Fusobacterium nucleatum transports noninvasive Streptococcus cristatus into human epithelial cells

Abstract

Analysis of human buccal epithelial cells frequently reveals an intracellular polymicrobial consortium of bacteria. Although several oral bacteria have been demonstrated to invade cultured epithelial cells, several others appear unable to internalize. We hypothesized that normally noninvasive bacteria may gain entry into epithelial cells via adhesion to invasive bacteria. Fusobacterium nucleatum is capable of binding to and invading oral epithelial cells. By contrast, Streptococcus cristatus binds weakly to host cells and is not internalized. F. nucleatum and S. cristatus coaggregate strongly via an arginine-sensitive interaction. Coincubation of KB or TERT-2 epithelial cells with equal numbers of F. nucleatum and S. cristatus bacteria led to significantly increased numbers of adherent and internalized streptococci. F. nucleatum also promoted invasion of KB cells by other oral streptococci and Actinomyces naeslundii. Dissection of fusobacterial or streptococcal adhesive interactions by using sugars, amino acids, or antibodies demonstrated that this phenomenon is due to direct attachment of S. cristatus to adherent and invading F. nucleatum. Inhibition of F. nucleatum host cell attachment and invasion with galactose, or fusobacterial-streptococcal coaggregation by the arginine homologue l-canavanine, abrogated the increased S. cristatus adhesion to, and invasion of, host cells. In addition, polyclonal antibodies to F. nucleatum, which inhibited fusobacterial attachment to both KB cells and S. cristatus, significantly decreased invasion by both species. Similar decreases were obtained when epithelial cells were pretreated with cytochalasin D, staurosporine, or cycloheximide. These studies indicate that F. nucleatum may facilitate the colonization of epithelial cells by bacteria unable to adhere or invade directly.

FIG. 1.

Percentages of CFU of S. cristatus (Sc; filled bars) or F. nucleatum (Fn; open bars) recovered from epithelial cells after 4 h of incubation (relative to the input) independently or in dual-incubation assays. Graphs indicate either total associated bacteria (adherent plus internalized; A and B) or those surviving incubation with antibiotics (internalized; C and D). Assays were performed with KB (A and C) or TERT-2 (B and D) epithelial cells. Error bars indicate the mean average ± the standard deviation of four independent experiments performed in triplicate. Values that differ from controls by a statistically significant amount are indicated (*).

FIG. 2.

Confirmation of adhesion of F. nucleatum or S. cristatus to KB cells by confocal microscopy. Fixed monolayers with adherent bacteria were stained with polyclonal antisera to F. nucleatum or S. cristatus and FITC- or TRITC-conjugated antibodies. F. nucleatum only, red in panel A; S. cristatus only, green or yellow in panel B; F. nucleatum and S. cristatus together, panels C and D. Scale bars represent 20 μm (A, B, and C) or 10 μm (D).

FIG. 3.

Assessment of internalization of F. nucleatum or S. cristatus in KB epithelial cells by confocal microscopy and SEM. Dual-antibody staining demonstrated that F. nucleatum could adhere to (yellow) and invade (red) KB cells (A). S. cristatus attached to but did not invade (B) unless in the presence of F. nucleatum, when internalization of both species occurred (C). SEM of KB cells incubated with F. nucleatum (D and G) confirmed internalization of this species and lack of internalization of S. cristatus (E). Examination of KB monolayers incubated with both F. nucleatum and S. cristatus revealed the direct attachment of streptococci to F. nucleatum bacteria that were in the process of internalization (H, F, and I). SEM of KB cells incubated in buffer only (J) demonstrated that cellular projections were not due to the presence of bacteria. Scale bars represent 20 μm (A and B), 2 μm (C), or 1 μm (D, E, F, G, H, and I).

FIG. 4.

Invasion of TERT-2 cells by F. nucleatum or S. cristatus. F. nucleatum readily invaded TERT-2 cells (A and B), although blebbing of the fusobacterial membrane was observed for the majority of the bacteria (A, B, E, and F). There was evidence of intercellular migration by F. nucleatum (C). S. cristatus was rarely observed to adhere directly to epithelial cells; attachment occurred via polar tufts of fibrils (D). In coincubation experiments, invading F. nucleatum bacteria were observed with attached S. cristatus (E and F). Bars represent 1 μm.

FIG. 5.

Adhesion to (A) and invasion of (D) KB cells by S. cristatus, F. nucleatum, or both species together were examined in the presence of anti-S. cristatus serum (a-Sc) or anti-F. nucleatum serum (a-Fn). Adhesion of or invasion by S. cristatus (filled bars) and F. nucleatum (open bars) is shown relative to the level obtained in the absence of either antiserum. The effects of increasing concentrations of galactose or l-canavanine on adhesion (B and C, respectively) or invasion (E and F, respectively) were assessed for S. cristatus only (open circles), F. nucleatum only (open squares), or S. cristatus and F. nucleatum together (filled circles and squares, respectively). Values that differ from controls (CTL) by a statistically significant amount are indicated (*). If all the values for a given concentration are statistically significantly different from the control value, this is indicated by the symbol †. Since S. cristatus internalization alone remained at the limit of detection in the presence of galactose or l-canavanine, those values are not shown.

FIG. 6.

Effect of a coaggregation-defective F. nucleatum isolate on S. cristatus adhesion or internalization. Adhesion to (A) or invasion of (B) KB cells by S. cristatus (filled bars) in the presence of wild-type F. nucleatum (WT, open bars) or isolate 21, a coaggregation-defective F. nucleatum isolate (21, open bars), was measured as described in Materials and Methods. Values are relative to those obtained with S. cristatus incubated in the presence of wild-type F. nucleatum.

FIG. 7.

Assessment of the effect of cytochalasin D (CD), staurosporine (STO), or cycloheximide (CX), relative to buffer only (control [CTL]), to inhibit invasion of KB cells by S. cristatus (filled bars) and F. nucleatum (open bars) in dual-incubation experiments. Relative percent inhibition is shown, and all values differed statistically significantly from those of untreated cells, except for S. cristatus in the presence of cycloheximide.

FIG. 8.

Effect of F. nucleatum on the internalization of various oral bacteria by KB cells. Percentages of recovered F. nucleatum (open bars) and other bacteria (closed bars) relative to the input were measured as described in Materials and Methods. Data from monoincubation (Mon) or coincubation (Co) assays are shown. Panels: A, S. cristatus ATCC 51110; B, S. sanguis SK36; C, S. gordonii DL-1; D, A. naeslundii ATCC 12104. Values that differ between invasion alone and in the presence of F. nucleatum by a statistically significant amount are indicated (*).