Host membrane trafficking for conveyance of intracellular oral pathogens
ATSUO AMANO, NOBUMICHI FURUTA & KAYOKO TSUDA
Entry of bacteria into host cells allows pathogens to occupy various niches within the human body, which is required for the successful establishment of bacterial infection. An intracellular location is considered to be advantageous for bacteria to escape immune surveillance by the host, as well as antibiotic pressure; this leads to intracellular persistence, multiplication and dissemination to adjacent tissues (14). Invasive bacterial pathogens and intracellular parasites are known to enter nonphagocytic cells by utilization of a diverse array of adhesive and invasive molecules (termed adhesins and invasins, respectively, or internalin), which are receptors able to exploit host-cell surface components. Recent results obtained from tissue culture assays and in vivo studies have revealed that many species of common bacteria which were previously considered to be extracellular etiological agents express an ability to invade nonprofessional phagocytic cells (reviewed in ref. 13). In earlier periodontal research studies, electron microscopy observations showed that a variety of plaque-forming bacteria were able to penetrate the epithelium of periodontal pockets in individuals with advanced periodontitis.
Furthermore, immunofluorescence and immunohistochemical techniques revealed the existence of
Porphyromonas gingivalis, Aggregatibacter (formally
Actinobacillus) actinomycetemcomitans, Prevotella intermedia and Actinomyces naeslundii in gingival tissues (reviewed in ref. 34). The persistent contact of subgingival bacterial biofilm with gingival crevices easily evokes the penetration of bacteria into periodontal tissues; however, results presented since 2000 have shown that periodontal bacteria can also enter periodontal cells. More recently, an optical sectioning technique that utilized confocal scanning laser microscopy showed the intracellular localization of several periodontal bacteria (34), while a method that used in situ hybridization with 16 ribosomal RNA probes and confocal scanning laser microscopy detected P. gingivalis, A. actinomycetemcomitans, Tannerella forsythia (forsythensis) and Treponema denticola within epithelial cells obtained from periodontal pockets, gingival crevices and buccal mucosa collected from subjects with or without chronic marginal periodontitis (12). Thus, most periodontal pathogens are probably able to enter periodontal cells, although it remains largely unknown how those microbes enter host cells, or if such intracellular bacteria can survive and multiply, or are killed. This article addresses the remarkable strategies used by periodontal pathogens to enter host cells.
Bacterial entry mechanisms
The plasma membrane of eukaryotic cells constitutes a dynamic boundary that isolates the cytoplasm from the surrounding environment (13). Traverse of this membrane by small molecules, such as ions and sugars, is easily mediated through various transmembrane channels and pumps in the bilayer.
By contrast, the transport of macromolecules through the plasma membrane requires endocytosis. In endocytosis, membrane domains invaginate and are then pinched off from the inner side of the plasma membrane and transported within the cell. The cellular endocytic machinery that engulfs bacteria is grouped into two major processes: phagocytosis and endocytosis. Phagocytosis is basically restricted to professional phagocytic cells, including macrophages, monocytes and neutrophils, which eliminate foreign substrates (including pathogens), while 84
Periodontology 2000, Vol. 52, 2010, 84–93
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PERIODONTOLOGY 2000 endocytosis is a process common to all cell types except red blood cells. Most invasive bacteria utilize endocytosis to become engulfed by the host cells.
Endocytosis encompasses several diverse mechanisms that cells use to internalize macromolecules and particles into transport vesicles derived from the plasma membrane. The endocytic pathways differ based on the size of the endocytic vesicle, for example macropinocytosis (<1 lm), clathrin-mediated endocytosis (120 nm), caveolae-mediated endocytosis (60 nm) and clathrin- ⁄ caveolin-independent endocytosis, which is either dependent or independent of lipid rafts (90 nm) (13). Macropinocytosis shares similarities with phagocytosis and is mediated by small GTPases that stimulate important actin polymerization and depolymerization events (13, 52). The membrane ruffles formed in this process then collapse onto the plasma membrane, where they fuse together and form a macropinosome that is larger than normal endocytic vesicles. Clathrinmediated endocytosis and caveolae-mediated endocytosis are characterized by the formation of their respective coats, which are protein complexes that form at the site where the plasma membrane begins to invaginate (9). These coat complexes assemble into a curved rigid scaffold in tight association with the plasma membrane and mediate the formation of endocytic carrier precursors (i.e. clathrin-coated or caveolin-coated pits), which remain connected to the extracellular milieu through constricted necks. In the case of clathrin-coated and caveolin-coated pits, dynamin – a fission-inducing protein – cuts off the necks of independent coated vesicles (15, 26).
Clathrin-mediated internalization is exploited to internalize molecules that bind to a receptor, such as the transferrin receptor, which is exposed on the external face of the plasma membrane. For example,
Listeria monocytogenes hijacks the clathrin-mediated endocytic machinery to permit its entry into host cells (67). The results of other reports have also shown that bacteria are often taken up via clathrindependent and ⁄ or caveolin-dependent pathways, probably because of bacterial size (0.5–2 lm). This method of bacterial entry requires dynamin activity to generate endocytic carriers (57, 62), as well as Rho family GTPases, such as Rac1 and Cdc42, for endocytic actin re-arrangement (6, 24).