Aggregation Substance Increases Adherence and Internalization, but Not Translocation, of Enterococcus faecalis through Different Intestinal Epithelial Cells In Vitro

Abstract

The aggregation substance of Enterococcus faecalis increased bacterial adherence to and internalization by epithelial cells originating from the colon and duodenum but not by cells derived from the ileum. However, enterococcal translocation through monolayers of intestinal epithelium was not observed.Enterococcus faecalis, a gram-positive facultative anaerobic bacterium, belongs to the normal flora of the intestinal tract and is also found in the vaginal vault and the oral cavity. Enterococci have increasingly gained attention as pathogens, since they have become the fourth leading cause of nosocomial infections in the United States (8). E. faecalis frequently causes local or systemic infections, such as urinary tract and abdominal infections, wound infections, bacteremia, and endocarditis (15). It is assumed that many enterococcal infections are endogenous, resulting from bacterial translocation from the intestinal lumen to extraintestinal sites (16,31).Surprisingly little is known about the virulence factors of E. faecalis. One of the potential virulence factors is the aggregation substance (AS), an adhesin encoded by inducible sex pheromone plasmids (for a review, see reference 5). The AS, a signal peptide-containing protein, appears as a hair-like structure on the cell surface and is incorporated primarily into the ''old'' parts of the cell wall (30). It has been demonstrated that this adhesin is responsible for bacterium-bacterium contact during conjugative transfer of sex pheromone plasmids (5).The AS contains two Arg-Gly-Asp motifs which are known to be recognized by integrins, a family of eukaryotic cell surface receptors (24). Since integrins have been reported to be expressed on intestinal epithelial cells (2), we hypothesized that the AS promotes adherence and possibly invasion into these cells, thereby enabling enterococci to translocate through the intestinal epithelial barrier.(This work was presented in part at the 98th General Meeting of the American Society for Microbiology, Atlanta, Ga., 17 to 21 May 1998.)Three different E. faecalis strains (Table 1), kindly provided by R. Wirth (Institute of Microbiology, University of Regensburg, Regensburg, Germany), were used to study the effect of the AS on adherence, internalization, and translocation through intestinal epithelial cells. The bacteria were cultured in Todd-Hewitt medium (Difco, Augsburg, Germany) supplemented with erythromycin (20 g/ml) for growth of the plasmid-containing strains (OG1X/pAM721 and OG1X/pAM944). Since E. faecalis strains expressing the AS spontaneously clump, all inocula were sonicated (Branson W-450 Sonifier; 80 W, 20 s, continuously) to obtain single-cell suspensions. Salmonella enterica serovar Typhimurium ATCC 14028 and Escherichia coli HB101 grown in Luria-Bertani medium were used as positive and negative controls, respectively. For the experiments described below, the bacteria were harvested in mid-log phase and suspended in tissue culture medium without supplements, which served as the infection medium. Appropriate ...

Translocation of Enterococcus faecalis Strains across a Monolayer of Polarized Human Enterocyte-Like T84 Cells

“…Although E. faecalis is included among the organisms that have shown translocation in animals, and such translocation is often assumed to occur in humans also, this phenomenon has not, to our knowledge, been demonstrated in an in vitro transcytosis system; indeed, the single study we could find on this subject reported failure of strain OG1X to show translocation across epithelial cell monolayers, and the authors concluded that the intact epithelial cell layer serves as a barrier for enterococci in vitro (18). That paper and others have, however, shown internalization of some E. faecalis organisms by T84 and other intestinal cell lines (14,18). In the present study, we first demonstrated that the E. faecalis strain OG1RF, unlike E. coli DH5␣, was able to translocate across a T84 monolayer, a model for intestinal epithelium transcytosis.…”

Characterization of Clinical Enterococcus faecalis Small-Colony Variants

“…The aggregation substance is encoded on the lowcopy-number 60-kb plasmid paD1, which also encodes a virulence-related cytolytic exotoxin (18,29). Cellular adherence of E. faecalis, internalization into mammalian cells, and intracellular survival in human macrophages have been found to be promoted by the aggregation substance (24,26). One may thus speculate that the SCV phenotype is associated with alterations in the aggregation substance.…”

Virulence Factor and Biofilm Formation in Clinical Enterococcal Isolates of the West of Iran

“…Gelatinase encoded by gelE gene is an extracellular zinc metalloprotease that hydrolyses gelatine, collagen, casein, haemoglobin, and antimicrobial peptides of the innate immune system (24,25). The asa1 is encoded by pheromoneresponsive plasmids, which often harbour antibiotic resistance genes (26,27). The protein causes clumping of E. faecalis cells and survival inside polymorphonuclear leucocytes, internalisation by intestinal cells, and increases in bacterial binding to cultured renal epithelial cells (28,29).…”