggregatibacter actinomycetemcomitans. Treatment with JTE013 enhanced vascular endothelial growth factor A (VEGFA), platelet derived growth factor subunit A (PDGFA), and growth differentiation factor 15 (GDF15) gene expression and increased transforming growth factor beta (TGFß)/Smad and Akt signaling. Eight-week-old male C57BL/6J mice were challenged with ligatures around the left maxillary 2nd molar for 15 days to induce inflammatory bone loss. After ligature removal, mice were treated with diluted DMSO or JTE013 in the periodontal tissues 3 times per week for 3 weeks. Calcein was also injected twice to measure bone regeneration. Micro-CT scanning of maxillary bone tissues and calcein imaging revealed that treatment with JTE013 enhanced alveolar bone regeneration. JTE013 also increased VEGFA, PDGFA, osteocalcin, and osterix gene expressions in the periodontal tissues compared to control. Histological examination of periodontal tissues revealed that JTE013 promoted angiogenesis in the periodontal tissues compared to control. Our findings support that inhibition of S1PR2 by JTE013 increased TGFß/Smad and Akt signaling; enhanced VEGFA, PDGFA, and GDF15 gene expression; and subsequently promoted angiogenesis and alveolar bone regeneration.

Porphyromonas gingivalis infection upregulates the endothelin (ET) system in brain microvascular endothelial cells.

Endothelin-1 (ET-1), the most potent vasoconstrictor identified to date, contributes to cerebrovascular dysfunction and brain ET-1 levels were shown to be related to Alzheimer's disease and related dementias (ADRD) progression. ET-1 also contributes to neuroinflammation, especially in infections of the central nervous system. Recent studies causally linked chronic periodontal infection with an opportunistic anaerobic bacterium Porphyromonas gingivalis (Coykendall et al.) Shah & Collins to AD development. Thus, the goal of the study was to determine the impact of P. gingivalis infection on the ET system and cell senescence in brain microvascular endothelial cells. Cells were infected with a multiplicity of infection 50 P. gingivalis with and without extracellular ATP-induced oxidative stress for 24 h. Cell lysates were collected for analysis of endothelin A receptor (ETA)/endothelin B receptor (ETB) receptor as well as senescence markers. ET-1 levels in cell culture media were measured with enzyme-linked immunosorbent assay. P. gingivalis infection increased ET-1 (pg/mL) secretion, as well as the ETA receptor expression, whereas decreased lamin A/C expression compared to control. Tight junction protein claudin-5 was also decreased under these conditions. ETA or ETB receptor blockade during infection did not affect ET-1 secretion or the expression of cell senescence markers. Current findings suggest that P. gingivalis infection may compromise endothelial integrity and activate the ET system.

Predominant Distribution of the RNAi Machinery at Apical Adherens Junctions in Colonic Epithelia Is Disrupted in Cancer.

The RNA interference (RNAi) machinery is an essential component of the cell, regulating miRNA biogenesis and function. RNAi complexes were thought to localize either in the nucleus, such as the microprocessor, or in the cytoplasm, such as the RNA-induced silencing complex (RISC). We recently revealed that the core microprocessor components DROSHA and DGCR8, as well as the main components of RISC, including Ago2, also associate with the apical adherens junctions of well-differentiated cultured epithelial cells. Here, we demonstrate that the localization of the core RNAi components is specific and predominant at apical areas of cell-cell contact of human normal colon epithelial tissues and normal primary colon epithelial cells. Importantly, the apical junctional localization of RNAi proteins is disrupted or lost in human colon tumors and in poorly differentiated colon cancer cell lines, correlating with the dysregulation of the adherens junction component PLEKHA7. We show that the restoration of PLEKHA7 expression at adherens junctions of aggressively tumorigenic colon cancer cells restores the junctional localization of RNAi components and suppresses cancer cell growth in vitro and in vivo. In summary, this work identifies the apical junctional localization of the RNAi machinery as a key feature of the differentiated colonic epithelium, with a putative tumor suppressing function.

Bacterial Protein Signatures Identified in Porphyromonas gingivalis Containing-Autophagic Vacuoles Reveal Co-Evolution Between Oral Red/Orange Complex Bacteria and Gut Bacteria.

Modern oral bacterial species present as a concoction of commensal and opportunistic pathogens originating from their evolution in humans. Due to the intricate colonization mechanisms shared amongst oral and gut bacteria, these bacteria have likely evolved together to establish and adapt in the human oro-digestive tract, resulting in the transfer of genetic information. Our liquid chromatography-with-tandem-mass-spectrometry (LC-MS-MS) analyses have revealed protein signatures, Elongation Factor Tu, RagB/SusD nutrient uptake outer membrane protein and DnaK, specifically from Porphyromonas gingivalis -containing autophagic vacuoles isolated from the infected human primary gingival epithelial cells. Interestingly, our Mass-Spectrometry analysis reported similar proteins from closely related oral bacteria, Tannerella forsythia and Prevotella intermedia . In our phylogenetic study of these key protein signatures, we have established that pathogenic oral bacteria share extensive relatedness to each other and gut resident bacteria. We show that in the virulence factors identified from gut bacteria, Elongation Factor Tu and DnaK, there are several structural similarities and conservations with proteins from oral pathogenic bacteria. There are also major similarities in the RagB/SusD proteins of oral bacteria to prominent gut bacteria. These findings not only highlight the shared virulence mechanisms amongst oral bacterial pathogens/pathobionts but also gut bacteria and elucidate their co-evolutions in the human host.

Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa.

Porphyromonas gingivalis , a major oral pathobiont, evades canonical host pathogen clearance in human primary gingival epithelial cells (GECs) by initiating a non-canonical variant of autophagy consisting of Microtubule-associated protein 1A/1B-light chain 3 (LC3)-rich autophagosomes, which then act as replicative niches. Simultaneously, P. gingivalis inhibits apoptosis and oxidative-stress, including extracellular-ATP (eATP)-mediated reactive-oxygen-species (ROS) production via phosphorylating Heat Shock Protein 27 (HSp27) with the bacterial nucleoside-diphosphate-kinase (Ndk). Here, we have mechanistically identified that P. gingivalis -mediated induction of HSp27 is crucial for the recruitment of the LC3 isoform, LC3C, to drive the formation of live P. gingivalis -containing Beclin1-ATG14-rich autophagosomes that are redox sensitive and non-degrading. HSp27 depletions of both infected GECs and gingiva-mimicking organotypic-culture systems resulted in the collapse of P. gingivalis -mediated autophagosomes, and abolished P. gingivalis -induced LC3C-specific autophagic-flux in a HSp27-dependent manner. Concurrently, HSp27 depletion accompanied by eATP treatment abrogated protracted Beclin 1-ATG14 partnering and decreased live intracellular P. gingivalis levels. These events were only partially restored via treatments with the antioxidant N-acetyl cysteine (NAC), which rescued the cellular redox environment independent of HSp27. Moreover, the temporal phosphorylation of HSp27 by the bacterial Ndk results in HSp27 tightly partnering with LC3C, hindering LC3C canonical cleavage, extending Beclin 1-ATG14 association, and halting canonical maturation. These findings pinpoint how HSp27 pleiotropically serves as a major platform-molecule, redox regulator, and stepwise modulator of LC3C during P. gingivalis -mediated non-canonical autophagy. Thus, our findings can determine specific molecular strategies for interfering with the host-adapted P. gingivalis ' successful mucosal colonization and oral dysbiosis.

Glutamate is a key nutrient for Porphyromonas gingivalis growth and survival during intracellular autophagic life under nutritionally limited conditions.

Porphyromonas gingivalis survives in special autophagic vacuoles that serve as major replicative habitats in human primary gingival epithelial cells (GECs). As an asaccharolytic strict anaerobe, P. gingivalis is dependent on amino acids and peptides for nutrient sources. However, it is largely unknown as to P. gingivalis' metabolic processing under the nutritionally limited intracellular environments such the vacuoles, especially the preferred amino acids and associated-metabolic machineries. Here we elucidate that a Glutamate (Glu) catabolic enzyme, glutamate dehydrogenase (GdhA) is highly enriched in the isolated P. gingivalis -containing vacuoles. Interestingly, we found that P. gingivalis induces conversion of intracellular glutamine pool to Glu determined by analyses of the P. gingivalis- containing vacuoles and the whole infected-GECs. Critically, exogenous Glu-Glu dipeptide, a simple precursor of Glu, significantly increases the size of isolated intact P. gingivalis containing-vacuoles and live wild-type P. gingivalis numbers in GECs. In contrast, the isogenic GdhA-deficient-strain, Δ gdhA displayed a significant growth defect with collapsed-vacuoles in GECs. Next, we confirmed that P. gingivalis uptakes 14 C-Glu and it preferentially utilizes Glu-Glu-dipeptide using a nutritionally reduced Tryptic-Soy-Broth (TSB) media supplemented with Glu-Glu. Contrary, Δ gdhA -strain showed no detectable growth especially in nutritionally reduced TSB media with Glu-Glu. Using Atomic-Force-Microscopy, we observed that, wild-type P. gingivalis but not Δ gdhA strain notably increased the cell volume upon Glu-Glu supplementation, an indicator of higher metabolism and growth. Utilization of a human gingiva-mimicking organoid-system further validated the importance of Glu as an essential nutrient for the intramucosal colonization of P. gingivalis via the protected replicative vacuoles in GECs. Importance: This study reveals that P. gingivalis heavily depends on preferential utilization of Glutamate (Glu) for autophagic vacuolar growth and survival in human GECs. Several novel observations are made to support this: (i) GdhA of P. gingivalis is highly enriched in these vacuoles, (ii) P. gingivalis induces a large conversion of intracellular glutamine to Glu, (iii) size of vacuoles are significantly increased in the presence of Glu-Glu in P. gingivalis wild-type strain infection which is opposite in a Δ gdhA strain, (iv) P. gingivalis uptakes 14 C-Glu and preferentially utilizes Glu-Glu dipeptide, (v) similarly, wild-type strain shows growth increase in a nutritionally reduced bacterial culture media, and (vi) finally, Glu-Glu supplementation increases bacterial cell-volume of P. gingivalis wild-type but not Δ gdhA strain, an indicator of higher metabolism and growth. Taken together, this study highlights the pathophysiological importance of Glu for P. gingivalis growth-rate, biomass induction and survival in nutritionally limited host subcellular environments.

Opportunistic pathogen Porphyromonas gingivalis targets the LC3B-ceramide complex and mediates lethal mitophagy resistance in oral tumors.

Mechanisms by which Porphyromonas gingivalis (P. gingivalis) infection enhances oral tumor growth or resistance to cell death remain elusive. Here, we determined that P. gingivalis infection mediates therapeutic resistance via inhibiting lethal mitophagy in cancer cells and tumors. Mechanistically, P. gingivalis targets the LC3B-ceramide complex by associating with LC3B via bacterial major fimbriae (FimA) protein, preventing ceramide-dependent mitophagy in response to various therapeutic agents. Moreover, ceramide-mediated mitophagy is induced by Annexin A2 (ANXA2)-ceramide association involving the E142 residue of ANXA2. Inhibition of ANXA2-ceramide-LC3B complex formation by wild-type P. gingivalis prevented ceramide-dependent mitophagy. Moreover, a FimA-deletion mutant P. gingivalis variant had no inhibitory effects on ceramide-dependent mitophagy. Further, 16S rRNA sequencing of oral tumors indicated that P. gingivalis infection altered the microbiome of the tumor macroenvironment in response to ceramide analog treatment in mice. Thus, these data provide a mechanism describing the pro-survival roles of P. gingivalis in oral tumors.

Characterization of Human Genes Modulated by Porphyromonas gingivalis Highlights the Ribosome, Hypothalamus, and Cholinergic Neurons.

Porphyromonas gingivalis, a bacterium associated with periodontal disease, is a suspected cause of Alzheimer's disease. This bacterium is reliant on gingipain proteases, which cleave host proteins after arginine and lysine residues. To characterize gingipain susceptibility, we performed enrichment analyses of arginine and lysine proportion proteome-wide. Genes differentially expressed in brain samples with detected P. gingivalis reads were also examined. Genes from these analyses were tested for functional enrichment and specific neuroanatomical expression patterns. Proteins in the SRP-dependent cotranslational protein targeting to membrane pathway were enriched for these residues and previously associated with periodontal and Alzheimer's disease. These ribosomal genes are up-regulated in prefrontal cortex samples with detected P. gingivalis sequences. Other differentially expressed genes have been previously associated with dementia (ITM2B, MAPT, ZNF267, and DHX37). For an anatomical perspective, we characterized the expression of the P. gingivalis associated genes in the mouse and human brain. This analysis highlighted the hypothalamus, cholinergic neurons, and the basal forebrain. Our results suggest markers of neural P. gingivalis infection and link the cholinergic and gingipain hypotheses of Alzheimer's disease.

In Situ Intraepithelial Localizations of Opportunistic Pathogens, Porphyromonas gingivalis and Filifactor alocis, in Human Gingiva.

Porphyromonas gingivalis and Filifactor alocis are fastidious oral pathogens and etiological agents associated with chronic periodontitis. Although previous studies showed increased levels of the two obligate anaerobic species in periodontitis patients, methodologies for this knowledge were primarily limited to sampling subgingival plaque, saliva, or gingival crevicular fluid. To evaluate the extent to which P. gingivalis and F. alocis may invade the periodontal tissues, an in situ cross-sectional study was comparatively conducted on the gingival biopsy specimens of patients diagnosed with periodontal health or chronic periodontitis. Immunostained tissue sections for each organism were imaged by Super-Resolution Confocal Scanning Microscopy to determine the relative presence and localization of target bacterial species. Fluorescence-in-situ-hybridization (FISH) coupled with species specific 16S rRNA method was utilized to confirm whether detected bacteria were live within the tissue. In periodontitis, P. gingivalis and F. alocis revealed similarly concentrated localization near the basement membrane or external basal lamina of the gingival epithelium. The presence of both bacteria was significantly increased in periodontitis vs. healthy tissue. However, P. gingivalis was still detected to an extent in health tissue, while only minimal levels of F. alocis were spotted in health. Additionally, the micrographic analyses displayed heightened formation of epithelial microvasculature containing significantly co-localized and metabolically active dual species within periodontitis tissue. Thus, this study demonstrates, for the first-time, spatial arrangements of P. gingivalis and F. alocis in both single and co-localized forms within the complex fabric of human gingiva during health and disease. It also exhibits critical visualizations of co-invaded microvascularized epithelial layer of the tissue by metabolically active P. gingivalis and F. alocis from patients with severe periodontitis. These findings collectively uncover novel visual evidence of a potential starting point for systemic spread of opportunistic bacteria during their chronic colonization in gingival epithelium.