Intracellular periodontal pathogen exploits recycling pathway to exit from infected cells

Although human gingival epithelium prevents intrusions by periodontal bacteria, Porphyromonas gingivalis, the most well‐known periodontal pathogen, is able to invade gingival epithelial cells and pass through the epithelial barrier into deeper tissues. We previously reported that intracellular P. gingivalis exits from gingival epithelial cells via a recycling pathway. However, the underlying molecular process remains unknown. In the present study, we found that the pathogen localized in early endosomes recruits VAMP2 and Rab4A. VAMP2 was found to be specifically localized in early endosomes, although its localization remained unclear in mammalian cells. A single transmembrane domain of VAMP2 was found to be necessary and sufficient for localizing in early endosomes containing P. gingivalis in gingival epithelial cells. VAMP2 forms a complex with EXOC2/Sec5 and EXOC3/Sec6, whereas Rab4A mediates dissociation of the EXOC complex followed by recruitment of RUFY1/Rabip4, Rab4A effector, and Rab14. Depletion of VAMP2 or Rab4A resulted in accumulation of bacteria in early endosomes and disturbed bacterial exit from infected cells. It is suggested that these novel dynamics allow P. gingivalis to exploit fast recycling pathways promoting further bacterial penetration of gingival tissues.     

Porphyromonas gingivalis induces the production of interleukin‐31 by human mast cells,resulting in dysfunction of the gingival epithelial barrier

Interleukin (IL)‐31 is important for innate immunity in mucosal tissues and skin, and increased IL‐31 expression participates in the pathogenesis of chronic inflammatory diseases affecting the skin, airways, lungs, and intestines. We investigated the contribution of mast cells to the induction of IL‐31 production following infection with the periodontal pathogen, Porphyromonas gingivalis. We found that oral infection with P. gingivalis increased IL‐31 expression in the gingival tissues of wild‐type mice but not in those of mast cell‐deficient mice. The P. gingivalis‐induced IL‐31 production by human mast cells occurred through the activation of the JNK and NF‐κB signalling pathways and was dependent on the P. gingivalis lysine‐specific protease gingipain‐K. P. gingivalis infection induced IL‐31 receptor α and oncostatin M receptor β expression in human gingival epithelial cells. Notably, the P. gingivalis‐induced IL‐31 production by mast cells led to the downregulation of claudin‐1, a tight junction molecule, in gingival epithelial cells, resulting in an IL‐31‐dependent increase in the paracellular permeability of the gingival epithelial barrier. These findings suggest that IL‐31 produced by mast cells in response to P. gingivalis infection causes gingival epithelial barrier dysfunction, which may contribute to the chronic inflammation observed in periodontitis.     

Lactobacillus helveticus SBT2171 upregulates the expression of β‐defensin and ameliorates periodontal disease caused by Porphyromonas gingivalis

Antimicrobial peptides play important roles in the innate immune system of various organisms, and they may also be considered to prevent the organisms from infections. In particular, β‐defensins, mainly produced in epithelial cells, are recognized as one of the major antimicrobial peptides in mammals, including humans. In this study, we showed that Lactobacillus helveticus SBT2171 (LH2171), one of the several species of lactic acid bacteria, upregulates the production of β‐defensins in oral epithelial cells in vitro. Moreover, LH2171 reduced the increase of proinflammatory cytokine expression, induced by Porphyromonas gingivalis stimulation, in gingival epithelial cells. These data suggested that LH2171 suppresses P. gingivalis‐induced inflammation by upregulating the expression of β‐defensins in gingival epithelial cells. We subsequently investigated the effects of LH2171 in vivo and revealed that β‐defensin expression was increased in the oral cavities of LH2171‐fed mice. Furthermore, LH2171 decreased alveolar bone loss, gingival inflammation, and amounts of P. gingivalis‐specific 16S ribosomal RNA in the gingiva of P. gingivalis‐inoculated mice. Taken together, our results showed that LH2171 upregulates the expression of β‐defensins in oral cavity, thereby decreasing the number of P. gingivalis consequently ameliorating the experimental periodontal disease.     

Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.     

Epithelial cell detachment by Porphyromonas gingivalis biofilm and planktonic cultures

Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated with epithelial cells in a 24-well plate for 4 h. Epithelial cell detachment was assessed using imaging. The activity of arginine-gingipain (Rgp) and gene expression profiles of P. gingivalis cultures were examined using a gingipain assay and quantitative PCR, respectively. P. gingivalis biofilms induced significantly higher cell detachment and displayed higher Rgp activity compared to the planktonic cultures. The genes involved in gingipain post-translational modification, but not rgp genes, were significantly up-regulated in P. gingivalis biofilms. The results underline the importance of including biofilms in the study of bacterial and host cell interactions.     

Regulation of protease‐activated receptor‐2 expression in gingival fibroblasts and Jurkat T cells by Porphyromonas gingivalis

Periodontal disease destroys the tooth‐supporting tissues as a result of chronic inflammation elicited by bacterial accumulation on tooth surfaces. Porphyromonas gingivalis is a major periodontal pathogen, with a significant capacity to perturb connective tissue homeostasis and immune responses in the periodontium, attributed to its virulence factors, including a group of secreted cysteine proteases (gingipains). PAR‐2 (protease‐activated receptor‐2) is a G‐protein‐coupled receptor activated upon proteolytic cleavage, mediating intracellular signalling events related to infection and inflammation, such as cytokine production. GF (gingival fibroblasts) and T cells have central roles in periodontal inflammation, which can potentially be mediated by PAR‐2. The aims of this study were to investigate the effects of P. gingivalis on PAR‐2 gene expression in human GF and Jurkat T cells, using quantitative real‐time PCR, and to evaluate the involvement of gingipains. After 6 h of challenge with ascending concentrations of P. gingivalis, PAR‐2 expression was up‐regulated in both cell types by approximately 5‐fold, compared with the control. The P. gingivalis concentration required for maximal PAR‐2 induction was 4‐fold greater in GF than Jurkat T cells. Heat inactivation or chemical inhibition of cysteine proteases abolished the capacity of P. gingivalis to induce PAR‐2 expression in Jurkat T cells. In conclusion, P. gingivalis can induce PAR‐2 expression in GF and Jurkat T cells, potentially attributed to its gingipains. These findings denote that P. gingivalis may perturb the host immune and inflammatory responses by enhancing PAR‐2 expression, thus contributing to the pathogenesis of periodontal disease.     

ATP‐dependent activation of an inflammasome in primary gingival epithelial cells infected by Porphyromonas gingivalis

Production of IL‐1β typically requires two‐separate signals. The first signal, from a pathogen‐associated molecular pattern, promotes intracellular production of immature cytokine. The second signal, derived from a danger signal such as extracellular ATP, results in assembly of an inflammasome, activation of caspase‐1 and secretion of mature cytokine. The inflammasome component, Nalp3, plays a non‐redundant role in caspase‐1 activation in response to ATP binding to P2X₇ in macrophages. Gingival epithelial cells (GECs) are an important component of the innate‐immune response to periodontal bacteria. We had shown that GECs express a functional P2X₇ receptor, but the ability of GECs to secrete IL‐1β during infection remained unknown. We find that GECs express a functional Nalp3 inflammasome. Treatment of GECs with LPS or infection with the periodontal pathogen, Porphyromonas gingivalis, induced expression of the il‐1β gene and intracellular accumulation of IL‐1β protein. However, IL‐1β was not secreted unless LPS‐treated or infected cells were subsequently stimulated with ATP. Conversely, caspase‐1 is activated in GECs following ATP treatment but not P. gingivalis infection. Furthermore, depletion of Nalp3 by siRNA abrogated the ability of ATP to induce IL‐1β secretion in infected cells. The Nalp3 inflammasome is therefore likely to be an important mediator of the inflammatory response in gingival epithelium.     

Differential Endosomal Sorting of a Novel P2Y12 Purinoreceptor Mutant

P2Y₁₂ receptor internalization and recycling play an essential role in ADP‐induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y₁₂ (P341A) whose P2Y₁₂ receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild‐type (WT) P2Y₁₂ recycling and investigated P2Y₁₂‐P341A receptor traffic. Treatment with ADP resulted in delayed Rab5‐dependent internalization of P341A when compared with WT P2Y₁₂. While WT P2Y₁₂ rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7‐positive endosomes with considerable agonist‐dependent accumulation in the trans‐Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT‐P2Y₁₂. Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y₁₂ receptor traffic and explain the compromised receptor function in the platelets of the P2Y₁₂‐P341A‐expressing patient.     

Drosophila exocyst components Sec5, Sec6, and Sec15 regulate DE-Cadherin trafficking from recycling endosomes to the plasma membrane

The E-Cadherin-catenin complex plays a critical role in epithelial cell-cell adhesion, polarization, and morphogenesis. Here, we have analyzed the mechanism of Drosophila E-Cadherin (DE-Cad) localization. Loss of function of the Drosophila exocyst components sec5, sec6, and sec15 in epithelial cells results in DE-Cad accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits DE-Cad delivery to the membrane. Furthermore, Rab11 and Armadillo interact with the exocyst components Sec15 and Sec10, respectively. Our results support a model whereby the exocyst regulates DE-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located.     

Regulation of ICAM‐1 expression in gingival fibroblasts infected with high‐glucose‐treated P. gingivalis

Porphyromonas gingivalis is a major pathogen in the initiation and progression of periodontal disease, which is recognized as a common complication of diabetes. ICAM‐1 expression by human gingival fibroblasts (HGFs) is crucial for regulating local inflammatory responses in inflamed periodontal tissues. However, the effect of P. gingivalis in a high‐glucose situation in regulating HGF function is not understood. The P. gingivalis strain CCUG25226 was used to study the mechanisms underlying the modulation of HGF ICAM‐1 expression by invasion of high‐glucose‐treated P. gingivalis (HGPg). A high‐glucose condition upregulated fimA mRNA expression in P. gingivalis and increased its invasion ability in HGFs. HGF invasion with HGPg induced increases in the expression of ICAM‐1. By using specific inhibitors and short hairpin RNA (shRNA), we have demonstrated that the activation of p38 MAPK and Akt pathways is critical for HGPg‐induced ICAM‐1 expression. Luciferase reporters and chromatin immunoprecipitation assays suggest that HGPg invasion increases NF‐κB‐ and Sp1‐DNA‐binding activities in HGFs. Inhibition of NF‐κB and Sp1 activations blocked the HGPg‐induced ICAM‐1 promoter activity and expression. The effect of HGPg on HGF signalling and ICAM‐1 expression is mediated by CXC chemokine receptor 4 (CXCR4). Our findings identify the molecular pathways underlying HGPg‐dependent ICAM‐1 expression in HGFs, providing insight into the effect of P. gingivalis invasion in HGFs.     

Porphyromonas gingivalis Gingipain-Dependently Enhances IL-33 Production in Human Gingival Epithelial Cells

The cytokine IL-33 is constitutively expressed in epithelial cells and it augments Th2 cytokine-mediated inflammatory responses by regulating innate immune cells. We aimed to determine the role of the periodontal pathogen, Porphyromonas gingivalis, in the enhanced expression of IL-33 in human gingival epithelial cells. We detected IL-33 in inflamed gingival epithelium from patients with chronic periodontitis, and found that P. gingivalis increased IL-33 expression in the cytoplasm of human gingival epithelial cells in vitro. In contrast, lipopolysaccharide, lipopeptide, and fimbriae derived from P. gingivalis did not increase IL-33 expression. Specific inhibitors of P. gingivalis proteases (gingipains) suppressed IL-33 mRNA induction by P. gingivalis and the P. gingivalis gingipain-null mutant KDP136 did not induce IL-33 expression. A small interfering RNA for protease-activated receptor-2 (PAR-2) as well as inhibitors of phospholipase C, p38 and NF-κB inhibited the expression of IL-33 induced by P. gingivalis. These results indicate that the PAR-2/IL-33 axis is promoted by P. gingivalis infection in human gingival epithelial cells through a gingipain-dependent mechanism.     

Rab11 is required for tubulogenesis of Malpighian tubules in Drosophila melanogaster

Intracellular vesicular trafficking is one of the important tools in maintaining polarity, adhesion, and shape of epithelial cells. Rab11, a subfamily of the Ypt/Rab gene family of ubiquitously expressed GTPases and a molecular marker of recycling endosomes, transports different components of plasma membrane. Here, we report that Rab11 affects tubulogenesis of Malpighian tubules (MTs). MTs are simple polarized epithelial tubular structures, considered as functional analogue of human kidney. Rab11 has pleiotropic effects on MTs development as down‐regulation of Rab11 in principal cells (PCs) of MTs from embryonic stages of development results in reduced endoreplication, clustering of cells, disorganized cytoskeleton, and disruption of polarity leading to shortening of MTs in third instar larvae. Rab11 is also required for proper localization of different transporters in PCs, essential for physiological activity of MTs. Collectively, our data suggest that Rab11 plays a key role in the process of tubulogenesis of MTs in Drosophila.     

Rab6 Is Required for Multiple Apical Transport Pathways but Not the Basolateral Transport Pathway in Drosophila Photoreceptors

Polarized membrane trafficking is essential for the construction and maintenance of multiple plasma membrane domains of cells. Highly polarized Drosophila photoreceptors are an excellent model for studying polarized transport. A single cross-section of Drosophila retina contains many photoreceptors with 3 clearly differentiated plasma membrane domains: a rhabdomere, stalk, and basolateral membrane. Genome-wide high-throughput ethyl methanesulfonate screening followed by precise immunohistochemical analysis identified a mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with normal basolateral transport. Rapid gene identification using whole-genome resequencing and single nucleotide polymorphism mapping identified a nonsense mutation of Rab6 responsible for the apical-specific transport deficiency. Detailed analysis of the trafficking of a major rhabdomere protein Rh1 using blue light-induced chromophore supply identified Rab6 as essential for Rh1 to exit the Golgi units. Rab6 is mostly distributed from the trans-Golgi network to a Golgi-associated Rab11-positive compartment that likely recycles endosomes or transport vesicles going to recycling endosomes. Furthermore, the Rab6 effector, Rich, is required for Rab6 recruitment in the trans-Golgi network. Moreover, a Rich null mutation phenocopies the Rab6 null mutant, indicating that Rich functions as a guanine nucleotide exchange factor for Rab6. The results collectively indicate that Rab6 and Rich are essential for the trans-Golgi network–recycling endosome transport of cargoes destined for 2 apical domains. However, basolateral cargos are sorted and exported from the trans-Golgi network in a Rab6-independent manner.     

The Anaplasma phagocytophilum‐occupied vacuole selectively recruits Rab‐GTPases that are predominantly associated with recycling endosomes

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell‐derived vacuole. The A. phagocytophilum‐occupied vacuole (ApV) fails to mature along the endocytic pathway and is non‐fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV's selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)‐tagged Rab GTPases in A. phagocytophilum‐infected HL‐60 cells. GFP‐Rab4A, GFP‐Rab10, GFP‐Rab11A, GFP‐Rab14, RFP‐Rab22A and GFP‐Rab35, which regulate endocytic recycling, and GFP‐Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome‐associated GFP‐Rabs and acquisition of GFP‐Rab5, GFP‐Rab7, and the lysosomal marker, LAMP‐1. Wild‐type and GTPase‐ deficient versions, but not GDP‐restricted versions of GFP‐Rab1, GFP‐Rab4A and GFP‐Rab11A, localize to the ApV. Strikingly, GFP‐Rab10 recruitment to the ApV is guanine nucleotide‐independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.     

Implication of Toll/IL-1 receptor domain containing adapters in Porphyromonas gingivalis-induced inflammation

Periodontitis is induced by periodontal dysbiosis characterized by the predominance of anaerobic species. TLRs constitute the classical pathway for cell activation by infection. Interestingly, the Toll/IL-1 receptor homology domain adapters initiate signaling events, leading to the activation of the expression of the genes involved in the host immune response. The aim of this study was to evaluate the effects of Porphyromonas gingivalis on the expression and protein-protein interactions among five TIR adapters (MAL, MyD88, TRIF, TRAM and SARM) in gingival epithelial cells and endothelial cells. It was observed that P. gingivalis is able to modulate the signaling cascades activated through its recognition by TLR4/2 in gingival epithelial cells and endothelial cells. Indeed, MAL-MyD88 protein-protein interactions associated with TLR4 was the main pathway activated by P. gingivalis infection. When transient siRNA inhibition was performed, cell viability, inflammation, and cell death induced by infection decreased and such deleterious effects were almost absent when MAL or TRAM were targeted. This study emphasizes the role of such TIR adapter proteins in P. gingivalis elicited inflammation and the precise evaluation of TIR adapter protein interactions may pave the way for future therapeutics in both periodontitis and systemic disease with a P. gingivalis involvement, such as atherothrombosis.     

Developmental expression of Rab11, a small GTP‐binding protein in Drosophila epithelia

Intracellular membrane trafficking regulates a wide variety of developmental processes, including cell and tissue morphogenesis. Here we report developmental expression of Drosophila Rab11, a small GTP‐binding protein, required for both endocytic recycling and exocytosis. Rab11 is expressed in the epithelial cell types of diverse lineages at all developmental stages, beginning from the cellular blastoderm in early embryos to adult primordia and adult tissues, like the columnar epithelia lining male ejaculatory bulb. A robust expression of Rab11 is seen both in the amnioserosa and in the lateral epidermis during embryonic dorsal closure, a morphogenetic event that involves spreading and fusion of the contra‐lateral sides of epidermis. Rab11 mutant embryos fail to display the characteristic morphological changes in these two epithelial tissues during dorsal closure, providing a strong basis to dissect the role of Rab11 in coordinated epithelial sheet movements. genesis 47:32–39, 2009. © 2008 Wiley‐Liss, Inc.