Inducible Expression of Enterococcus faecalis Aggregation Substance Surface Protein Facilitates Bacterial Internalization by Cultured Enterocytes

Abstract

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...

Sex Pheromone Response, Clumping, and Slime Production in Enterococcal Strains Isolated from Occluded Biliary Stents

“…In addition to promoting conjugation processes, the presence of AS also increases the ability of enterococci to adhere to host surfaces. The reported increase in uptake of AS-expressing cells into intestinal epithelial cells (20,26,31) suggests the possible involvement of AS in the translocation of E. faecalis from the intestine to the bloodstream. On the other hand, AS of E. faecalis has been implicated as a virulence factor in several model systems (21), and its in vivo induction has recently been demonstrated (17).…”

In Vivo Induction of Virulence and Antibiotic Resistance Transfer in Enterococcus faecalis Mediated by the Sex Pheromone-Sensing System of pCF10

“…Recently, it was reported that AS promotes the adhesion of E. faecalis to fibrin (24) as well as to the extracellular matrix proteins fibronectin, vitronectin, collagen type I, and thrombospondin (36). In direct interaction with cultured mammalial cells, AS-mediated uptake was demonstrated with intestinal epithelial cells (34,43) and a colon cell line (26). Increased adhesion to renal tubular cells was demonstrated (28).…”

Characterization of the Pheromone Response of theEnterococcus faecalisConjugative Plasmid pCF10: Complete Sequence and Comparative Analysis of the Transcriptional and Phenotypic Responses of pCF10-Containing Cells to Pheromone Induction

“…The stability of Asc10 on the cell surface opens the possibility that only a brief induction event-resulting from a recipient cell or an environmental cue-is necessary to present AS on the surface for a considerable amount of time. This could allow pCF10-containing cells to adhere to different host cell types (31,46,53), to withstand the attack of phagocytic cells (43,50), or to bind to potential enterococcal recipient cells that might only be transiently passing through a particular ecological niche, such as the intestinal lumen. This tethering of potential recipients in close proximity could facilitate a pheromone response by the donor cells, ultimately increasing plasmid transfer.…”