scholarly journals Porphyromonas gingivalis Vesicles Enhance Attachment, and the Leucine-Rich Repeat BspA Protein Is Required for Invasion of Epithelial Cells by “Tannerella forsythia”

2006 ◽  
Vol 74 (9) ◽  
pp. 5023-5028 ◽  
Author(s):  
Satoru Inagaki ◽  
Shinsuke Onishi ◽  
Howard K. Kuramitsu ◽  
Ashu Sharma
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Porphyromonas Gingivalis ◽  
Cell Attachment ◽  
Membrane Vesicles ◽  
Defense Responses ◽  
Outer Membrane Vesicles ◽  
Cell Adherence ◽  
Tannerella Forsythia ◽  
Select Group

ABSTRACTThe human oral cavity harbors more than 500 species of bacteria. Periodontitis, a bacterially induced inflammatory disease that leads to tooth loss, is believed to result from infection by a select group of gram-negative periodontopathogens that includesPorphyromonas gingivalis,Treponema denticola, and “Tannerella forsythia” (opinion on name change fromTannerella forsythensispending; formerlyBacteroides forsythus). Epithelial cell invasion by periodontopathogens is considered to be an important virulence mechanism for evasion of the host defense responses. Further, the epithelial cells with invading bacteria also serve as reservoirs important in recurrent infections. The present study was therefore undertaken to address the epithelial cell adherence and invasion properties ofT. forsythiaand the role of the cell surface-associated protein BspA in these processes. Further, we were interested in determining ifP. gingivalis, one of the pathogens frequently found associated in disease, or its outer membrane vesicles (OMVs) could modulate the epithelial cell adherence and invasion abilities ofT. forsythia. Here we show that epithelial cell attachment and invasion byT. forsythiaare dependent on the BspA protein. In addition,P. gingivalisor its OMVs enhance the attachment and invasion ofT. forsythiato epithelial cells. Thus, interactions between these two bacteria may play important roles in virulence by promoting host cell attachment and invasion.

scholarly journals Porphyromonas gingivalis Outer Membrane Vesicles Enter Human Epithelial Cells via an Endocytic Pathway and Are Sorted to Lysosomal Compartments

2009 ◽  
Vol 77 (10) ◽  
pp. 4187-4196 ◽  
Author(s):  
Nobumichi Furuta ◽  
Kayoko Tsuda ◽  
Hiroko Omori ◽  
Tamotsu Yoshimori ◽  
Fuminobu Yoshimura ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Outer Membrane ◽  
Porphyromonas Gingivalis ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Periodontal Pathogen ◽  
Dependent Manner ◽  
Human Epithelial Cells

ABSTRACT Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including major fimbriae and proteases termed gingipains, although it is not confirmed whether MVs enter host cells. In this study, we analyzed the mechanisms involved in the interactions of P. gingivalis MVs with human epithelial cells. Our results showed that MVs swiftly adhered to HeLa and immortalized human gingival epithelial cells in a fimbria-dependent manner and then entered via a lipid raft-dependent endocytic pathway. The intracellular MVs were subsequently routed to early endosome antigen 1-associated compartments and then were sorted to lysosomal compartments within 90 min, suggesting that intracellular MVs were ultimately degraded by the cellular digestive machinery. However, P. gingivalis MVs remained there for over 24 h and significantly induced acidified compartment formation after being taken up by the cellular digestive machinery. In addition, MV entry was shown to be mediated by a novel pathway for transmission of bacterial products into host cells, a Rac1-regulated pinocytic pathway that is independent of caveolin, dynamin, and clathrin. Our findings indicate that P. gingivalis MVs efficiently enter host cells via an endocytic pathway and survive within the endocyte organelles for an extended period, which provides better understanding of the role of MVs in the etiology of periodontitis.

scholarly journals Entry of Porphyromonas gingivalis Outer Membrane Vesicles into Epithelial Cells Causes Cellular Functional Impairment

2009 ◽  
Vol 77 (11) ◽  
pp. 4761-4770 ◽  
Author(s):  
Nobumichi Furuta ◽  
Hiroki Takeuchi ◽  
Atsuo Amano
Keyword(s):  
Epithelial Cells ◽  
Outer Membrane ◽  
Porphyromonas Gingivalis ◽  
Virulence Factors ◽  
Functional Impairment ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Cellular Migration ◽  
Host Cells ◽  
Periodontal Pathogen

ABSTRACT Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including proteases termed gingipains (Arg-gingipain [Rgp] and Lys-gingipain [Kgp]). We recently showed that P. gingivalis MVs swiftly enter host epithelial cells via an endocytosis pathway and are finally sorted to lytic compartments. However, it remains unknown whether MV entry impairs cellular function. Herein, we analyzed cellular functional impairment following entry of P. gingivalis into epithelial cells, including HeLa and immortalized human gingival epithelial (IHGE) cells. After being taken up by endocytic vacuoles, MVs degraded the cellular transferrin receptor (TfR) and integrin-related signaling molecules, such as paxillin and focal adhesion kinase (FAK), which resulted in depletion of intracellular transferrin and inhibition of cellular migration. Few Rgp-null MVs entered the cells, and these negligibly degraded TfR, whereas paxillin and FAK degradation was significant. In contrast, Kgp-null MVs clearly entered the cells and degraded TfR, while they scarcely degraded paxillin and FAK. In addition, both wild-type and Kgp-null MVs significantly impaired cellular migration, whereas the effect of Rgp-null MVs was limited. Our findings suggest that, following entry of P. gingivalis MVs into host cells, MV-associated gingipains degrade cellular functional molecules such as TfR and paxillin/FAK, resulting in cellular impairment, indicating that P. gingivalis MVs are potent vehicles for transmission of virulence factors into host cells and are involved in the etiology of periodontitis.

scholarly journals Bioengineered airway epithelial grafts with mucociliary function based on collagen IV- and laminin-containing extracellular matrix scaffolds

2020 ◽  
Vol 55 (6) ◽  
pp. 1901200 ◽  
Author(s):  
Nick J.I. Hamilton ◽  
Dani Do Hyang Lee ◽  
Kate H.C. Gowers ◽  
Colin R. Butler ◽  
Elizabeth F. Maughan ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Airway Epithelium ◽  
Airway Epithelial Cell ◽  
Collagen Iv ◽  
Airway Epithelial ◽  
Cell Adherence ◽  
Bronchial Epithelial ◽  
Mucociliary Function

Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease.Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. In vitro, primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence.Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin α2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air–liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using in vivo chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation.Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models.

scholarly journals Porphyromonas gingivalis Fimbriae Bind to Cytokeratin of Epithelial Cells

2002 ◽  
Vol 70 (1) ◽  
pp. 96-101 ◽  
Author(s):  
Hakimuddin T. Sojar ◽  
Ashu Sharma ◽  
Robert J. Genco
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Porphyromonas Gingivalis ◽  
Polyclonal Antibodies ◽  
Cell Extract ◽  
Host Cells ◽  
Amino Terminal ◽  
A Minor ◽  
Sepharose 4B ◽  
Overlay Assay

ABSTRACT The adherence of Porphyromonas gingivalis to host cells is likely a prerequisite step in the pathogenesis of P. gingivalis-induced periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are shown to be involved in this process. Little is known regarding epithelial receptor(s) involved in binding of P. gingivalis fimbriae. Using an overlay assay with purified P. gingivalis fimbriae as a probe, two major epithelial cell proteins with masses of 50 and 40 kDa were identified by immunoblotting with fimbria-specific antibodies. Iodinated purified fimbriae also bound to the same two epithelial cell proteins. An affinity chromatography technique was utilized to isolate and purify the epithelial components to which P. gingivalis fimbriae bind. Purified fimbriae were coupled to CNBr-activated Sepharose-4B, and the solubilized epithelial cell extract proteins bound to the immobilized fimbriae were isolated from the column. A major 50-kDa component and a minor 40-kDa component were purified and could be digested with trypsin, suggesting that they were proteins. These affinity-eluted 50- and 40-kDa proteins were then subjected to amino-terminal sequencing, and no sequence could be determined, suggesting that these proteins have blocked amino-terminal residues. CNBr digestion of the 50-kDa component resulted in an internal sequence homologous to that of Keratin I molecules. Further evidence that P. gingivalis fimbriae bind to cytokeratin molecule(s) comes from studies showing that multicytokeratin rabbit polyclonal antibodies cross-react with the affinity-purified 50-kDa epithelial cell surface component. Also, binding of purified P. gingivalis fimbriae to epithelial components can be inhibited in an overlay assay by multicytokeratin rabbit polyclonal antibodies. Furthermore, we showed that biotinylated purified fimbriae bind to purified human epidermal keratin in an overlay assay. These studies suggest that the surface-accessible epithelial cytokeratins may act as receptor(s) for P. gingivalis fimbriae. We hypothesize that adherence of P. gingivalis fimbriae to cytokeratin may be important for colonization of oral mucous membranes and possibly also for activation of epithelial cells.

scholarly journals Cyclodextrins reduce the ability of Pseudomonas aeruginosa outer-membrane vesicles to reduce CFTR Cl− secretion

2019 ◽  
Vol 316 (1) ◽  
pp. L206-L215 ◽  
Author(s):  
Roxanna Barnaby ◽  
Katja Koeppen ◽  
Bruce A. Stanton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Outer Membrane ◽  
Membrane Vesicles ◽  
Airway Epithelial Cells ◽  
Outer Membrane Vesicles ◽  
Airway Epithelial ◽  
Ussing Chambers ◽  
Planktonic Growth ◽  
Apical Side

Pseudomonas aeruginosa secretes outer-membrane vesicles (OMVs) that fuse with cholesterol-rich lipid rafts in the apical membrane of airway epithelial cells and decrease wt-CFTR Cl− secretion. Herein, we tested the hypothesis that a reduction of the cholesterol content of CF human airway epithelial cells by cyclodextrins reduces the inhibitory effect of OMVs on VX-809 (lumacaftor)-stimulated Phe508del CFTR Cl− secretion. Primary CF bronchial epithelial cells and CFBE cells were treated with vehicle, hydroxypropyl-β-cyclodextrin (HPβCD), or methyl-β-cyclodextrin (MβCD), and the effects of OMVs secreted by P. aeruginosa on VX-809 stimulated Phe508del CFTR Cl− secretion were measured in Ussing chambers. Neither HPβCD nor MβCD were cytotoxic, and neither altered Phe508del CFTR Cl− secretion. Both cyclodextrins reduced OMV inhibition of VX-809-stimulated Phe508del-CFTR Cl− secretion when added to the apical side of CF monolayers. Both cyclodextrins also reduced the ability of P. aeruginosa to form biofilms and suppressed planktonic growth of P. aeruginosa. Our data suggest that HPβCD, which is in clinical trials for Niemann-Pick Type C disease, and MβCD, which has been approved by the U.S. Food and Drug Administration for use in solubilizing lipophilic drugs, may enhance the clinical efficacy of VX-809 in CF patients when added to the apical side of airway epithelial cells, and reduce planktonic growth and biofilm formation by P. aeruginosa. Both effects would be beneficial to CF patients.

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