Specific Control of Endogenous cCF10 Pheromone by a Conserved Domain of the pCF10-Encoded Regulatory Protein PrgY inEnterococcus faecalis

Abstract

Conjugative transfer of Enterococcus faecalis plasmid pCF10 is induced by the heptapeptide pheromone cCF10. cCF10 produced by plasmid-free recipient cells is detected by pCF10-containing donor cells, which respond by induction of plasmid-encoded transfer functions. The pCF10-encoded membrane protein PrgY is essential to prevent donor cells from responding to endogenously produced pheromone while maintaining the ability to respond to pheromone from an exogenous source; this function has not been identified in any nonenterococcal prokaryotic signaling system. PrgY specifically inhibited endogenous cCF10 and cPD1 (a pheromone that induces transfer of closely related plasmid pPD1) but not cAD1 (which is specific for less-related plasmid pAD1). Ectopic expression of PrgY in plasmid-free recipient cells reduced pheromone activity in culture supernatants and reduced the ability of these cells to acquire pCF10 by conjugation but did not have any effect on the interaction of these cells with exogenously supplied cCF10. The cloned prgY gene could complement a pCF10 prgY null mutation, and complementation was used to identify point mutations impairing PrgY function. Such mutations also abolished the inhibitory effect of PrgY expression in recipients on pheromone production and on acquisition of pCF10. Most randomly generated point mutations identified in the genetic screen mapped to a predicted extracellular domain in the N terminus of PrgY that is conserved in a newly identified family of related proteins from disparate species including Borrelia burgdorferi, Archaeoglobus fulgidus, Arabidopsis thaliana, and Homo sapiens. The combined genetic and physiological data suggest that PrgY may sequester or inactivate cCF10 as it is released from the membrane.

A paracrine peptide sex pheromone also acts as an autocrine signal to induce plasmid transfer and virulence factor expressionin vivo

“…For Asc10, cell extracts were prepared and analyzed as described (11) except that cells were grown overnight in M9-YE medium, then diluted 1:10 into fresh M9, plasma, or M9 ϩ 1 ng͞ml cCF10 and grown for 2 hr at 37°C before harvesting. SDS͞PAGE and Western blot analyses were performed as described in ref.…”

“…PrgY is a membrane protein that is essential to block self-induction by pCF10-carrying donors. PrgY has been shown to reduce the amount of both excreted and cell-associated pheromone in donor cells, possibly by binding or degrading cCF10 as it is released from the outer surface of the membrane (5,11). Control of the residual donor pheromone activity that is not blocked by PrgY is mediated by an inhibitor peptide, iCF10.…”

Pheromone-inducible conjugation in Enterococcus faecalis: A model for the evolution of biological complexity?

“…1A). The PrgY membrane protein reduces the production of endogenous pheromone activity by donor cells (Buttaro et al, 2000;Chandler et al, 2005a), without affecting the donor cell interaction with exogenous cCF10. The iCF10 inhibitor peptide neutralizes the residual excreted cCF10 from donor cells that escapes PrgY control.…”

Enterococcal Sex Pheromones: Evolutionary Pathways to Complex, Two-Signal Systems

“…In the case of pCF10, the C peptide is produced by processing the cleaved signal peptide of a predicted secreted lipoprotein CcfA, whose function has not been demonstrated (15); likewise, all known pheromone-responsive plasmids analyzed to date encode a response to a specific peptide encoded by one of the Ͼ50 potential lipoprotein genes in the organism (16). As indicated in step II, the system acquired additional components that recognize C; PrgY prevents self-induction of donors by decreasing the amount of mature C released (36), and PrgZ binds both C and I and facilitates their import into the cell via a chromosomally encoded peptide transporter (37,38). PrgX also needed to evolve to recognize C and I.…”

“…At the mechanistic level, the C peptide competes with I, which functions as a classic quorum-sensing signal of donor density (self-sensing) (64). Evolution of the ability to differentially respond to these two antagonistic peptides was accompanied by the acquisition of genes encoding an oligopeptide binding protein, PrgZ, which binds both C and I with high affinity and increases their import via the Opp ABC transporter (37,38), and PrgY, a predicted membrane peptidase that reduces the production of endogenous C by the host cell (36). III depicts the acquisition of T4SS and Dtr genes conferring conjugative transfer ability.…”