Aggregation substance (AS) is anEnterococcus faecalis surface protein that may contribute to virulence. Using a recently described system for controlled expression of AS in E. faecalis and the heterologous host Lactococcus lactis, experiments were designed to assess the effect of AS on bacterial internalization by HT-29 and Caco-2 enterocytes. AS expression was associated with increased internalization of E. faecalis by HT-29 enterocytes and of L. lactis by HT-29 and Caco-2 enterocytes. Compared to enterocytes cultivated under standard conditions, either cultivation in hypoxia or 1-h pretreatment of enterocytes with calcium-free medium resulted in increased internalization of both E. faecalis and L. lactis (with and without AS expression). Also, AS expression augmented these increases when E. faecalis was incubated with pretreated HT-29 enterocytes and when L. lactis was incubated with pretreated Caco-2 and HT-29 enterocytes. These data indicated that AS might facilitate E. faecalis internalization by cultured enterocytes.Although Enterococcus faecalis is a component of the normal human intestinal flora, enterococci number among the top three nosocomial microbial pathogens (8), and strains resistant to all useful antimicrobial agents are increasingly involved in fatal infections (12). Thus, it is important to clarify the mechanisms involved in extraintestinal dissemination of enterococci. Aggregation substance (AS) protein may be involved in virulence and is expressed on the surface of E. faecalis. AS molecules are encoded by different pheromone-responsive plasmids; e.g., Asc10 is encoded by pCF10, and Asa1 is encoded by pAD1 (6). Pheromones produced by potential recipients induce expression of AS on the surfaces of plasmid-containing donor cells. AS facilitates aggregation of donor and recipient bacteria and aids conjugative plasmid transfer (6).The gene for Asc10 was recently cloned in a vector containing a nisin-inducible promoter, resulting in surface expression of Asc10 on E. faecalis and the heterologous host Lactococcus lactis. E. faecalis OG1SSp and L. lactis NZ9800 were transformed with plasmid pMSP7517 that encodes Asc10 (9). We have used these transformants to clarify the effect of AS on bacterium-enterocyte interactions. Because pMSP7517 contains a gene for erythromycin resistance, bacteriological media were supplemented with 10 g of erythromycin (Sigma Chemical Co., St. Louis, Mo.) per ml. For experiments, E. faecalis was cultivated overnight in Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) in the absence of nisin or in broth supplemented with 25 ng of nisin (Sigma) per ml; nisin was present either throughout the incubation period or only during the final 2 h. Following incubation at 35°C with nisin, E. faecalis cells clumped, confirming Asc10 expression (6, 9). To obtain single-cell suspensions (verified by light microscopy), these inocula were sonicated, typically with 20 W for 10 s using a 40-W high-intensity ultrasonic processor (Sonics and Materials, Danbury, Conn.). L. lactis was cult...