Fluorescence image analysis of the association between Porphyromonas gingivalis and gingival epithelial cells

Belton, Carol M.; Izutsu, Kenneth T.; Goodwin, Paul C.; Park, Yoonsuk; Lamont, Richard J.

doi:10.1046/j.1462-5822.1999.00022.x

Cited by 117 publications

(155 citation statements)

References 34 publications

(46 reference statements)

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“…18,21,[43][44][45] In gingival epithelial cells (GEC), P. gingivalis accumulates in the perinuclear area and neither apoptosis nor necrotic cell death is observed. 43,46,47 However, in KB cells the bacteria are found free in the cytosol or within single-membrane bound vacuoles. 19,[48][49][50] P. gingivalis has been shown to promote cell death in KB cells as well as in gingival fibroblasts.…”

Section: Bacterial Persistence and Host Cell Survivalmentioning

confidence: 99%

Autophagy: A Highway for Porphyromonas gingivalis in Endothelial Cells

Bélanger

Rodrigues²,

Dunn³

et al. 2006

Autophagy

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Section: Bacterial Persistence and Host Cell Survivalmentioning

confidence: 99%

Autophagy: A Highway for Porphyromonas gingivalis in Endothelial Cells

Bélanger

Rodrigues²,

Dunn³

et al. 2006

Autophagy

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“…P. gingivalis can attach to a variety of oral surfaces and can induce its internalization into the normally non-phagocytic gingival epithelial cells (GECs). The invasion of P. gingivalis into GECs is completed in under 20 min, and P. gingivalis accumulates in high numbers in the perinuclear region (Belton et al, 1999). Intracellularly, the organism remains viable and capable of replication.…”

Section: Introductionmentioning

confidence: 99%

“…Intracellularly, the organism remains viable and capable of replication. The GECs exhibit morphological changes after prolonged (24 h) association with intracellular P. gingivalis; however, they do not undergo apoptosis and maintain physiological integrity for extended periods (Lamont et al, 1995;Belton et al, 1999;Nakhjiri et al, 2001). Invasion is associated with phosphorylation of c-jun N-terminal kinase (JNK) and down-regulation of extracellular signal-regulated kinase (ERK1/2) .…”

Section: Introductionmentioning

confidence: 99%

Gingival epithelial cell signalling and cytoskeletal responses to Porphyromonas gingivalis invasion

et al. 2003

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Porphyromonas gingivalis, an oral pathogen, can internalize within primary gingival epithelial cells (GECs) through an invasion mechanism mediated by interactions between P. gingivalis fimbriae and integrins on the surface of the GECs. Fimbriae-integrin-based signalling events were studied by fluorescence microscopy, and the subcellular localization of integrin-associated signalling molecules paxillin and focal adhesion kinase (FAK), and the architecture of the actin and microtubule cytoskeleton were examined. GECs infected with P. gingivalis for 30 min demonstrated significant redistribution of paxillin and FAK from the cytosol to cell peripheries and assembly into focal adhesion complexes. In contrast, a fimbriae-deficient mutant of P. gingivalis did not contribute substantially to activation of paxillin or FAK. After 24 h, the majority of paxillin and FAK had returned to the cytoplasm with significant co-localization with P. gingivalis in the perinuclear region. Wild-type P. gingivalis induced nucleation of actin filaments forming microspike-like protrusions and long stable microfilaments distributed throughout the cells. Fimbriae mutants promoted a rich cortical actin meshwork accompanied by membrane ruffling dispersed along the cell membrane. Remarkable disassembly and nucleation of the actin and microtubule filamentous network was observed following 24 h infection with either wild-type or fimbriae-deficient mutants of P. gingivalis. The results show that fimbriated P. gingivalis cells induce formation of integrin-associated focal adhesions with subsequent remodelling of the actin and tubulin cytoskeleton.

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“…It produces a large number of virulence factors that are active against a broad spectrum of host proteins and provides mechanisms for evasion of host defenses 11 . It is also capable of invading epithelial, endothelial, fibroblastic, and periodontal ligament cells in vitro [12][13][14] and in vivo allows the bacterium to escape host defenses but also serves as a reservoir for future re-infection as well as alters the host cell. Studies of the molecular mechanisms involved in adhesion and internalization of the bacterium by host cells are needed.…”

Section: Introductionmentioning

confidence: 99%

<em>Porphyromonas gingivalis</em> as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Wunsch

Lewis

2015

JoVE

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Citation: Wunsch, C.M., Lewis, J.P. Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells. J. Vis. Exp. (106), e53408, doi:10.3791/53408 (2015). AbstractAnaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth. The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type. Video LinkThe video component of this article can be found at

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Fluorescence image analysis of the association between Porphyromonas gingivalis and gingival epithelial cells

Cited by 117 publications

References 34 publications

Autophagy: A Highway for Porphyromonas gingivalis in Endothelial Cells

Autophagy: A Highway for Porphyromonas gingivalis in Endothelial Cells

Gingival epithelial cell signalling and cytoskeletal responses to Porphyromonas gingivalis invasion

<em>Porphyromonas gingivalis</em> as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

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