No abstract

The invasion process of Actinobacillus actinomycetemcomitans, a periodontopathogen, was studied with microscopy and viable quantitative assays using both KB and Madin-Darby canine kidney (MDCK) epithelial cells. Microscopy revealed that the events associated with the A. actinomycetemcomitans invasion process occurred rapidly. Scanning electron micrographs revealed A. actinomycetemcomitans associated with craters on the KB cell surface and others entering the KB cells through apertures with lip-like rims within 30 min of infection. Both transmission electron and immunofluorescence micrographs demonstrated that by this time some bacteria had, in fact, already entered, replicated, and exited host cells. Scanning electron micrographs revealed that infected KB cells exhibited fibrillar protrusions which contained bulges with the conformation of bacteria. Some protrusions formed intercellular connections between KB cells. Immunofluorescence micrographs revealed protrusions which harbored A. actinomycetemcomitans. The spread of internalized A. actinomycetemcomitans from one MDCK epithelial cell monolayer to another was demonstrated using a sandwich assay developed in our laboratory. Transcytosis of A. actinomycetemcomitans through polarized MDCK cells was also demonstrated. This study indicates that soon after entry of A. actinomycetemcomitans bacteria into epithelial cells, they undergo rapid multiplication and may subsequently be found in protrusions which sometimes extend between neighboring epithelial cells. The protrusions are thought to mediate the cell-to-cell spread of A. actinomycetemcomitans. Cell-to-cell spread may also occur by the endocytosis of A. actinomycetemcomitans bacteria which have been released into the medium via rudimentary protrusions which do not interconnect epithelial cells. The finding that the A. actinomycetemcomitans invasion process is so dynamic sheds significant new light on the interaction of this periodontopathogen with mammalian cells.

This pilot study successfully demonstrated that pranayama was associated with improved exercise tolerance in patients with COPD. Lay personnel were able to adequately teach patients to practice pranayama. These results suggest that pranayama may have significant clinical benefits for symptomatic patients with COPD, a concept that needs to be confirmed in future, larger clinical trials.

Actinobacillus actinomycetemcomitans SUNY 465, the invasion prototype strain, enters epithelial cells by an actin-dependent mechanism, escapes from the host cell vacuole, and spreads intracellularly and to adjacent epithelial cells via intercellular protrusions. Internalized organisms also egress from host cells into the assay medium via protrusions that are associated with just a single epithelial cell. Here we demonstrate that agents which inhibit microtubule polymerization (e.g., colchicine) and those which stabilize polymerized microtubules (e.g., taxol) both increase markedly the number of intracellular A. actinomycetemcomitansorganisms. Furthermore, both colchicine and taxol prevented the egression of A. actinomycetemcomitans from host cells into the assay medium. Immunofluorescence microscopy revealed that protrusions that mediate the bacterial spread contain microtubules.A. actinomycetemcomitans SUNY 465 and 652, strains that are both invasive and egressive, interacted specifically with the plus ends (growing ends) of the filaments of microtubule asters in a KB cell extract. By contrast, neither A. actinomycetemcomitans 523, a strain that is invasive but not egressive, nor Haemophilus aphrophilus, a noninvasive oral bacterium with characteristics similar to those of A. actinomycetemcomitans, bound to microtubules. Together these data suggest that microtubules function in the spread and movement of A. actinomycetemcomitans and provide the first evidence that host cell dispersion of an invasive bacterium may involve the usurption of host cell microtubules.

DNA adenine methyltransferase (DAM) plays critical roles in diverse biological pathways in gram-negative bacteria, and specifically in regulating the expression of virulence genes in several organisms. Actinobacillus actinomycetemcomitans plays an important role in the pathogenesis of juvenile and adult periodontal disease, yet little is known about its mechanisms of gene regulation. DAM is shown here to directly or indirectly affect well-known A. actinomycetemcomitans virulence factors. A mutant A. actinomycetemcomitans strain lacking the dam gene was created by homologous recombination and shows normal growth phenotypes when grown exponentially. This mutant strain has four sixfold increased levels of extracellular leukotoxin, altered cellular levels of leukotoxin, and significant changes in bacterial invasion of KB oral epithelial cells. These results provide a basis for further characterization of regulatory mechanisms that control A. actinomycetemcomitans virulence.