Characterization of the sequence specificity determinants required for processing and control of sex pheromone by the intramembrane protease Eep and the plasmid-encoded protein PrgY

Abstract

Conjugative transfer of the Enterococcus faecalis plasmid pCF10 is induced by the peptide pheromone cCF10 when recipient-produced cCF10 is detected by donors. cCF10 is produced by proteolytic processing of the signal sequence of a chromosomally encoded lipoprotein (CcfA). In donors, endogenously produced cCF10 is carefully controlled to prevent constitutive expression of conjugation functions, an energetically wasteful process, except in vivo, where endogenous cCF10 induces a conjugation-linked virulence factor. Endogenous cCF10 is controlled by two plasmid-encoded products; a membrane protein PrgY reduces pheromone levels in donors, and a secreted inhibitor peptide iCF10 inhibits the residual endogenous pheromone that escapes PrgY control. In this study we genetically determined the amino acid specificity determinants within PrgY, cCF10, and the cCF10 precursor that are necessary for cCF10 processing and for PrgY-mediated control. We showed that amino acid residues 125 to 241 of PrgY are required for specific recognition of cCF10 and that PrgY recognizes determinants within the heptapeptide cCF10 sequence, supporting a direct interaction between PrgY and mature cCF10. In addition, we found that a regulated intramembrane proteolysis (RIP) family pheromone precursor-processing protein Eep recognizes amino acids N-terminal to cCF10 in the signal sequence of CcfA. These results support a model where Eep directly targets pheromone precursors for RIP and PrgY interacts directly with the mature cCF10 peptide during processing. Despite evidence that both PrgY and Eep associate with cCF10 in or near the membrane, results presented here indicate that these two proteins function independently.

FIG. 1.

(A) Model of pheromone response and control in a donor cell. cCF10 is imported into the cytoplasm, where it interacts with PrgX, causing derepression of P_Q and subsequent induction of downstream conjugation genes. PrgY reduces endogenous cCF10, and iCF10 neutralizes remaining cCF10. (B) Predicted proteolytic processing of cCF10. cCF10 is processed from CcfA in several steps. SPII, signal peptidase II; Eep, membrane protease; C.P., carboxy peptidase. Triangles indicate cleavage sites.

FIG. 2.

iCF10 is insufficient to control endogenous pheromone in the absence of PrgY. Pheromone induction by endogenous pheromone was measured in OG1RF(pBK2) strains coharboring a plasmid expressing PrgZ+PrgY (pMSP6043), PrgY (pMSP6049), PrgZ (pMSP6043-1), or a vector control (pMSP6043-2). Cultures grown overnight in M9 medium were diluted 1:5, and β-galactosidase levels were determined after 2.5 h of growth. iCF10 was added at 0, 50, 500, or 5,000 ng/ml to overnight cultures and again to diluted cultures. The data shown are representative of three independent experiments, each performed in duplicate.

FIG. 3.

P_Q-driven peptide expression constructs used in these studies. (A) Diagram of the peptide-coding portion of the plasmids. Transcription of each peptide is driven by the prgQ promoter (P_Q), shown at the left. Within the 22- to 23-amino-acid peptide of each plasmid is an N-terminal peptide sequence derived from the iCF10 or cAM373 preprocessed sequence (N-terminal peptide) and a pheromone or inhibitor at the C-terminal end (in gray). (B) N-terminal peptide and pheromone/inhibitor that is encoded within each plasmid. For details regarding the construction of these plasmids, see Materials and Methods.

FIG. 4.

(A) PrgY reduces the activity of cCF10 processed from both the iCF10 and the cAM373 signal peptides. Supernatant from JRC107 or JRC110 (Eep⁻) strains harboring pPCR4 or pJRC3 and pMSP3545Y (expressing PrgY) or pMS3545 (vector) were used to induce exponentially growing OG1RF(p043lacZ) strains, and the ability of supernatant cCF10 to induce LacZ expression of this strain was measured by β-galactosidase assay and expressed in Miller units. The PrgY-dependent percent reduction of cCF10 activity (+PrgY/Vector) was averaged from three independent experiments (error bars represent the standard deviation of three independent experiments). (B) PrgY does not decrease endogenous cAM373 activity. Cell culture supernatants prepared as for Table 3 were diluted twofold, and the pheromone activity is represented as the inverse of the largest dilution able to aggregate an OG1RF(pCF10) or OG1RF(pAM373::pAD2) indicator strain. The titers represent the results of two independent experiments; the same results were seen for both assays.

FIG. 5.

PrgY targets L4 of cCF10. Wild-type (WT) cCF10 peptide from pPCR4 or cCF10 variants were coexpressed with PrgY (pMSP3545Y) or a vector control (pMSP3545). The peptide produced by each strain during overnight growth in THB was collected in the supernatant and used to induce an exponentially growing responder strain OG1RF(p043lacZ) for 1.5 h, and the β-galactosidase activity was subsequently determined. Cell culture supernatants were added to the responder strain at a 1:100 dilution (for three cCF10 variants with low activity [V2L, L4V, and F6Y]) or at a 1:200 dilution (for all others). (A) Absolute values and (B) the PrgY-dependent percent reduction of cCF10 activity are shown. For both panels A and B, the error range represents the standard deviation of two independent experiments. MU, Miller units.

FIG. 6.

Specific reduction of cCF10 activity is conferred by PrgY amino acids 125 to 241. (A) cCF10 or cPD1 activity was measured in supernatants of TX5128 strains harboring the indicated construct. Cell culture supernatants (grown 6 h from a 10% overnight inoculum induced with 25 ng of nisin/ml) were diluted twofold, and the pheromone activity was determined in a clumping induction assay using OG1RF(pCF10) or (pPD1) indicator strains. The PrgY-dependent percent reduction of cCF10 activity averaged from two independent experiments (the error represents the standard deviation of two independent experiments; the cCF10 titer in the absence of PrgY, TraB, or a chimeric protein was 16 in the first experiment and 32 in the second). cPD1 was similarly assayed twice with the same results each time; the cPD1 titer was 4 in the absence of either PrgY or TraB, and all of the chimeras completely reduced all cPD1 activity. (B) The expression of pPD1 TraB (P), PrgY (Y) and TraB/PrgY chimeras 1 to 6, 9, and 10 (labeled 1 to 6, 9, and 10) in whole-cell extracts was analyzed by Western blotting with a polyclonal anti-PrgY antibody (8). TX5128 was the strain background in these experiments. The arrow at 43 kDa corresponds to the expected size of PrgY. All strains were treated with 25 ng of nisin/ml for 2.5 h prior to harvesting. An equivalent amount of protein was loaded for each sample.

FIG. 7.

PrgY reduces the activity of cCF10 in the Eep-deficient strain JRC110. (A) cCF10 activity produced by pPCR4 expressed with pMSP3545Y (PrgY) or pMSP3545 (vector), as indicated, in strain JRC110 was measured by clumping assay as described in Table 3 and Materials and Methods. (B) PrgY-dependent percent reduction of cCF10 activity for JRC110 strains harboring pPCR4 (cCF10 processed from the iCF10 leader) or pJRC3 (cCF10 processed from the cAM373 leader). For both panels A and B, the results are averaged from three independent experiments done in duplicate (error bars represent the standard deviation of three independent experiments).

FIG. 8.

(A) Linear diagram of PrgY denoting the location of functional and specificity domains. The functional domain was identified through random and site-directed mutagenesis strategies (12) (random mutations are shown), and the specificity domain (amino acids 125 to 241) was identified in the present study (Fig. 5). Predicted transmembrane domains (TM1 to TM4) are noted. (B) Model of cCF10 processing and export in a donor cell. Mature cCF10 is encoded within the signal sequence of host-encoded lipoprotein CcfA (3). The CcfA signal peptide is most likely removed by signal peptidase II (SPII). The membrane protease Eep then recognizes the N-terminal domain of the signal peptide and further processes it to enable the release of mature cCF10. Active export or further proteolytic processing by host-encoded proteins may also occur at this stage. The topology of PrgY (center) is shown as predicted by the HMMTOP program (http://www.enzim.hu/hmmtop/). PrgY specifically targets cCF10 through amino acids 125 to 241 in PrgY and inhibits the activity of cCF10 through an N-terminal functional domain (12); this inhibition may occur through degrading or modifying newly processed or preprocessed cCF10 in the cell wall (as shown) or by blocking release into the cell medium.