The aggregation domain of aggregation substance, not the RGD motifs, is critical for efficient internalization by HT-29 enterocytes

Abstract

Aggregation substance (AS), a surface protein encoded on the pheromone-inducible plasmids of Enterococcus faecalis, has been shown to increase adherence and internalization into a number of different cell types, presumably through integrin binding mediated by the N-terminal RGD motif of AS. Here, defined mutations constructed in Asc10, the AS encoded by the plasmid pCF10, are analyzed for their ability to promote increased internalization levels into HT-29 enterocytes. The results clearly show that the previously identified Asc10 functional domain, not the RGD motifs, is critical for Asc10-directed internalization of E. faecalis into HT-29 enterocytes. Also, expression of Asc10 in the nonaggregating E. faecalis strain INY3000 is unable to mediate HT-29 internalization. However, Asc10-expressing E. faecalis cells are not internalized as bacterial aggregates, suggesting bacterial aggregation is not a prerequisite for HT-29 internalization. These data show that Asc10 directs internalization of E. faecalis into HT-29 enterocytes through a non-RGD-dependent mechanism.

FIG. 1.

The aggregation domain of Asc10 is required for efficient HT-29 internalization. (A) The numbers of viable, internalized OG1RF cells expressing wild-type Asc10 (7517), the vector control (3535), or 10 different insertion mutants from stationary-phase cultures into HT-29 enterocytes under standard conditions is plotted on a log₁₀ scale. Note that log₁₀ 1.7 is the minimum number of bacteria that can be measured in this assay. (B) Internalization of the Asc10 insertion mutants Ω1419, Ω1551, and Ω1638 grown to exponential phase is shown, as described for panel A. Error bars indicate the standard errors of the means.

FIG. 2.

Asc10 expression in strains used in this study. Lysozyme surface extractions were electrophoresed in an SDS-7.5% PAGE gel and Western blotted with an anti-Asc10 polyclonal antibody. Lanes 1 to 7 are from stationary-phase cultures grown overnight, while lanes 8 to 12 are exponential-phase cultures as indicated in Materials and Methods. Lanes: 1 = INY3000(7517); 2 = INY3000(3535); 3 and 8 = OG1RF(7517); 4 and 9 = OG1RF(3535); 5 and 10 = OG1RF(Ω1419); 6 and 11 = OG1RF(Ω1551); 7 and 12 = OG1RF(Ω1638). Molecular mass markers are indicated to the left of the blot. The arrow indicates full-length Asc10.

FIG. 3.

Mutation of the RGD motifs had no effect on aggregation. Overnight nisin-induced cultures of OG1RF expressing wild-type Asc10 (7517), the vector control (3535), the N-terminal RGD→RAD (3607) mutation, C-terminal RGD→RAD (3608) mutation, or the double RGD→RAD mutations (3609) allowed to settle for 1 min are shown. The numbers below the pictures indicate the optical density readings at 600 nm of the settled cultures.

FIG. 4.

Mutations in the RGD motifs of Asc10 did not affect internalization into HT-29 enterocytes. Overnight cultures of OG1RF expressing wild-type Asc10 (7517), the vector control (3535), the N-terminal RGD→RAD (3607) mutation, C-terminal RGD→RAD (3608) mutation, or the double RGD→RAD mutations (3609) were analyzed for the ability to promote internalization into HT-29 enterocytes under both standard and calcium-free conditions. Invasion levels mediated by the RGD mutant proteins did not show any significant reduction in HT-29 internalization under either condition. The data are plotted as the log₁₀ value of internalized E. faecalis (1.7 is the minimum value of the assay). The error bars indicate the standard errors of the means.

FIG. 5.

Expression of Asc10 in INY3000 did not promote internalization into HT-29 enterocytes. (A) Asc10 was expressed in the wild-type E. faecalis strain OG1RF or the nonaggregating E. faecalis mutant strain INY3000 and analyzed for internalization into HT-29 enterocytes grown under standard conditions. The data are plotted as the log₁₀ value of viable internalized E. faecalis (1.7 is the minimum value of the assay). (B) The same four strains shown in panel A were pelleted onto HT-29 monolayers (182 × g for 10 min), and a standard internalization assay was performed. Error bars for both graphs represent the standard errors of the means.

FIG. 6.

Multicellular bacterial aggregates do not invade HT-29 enterocytes. (A) OG1RF(7517) was mixed with OG1SSp(3535) or INY3000(3535) at ratios of 4:1, 1:1, and 1:4 after adjustment of each culture to 10⁹ CFU/ml. The mixed cultures were then added to HT-29 monolayers, and a standard internalization experiment was performed. The data are plotted as the log₁₀ value of viable internalized E. faecalis organisms. Error bars represent the standard errors of the means. (B and C) An FL-1 (y axis), FSC (x axis) FACS quadrant analysis of OG1SSp(3535) labeled with FITC unmixed (B) or mixed at a 1:1 ratio with unlabeled OG1RF(7517) (C) is shown. (D) The percentage of each mixed population that forms mixed aggregates (high FL-1 and FSC) of FITC-labeled OG1SSp(3535) and INY3000(3535) unmixed or mixed with unlabeled OG1RF(7517) at the ratio of 4:1, 1:1, or 1:4 is indicated. 7517 = Asc10⁺; 3535 = Asc10⁻.