Microtubules are associated with intracellular movement and spread of the periodontopathogen Actinobacillus actinomycetemcomitans

Abstract

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. actinomycetemcomitans organisms. 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.

FIG. 1

Taxol, nocodazole, and colchicine increased the number of intracellular organisms recovered from A. actinomycetemcomitans-infected KB cells. Taxol (10 μM), nocodazole (10 μg/ml), and colchicine (5 μg/ml) were added to monolayers 15 min prior to the addition of A. actinomycetemcomitans and were present in the assay medium at these concentrations throughout the assay. The bars represent ratios of internalized A. actinomycetemcomitans in the presence of the microtubule modulator to internalized A. actinomycetemcomitans in the absence of the modulator. Values are the means for quadruplicate samples from a typical experiment. The standard deviation was less than 25% of the mean in all cases.

FIG. 2

Immunofluorescence microscopy of taxol-treated KB cells infected with A. actinomycetemcomitans SUNY 465. (a) Untreated KB cells; (b) taxol (10 μM)-treated KB cells. Taxol was added to KB cells 15 min prior to infection to stabilize the microtubules. The staining and filter set used allows visualization of only internalized bacteria (arrows) and not KB cells; thus, the KB cells (grey) are not well defined. Note that the number of intracellular A. actinomycetemcomitans organisms is increased greatly and bacteria occur in clusters in KB cells treated with taxol.

FIG. 3

Effects of inhibitors on the egression of A. actinomycetemcomitans SUNY 465 from KB cells. (a) Kinetics of A. actinomycetemcomitans accumulation into the assay medium. A. actinomycetemcomitans egresses from control cells and accumulates in the medium (). Bacteria do not accumulate in the assay medium if either taxol (□) or colchicine (■) is present. Bars represent additive values (i.e., the 60-min bar represents the 40 × 10³ CFU recovered at 30 min plus 20 × 10³ CFU recovered during the next 30 min, and so on) from a typical experiment carried out in quadruplicate. The standard deviation was less than 20% of the mean in all cases. The inset shows SUNY 465 in untreated and taxol-treated cells at time zero (□) and 180 min (). (b) SUNY 465 recovered from KB cells after treatment with cytochalasin D or brefeldin A. ■, control cells; , treated cells. KB cells were treated with brefeldin A prior to infection, whereas cytochalasin D was added 90 min after infection.

FIG. 4

Immunofluorescence micrographs of KB cells 60 min after infection with A. actinomycetemcomitans SUNY 465. KB cell microtubules were labeled with mouse monoclonal α-tubulin and TRITC-conjugated anti-mouse IgG. Bacteria were labeled with rabbit immune serum specific for SUNY 465 and secondary IgG conjugated to either FITC or TRITC. Bacteria were differentially stained by a double-labeling technique, and internal and external organisms were distinguished by comparing fields with red (TRITC) and green (FITC) fluores cence filters. Internalized organisms are visualized only with the FITC filter, whereas external organisms are visualized with both FITC and TRITC filters; i.e., if an organism is visualized with both filters, it is external. Microtubules are visualized only with the TRITC filter. The micrographs shown here were taken with a multiuse filter which allows the simultaneous visualization of both bacteria and microtubules. (a) Microtubules (white arrowheads) were dispersed throughout the cytoplasm of KB cells. Internalized A. actinomycetemcomitans (black arrows) localized to the same region of the cytoplasm. (b and c) Microtubules also occurred in both cell-to-cell (b) and rudimentary (c) protrusions. Bacteria in protrusions are indicated by the arrow in panel b and by arrowheads in panel c.

FIG. 5

Immunofluorescence micrographs of the interaction of bacteria with taxol-induced microtubule asters. A. actinomycetemcomitans SUNY 465 (a) and 652 (b) and L. monocytogenes (c), organisms that can invade and egress from host cells, all bound primarily to the plus ends of microtubules in asters. S. flexneri, another organism that can invade and egress, bound to the minus ends (d). The noninvasive organisms H. aphrophilus (e) and E. coli HB101 (f) did not bind. H. influenzae (g) and A. actinomycetemcomitans SUNY 523 (h), organisms that invade but do not egress from the host cell, did not bind to the asters either. (i) Asters not subjected to bacteria.