Cell-associated pheromone peptide (cCF10) production and pheromone inhibition in Enterococcus faecalis

Abstract

In Enterococcus faecalis, the peptide cCF10 acts as a pheromone, inducing transfer of the conjugative plasmid pCF10 from plasmid-containing donor cells to plasmid-free recipient cells. In these studies, it was found that a substantial amount of cCF10 associates with the envelope of the producing cell. Pheromone activity was detected in both wall and membrane fractions, with the highest activity associated with the wall. Experiments examining the effects of protease inhibitor treatments either prior to or following cell fractionation suggested the presence of a cell envelope-associated pro-cCF10 that can be processed to mature cCF10 by a maturase or protease. A pCF10-encoded membrane protein, PrgY, was shown to prevent self-induction of donor cells by reducing the level of pheromone activity in the cell wall fraction.

FIG. 1

Analysis of PrgY in cell fractions of cells used in this study. The presence of PrgY in cellular fractions was analyzed for OG1RF, OG1RF(pCF10) (pCF10 encodes PrgY), and OG1RF(pCF389) (a transposon insert in pCF10 disrupts PrgY). Cellular lysates were prepared by lysozyme-mutanolysin lysis, cell walls were recovered by low-speed centrifugation, and cell membranes were recovered sequentially by high-speed centrifugation. Equal amounts of collected material were electrophoresed through a sodium dodecyl sulfate–7.5% polyacrylamide gel and electroblotted onto a nylon membrane. PrgY was detected by Western blotting with a polyclonal PrgY antibody raised to PrgY peptides. Bands were visualized as described previously (12). The strain used is indicated above each lane, and the fraction analyzed is indicated beneath each group of lanes. The size of the detected band corresponded to the previously predicted size of PrgY (28). The bands detected at the side of the left lane are due to the strong reaction of the premarked molecular weight markers.

FIG. 2

Presence of cCF10 activity in cellular fractions of OG1RF and OG1RF(pCF10) cells. Cellular lysates were prepared by lysozyme-mutanolysin lysis, cell walls were recovered by low-speed centrifugation, and cell membranes were recovered sequentially by high-speed centrifugation. Proteins were recovered from the whole envelope and cell membrane and cell wall fractions by TCA precipitation. For this experiment, as well as those described in subsequent figures, the extracted and concentrated culture supernatant and subcellular fractions were all resuspended in the same final volume so that relative activities in each fraction could be directly compared. The cCF10 activity was detected by microtiter clumping assays; the titers reported represent the reciprocal of the highest dilution that induced clumping (see Materials and Methods). While the absolute amount of cCF10 recovered varied somewhat between experiments, the relative amount of cCF10 in each fraction remained the same. The relative amounts of cCF10 in each fraction shown are representative of at least two independent experiments.

FIG. 3

Production of cCF10 activity in subcellular fractions of OG1RF cells in the presence and absence of protease inhibitors. Cellular lysates were prepared by lysozyme-mutanolysin lysis. The lysis steps were carried out either in the presence or absence of protease inhibitors (see Materials and Methods). Cell walls were recovered by low-speed centrifugation, and cell membranes were recovered sequentially by high-speed centrifugation. Proteins were recovered from cell membranes or cell wall fractions by chloroform-methanol lipid extraction followed by TCA precipitation. The presence of cCF10 was detected by microtiter clumping assay, as described above. The relative amounts of cCF10 in each fraction shown are representative of at least two independent experiments.

FIG. 4

Comparison of cCF10 production by cell fractions of OG1RF(pCF10) and the prgY-negative derivative OG1RF(pCF389). Cellular lysates were fractionated as described above. Pheromone activity recovered from whole-cell lysates, cell membranes, or cell wall fractions by chloroform-methanol extraction followed by TCA precipitation was detected by microtiter clumping assays. In some experiments, protease inhibitors were present during cell lysis and fractionation (see Materials and Methods). The relative amounts of cCF10 in each fraction shown above are representative of at least two independent experiments.

FIG. 5

Production of cCF10 in the growth medium by OG1RF cells containing no plasmid, pCF10, or pCF389. Overnight cultures were diluted 1:10 in fresh medium. (A) Aliquots were removed at various times during growth, and bacterial numbers were determined by viable plate counting. (B) cCF10 was recovered by cell lysis, chloroform-methanol extraction, and TCA precipitation. cCF10 was separated from iCF10 by HPLC, and the amount of cCF10 was determined by performing a microtiter OG1RF(pCF10) clumping. The amount of cCF10 present was calculated following the method of Nakayama et al. (36) and is reported as nanograms of cCF10/CFU.

FIG. 6

Inability of iCF10 to suppress the constitutively clumpy phenotype of a prgY mutant. OG1RF(pCF10) or OG1RF(pCF389) derivatives were grown in M9-YE medium or M9-YE medium containing synthetic iCF10 (6 × 10⁻⁸ M) and clumping was scored. The wells shown represent the appearance of nonclumpy [OG1RF(pCF10)] or clumpy [OG1RF(pCF389)] strains in liquid culture. The strains exhibiting each phenotype are listed below the appropriate wells. The relevant genes contained in each plasmid are shown above the wells. pCF389 is the pCF10 plasmid containing a transposon insert in prgY at amino acid 310 of 363. pMSP5011 represents the cloned iCF10 structural gene (prgQ).

FIG. 7

Model for pheromone production and control of endogenous pheromone activity by PrgY and iCF10. On the left is the cell envelope of a plasmid-free cell producing cCF10. The synthesis of mature cCF10 is proposed to occur via processing of a propheromone, now believed to represent the cleaved signal peptide from a secreted lipoprotein (19). The Eep protein described by An et al. (3) is proposed to be a membrane protease of the RIP family (11). Some mature pheromone is released into the medium, but a substantial portion remains associated with the cell wall of the organism. In a cell carrying pCF10 (right), both pheromone synthesis and pheromone response functions are present. The plasmid-encoded iCF10 peptide effectively neutralizes the cCF10 released into the medium, while PrgY interferes with a potential autocrine circuit where cell-associated pheromone is immediately reinternalized by the concerted action of the PrgZ binding protein and the chromosomal oligopeptide permease system (33), resulting in self-induction. It is not yet clear whether PrgY acts by sequestering or degrading cell-associated cCF10, by interfering with its interaction with PrgZ, or by some other mechanism.