PrgU: a suppressor of sex pheromone toxicity in Enterococcus faecalis

Abstract

Upon sensing of the peptide pheromone cCF10, Enterococcus faecalis cells carrying pCF10 produce three surface adhesins (PrgA, PrgB or Aggregation Substance, PrgC) and the Prg/Pcf type IV secretion system and, in turn, conjugatively transfer the plasmid at high frequencies to recipient cells. Here, we report that cCF10 induction is highly toxic to cells sustaining a deletion of prgU, a small orf located immediately downstream of prgB on pCF10. Upon pheromone exposure, these cells overproduce the Prg adhesins and display impaired envelope integrity, as evidenced by antibiotic susceptibility, misplaced division septa and cell lysis. Compensatory mutations in regulatory loci controlling expression of pCF10-encoded prg/pcf genes, or constitutive PrgU overproduction, block production of the Prg adhesins and render cells insensitive to pheromone. Cells engineered to overproduce PrgB, even independently of other pCF10-encoded proteins, have severely compromised cell envelopes and strong growth defects. PrgU has an RNA-binding fold, and prgB-prgU gene pairs are widely distributed among E. faecalis isolates and other enterococcal and staphylococcal species. Together, our findings support a model in which PrgU proteins represent a novel class of RNA-binding regulators that act to mitigate toxicity accompanying overproduction of PrgB-like adhesins in E. faecalis and other clinically-important Gram-positive species.

FIG. 1

Effects of PrgU deletion and overproduction on Prg/Pcf protein synthesis and plasmid transfer. A) Schematic of the P_Q regulatory region and prgQ operon carried by pCF10. The pheromone-responsive P_Q promoter is regulated by flanking regulatory functions and directs expression of the downstream-encoded adherence and plasmid transfer functions. The P_X promoter controls expression of prgX, which encodes the PrgX transcriptional regulator. Another promoter in the prgR/S region constitutively expresses a short prgA transcript at low levels. Plasmid p10-mini carries the ~17-kb fragment shown. B) Transfer frequencies of pCF10 plasmids in filter matings carried out for the durations indicated. Strains: OG1RF with pCF10 or pCF10ΔprgU (denoted ΔprgU) plasmids alone or additionally with the vector plasmid (P₂₃; pDL278p23) or the P₂₃:prgU expression plasmid (denoted P₂₃::U; pMB11). Transfer frequencies are presented as the number of transconjugants per donor cell (Tc’s/Donor). Experiments were repeated at least three times in duplicate, and results from a representative experiment are shown. C) Steady-state levels of Prg/Pcf proteins in strains induced for 1 h with cCF10 pheromone (10 ng ml⁻¹). OG1RF with plasmids indicated. Upper line: pCF10, ΔprgU (pCF10ΔprgU). Lower line: P₂₃ (pDL278p23), P₂₃::U (pMB11), - (no P₂₃ plasmid). Immunoblots were developed with antibodies to the Prg/Pcf proteins shown or to RNA polymerase β subunit as a loading control. Protein sizes (in kilodaltons, kDa) are listed at the right. Protein extracts were loaded on a per-cell equivalent basis. Numbers below each panel correspond to relative protein abundance compared to levels detected in OG1RF(pCF10) cell extracts (set to 1), as determined by densitometry tracing using Image J software.

FIG. 2

cCF10 pheromone suppresses growth of E. faecalis cells carrying the pCF10ΔprgU plasmid. A) Serial dilution plate assay. E. faecalis overnight cultures were diluted 1:100 in fresh BHI and incubated for 1 h at 37°C in the absence (−) or presence (+) of cCF10 (10 ng ml⁻¹). Tenfold serial dilutions were inoculated onto BHI medium and assessed for growth. Strains: OG1RF with pCF10 or ΔprgU (pCF10ΔprgU) alone, or additionally with the P₂₃ vector plasmid (pDL278P₂₃) or the P₂₃::prgU expression plasmid (denoted P₂₃::U; pMB11). B) Pheromone spot assay. Overnight cultures listed at the left in Panel A were diluted 1:100 in fresh BHI and incubated for 1 h at 37°C in the absence of pheromone. Cultures were spread on BHI media, allowed to dry, and cCF10 pheromone (10 ng ml⁻¹) was added to the center of the plate. Plates were incubated overnight at 37 °C and assessed for growth. cCF10 pheromone is solubilized in DMSO, which inhibits E. faecalis growth and causes small zones of clearance independently of cCF10-induced toxicity; for example, see spot assay for OG1RF(pCF10). C) Growth curve assay. Overnight cultures were diluted 1:50 in BHI and incubated for 1 h at 37 °C. The cells were normalized to an OD₆₀₀ of 0.1 and cultures were incubated in the absence or presence of cCF10 (10 ng ml⁻¹) at 37°C. Aliquots were removed at specified time points, 10 μl of 0.5 M EDTA was added to disaggregate the cells, and the OD₆₀₀ was measured.

FIG. 3

The ΔU-Res R1 compensatory mutation maps to the putative regulatory gene prgR.. A) Schematic of the prgQ regulatory region showing the locations of the P_Q and P_X promoters, inverted repeats IRS1 and IRS2 predicted to form stem-loop transcription terminators, and the putative regulatory genes prgR and prgS. Numbers refer to distances (in base pairs, bp’s) from the P_Q promoter start-site. Below: The ΔU-Res R1 mutation is a 1 bp deletion at position 271 in the 318-bp prgR sequence. B) Modulation of PrgA and PrgB levels by the P₂₃::prgR,prgS expression plasmid. Steady-state levels of PrgA and PrgB in strains induced for 1 h with cCF10 pheromone (10 ng ml⁻¹). OG1RF with plasmids indicated. Upper line: pCF10, ΔprgU (pCF10ΔprgU), ΔU-Res R1 (pCF10ΔprgU,prgRΔ271). Lower line: - (no P₂₃ plasmid), P₂₃::RS (P₂₃::prgR,prgS expression plasmid, pMC003). Immunoblots were developed with antibodies to PrgA or PrgB. Protein extracts were loaded on a per-cell equivalent basis with RNA polymerase β subunit as a loading control. C) Pheromone spot and mating assays. Strains analyzed are the same as in panel B. Transfer frequencies of pCF10 and its derivatives are presented as the number of transconjugants per donor cell (Tc’s/Donor).

FIG. 4

ΔprgU mutation confers defects in cell envelope integrity and cell growth. A) Growth defect of the ΔprgU mutant in the presence of subinhibitory concentrations of antibiotics. Strains: OG1RF with pCF10, ΔprgU (pCF10ΔprgU) alone or with the P₂₃::prgU expression plasmid (P₂₃::U, pMB11). Strains were inoculated from glycerol stocks into BHI lacking (−) or containing (+) cCF10 and in the absence (No AB) or presence of the antibiotics at final concentrations (in μg ml⁻¹) listed. Cultures were incubated overnight at 37°C without shaking and culture densities (OD₆₀₀) were measured. B) Disc diffusion assay showing that a combination of pheromone (10 ng ml⁻¹) and antibiotics at the indicated concentrations suppressed the appearance of ΔU-Res variants. C) E. faecalis strains containing the constitutive lacZ reporter construct pCJK205 were cultured in BHI broth supplemented with erythromycin and 40 μg ml⁻¹ CPRG, and incubated in the absence or presence of cCF10 at 37°C. Samples were removed at the times indicated, and CPRG hydrolysis was quantitated by measuring the absorbance (OD₅₇₀) of the bacterium-free culture supernatant and normalizing to CFU. The experiment was performed at least two times, and the data are represented as the mean +/− standard deviation. Statistical significance was evaluated by t test, *, P<0.05 versus cCF10-untreated culture. D) Morphological aberrations of pheromone-exposed ΔprgU mutant cells. Overnight cultures were diluted 1:100 in fresh BHI supplemented with pheromone (10 ng ml⁻¹) and incubated without shaking for 1 h at 37°C. Cells were processed for imaging as described in the Experimental procedures.

FIG. 5

Constitutive overproduction of PrgB confers toxicity in the absence of other pCF10-encoded factors. A) Plasmid curing assay. Colonies from transformation plates were inoculated into antibiotic-free BHI and incubated without shaking overnight at 37°C. Overnight cultures were serially diluted and spotted onto BHI agar plates containing or lacking spectinomycin (500 μg ml⁻¹) to which the P₂₃ plasmid confers resistance. B) PrgB overproduction confers severe growth defects. Freshly transformed cells were inoculated in fresh BHI supplemented with pheromone (10 ng ml⁻¹) and incubated without shaking for 1 h at 37°C. Cells were processed for imaging as described in the Experimental procedures. Strains: OG1RF alone or harboring the P₂₃::prgA, P₂₃::prgB, or P₂₃::prgC expression plasmids.