A new quorum-sensing system (TprA/PhrA) for Streptococcus pneumoniae D39 that regulates a lantibiotic biosynthesis gene cluster

Abstract

The Phr peptides of the Bacillus species mediate quorum sensing, but their identification and function in other species of bacteria have not been determined. We have identified a Phr peptide quorum-sensing system (TprA/PhrA) that controls the expression of a lantibiotic gene cluster in the Gram-positive human pathogen, Streptococcus pneumoniae. Lantibiotics are highly modified peptides that are part of the bacteriocin family of antimicrobial peptides. We have characterized the basic mechanism for a Phr-peptide signaling system in S. pneumoniae and found that it induces the expression of the lantibiotic genes when pneumococcal cells are at high density in the presence of galactose, a main sugar of the human nasopharynx, a highly competitive microbial environment. Activity of the Phr peptide system is not seen when pneumococcal cells are grown with glucose, the preferred carbon source and the most prevalent sugar encountered by S. pneumoniae during invasive disease. Thus, the lantibiotic genes are expressed under the control of both cell density signals via the Phr peptide system and nutritional signals from the carbon source present, suggesting that quorum sensing and the lantibiotic machinery may help pneumococcal cells compete for space and resources during colonization of the nasopharynx.

Fig 1. TprA serves as an inhibitor of phrA expression

(A) The effect of ΔtprA and ΔphrA mutations on phrA-lacZ expression. Strains: Spn007, “wild-type” parental strain used in this experiment; Spn013, ΔtprA; Spn019, ΔphrA; Spn195, ΔtprA CEP::TprA (complemented strain). Results shown are averages of 2-5 independent experiments and error bars depict the standard error of the mean. ***, significant at P < 0.001 compared to “wild type.” +++, significant at P < 0.001 compared to ΔtprA. (B) phrA mRNA levels in a ΔtprA mutant strain. Strains: IU1781 & Spn049, “wild-type” parental strains used in this experiment; Spn052, ΔtprA; Spn197, ΔtprA CEP::TprA (complemented strain). mRNA levels were normalized to 16S RNA levels, from 2 independent experiments, and are shown as a ratio relative to the wild-type levels. Error bars depict the standard error of the mean. *, significant at P < 0.05 compared to “wild type.” +, significant at P < 0.05 compared to ΔtprA. Different parent strains were used in these approaches containing wild-type alleles for the genes of interest.

Fig 2. Identification of the minimal PhrA-signaling peptide

(A) phrA-lacZ reporter expression is elevated when the full length phrA gene is overexpressed. Strains: Spn065, full length PhrA; Spn191, PhrAΔ42-56; Spn189, PhrAΔ47-56; Spn187, PhrAΔ52-56; Spn243, PhrAΔ56. Cells were grown in BHI or BHI+1% fucose (inducer) to mid-exponential phase (OD₆₂₀ of between 0.15 to 0.35) when samples were removed for β-galactosidase activity assays. Results shown are the averages of at least 3 independent replicates and error bars indicate the standard error of the mean for each set. ***, significant at P < 0.001 compared to uninduced strain containing the full-length PhrA construct. (B) Synthetic peptides corresponding to the C-terminus of PhrA used in (C) below. (C) Induction of the phrA-lacZ reporter was observed when cells were treated with the last 6, 7, or 10 amino acids of PhrA. Early exponential phase (OD₆₂₀ of ~0.1) wild-type cells (Spn007) were incubated with synthetic peptides at a final concentration of 5 μM or peptide-resuspension buffer for two hours prior to analysis by β-galactosidase assays. Results shown are the averages of at least 3 independent replicates and error bars indicate the standard error of the mean for each set. *, significant at P < 0.05 and **, significant at P < 0.01 compared to the “wild type” strain incubated with buffer.

Fig 3. Oligopeptide permease is required for induction of phrA-lacZ in response to synthetic peptide

Strains lacking amiC in a wild-type or a ΔtprA mutant background were tested for their ability to induce phrA-lacZ expression in response to the 10-residue PhrA peptide. Strains: Spn007, “wild-type” parental strain used in this experiment; Spn013, ΔtprA; Spn141, ΔamiC; Spn165, ΔtprA ΔamiC. Early exponential phase cells (OD₆₂₀ of ~0.1) grown in BHI were incubated with 5 μM synthetic peptide or peptide-resuspension buffer for two hours prior to analysis by β-galactosidase activity assays. Results shown are the average of at least two independent trials, and error bars represent the standard error of the mean. ***, significant at P < 0.001 compared to the strain treated with buffer.

Fig 4. PhrA can signal between cells when grown to high cell density in media containing galactose

Cells (Spn007, wild-type) grown in CDM-glucose (closed squares) or CDM-galactose (open circles). Panel A shows a representative growth curve of these cells on these media. Note that after inoculation of the cultures several hours pass before there is a measurable level of cells, and this lag phase is longer in CDM-galactose. Panel B shows expression of phrA-lacZ in the Spn007 cells. At least two independent experiments were performed; the results from one representative experiment are shown. Panel C shows induction of phrA-lacZ when these were resuspended in conditioned media from wild-type cells (IU1781) compared to untreated media, and no induction was observed in conditioned media from cells lacking phrA (IU4957). The results shown are the average of at least two independent trials and the error bars depict the standard error of the mean. *, significant at P < 0.05 compared to untreated media. +, significant at P < 0.05 compared to wild-type conditioned media.

Fig 5. The TprA/PhrA system regulates a putative lantibiotic biosynthesis operon

ORFs are represented by dark grey arrows (in the case of the TprA/PhrA system) or light gray arrows (for the putative lantibiotic biosynthesis genes) and the D39 gene identification numbers are indicated in the arrows, with genes spd1747 and spd1748 shorten to 47 and 48, respectively. Known or predicted functions of each gene are indicated above the arrows, and -- indicates that the function of the genes is unknown. The putative promoters, predicted by the results with RNA-seq, are represented by bent black arrows, and putative promoters predicted by the DOOR database are shown as bent gray arrows. Small black boxes are predicted CRE-binding sites of CcpA (Carvalho et al., 2011). Genes whose expression has been found to be increased in either a ΔtprA mutant or by the addition of the PhrA peptide are denoted by + under the gene.

Fig 6. Model for the mechanism by which PhrA and TprA control gene expression in S. pneumoniae

The mature PhrA peptide is encoded by phrA producing a precursor protein in the absence of glucose. Glucose repression occurs through a CRE element that is in phrA promoter region. The PhrA precursor is exported and processed to release the mature PhrA peptide (dark gray, small ovals). When at a sufficient concentration, the PhrA peptide interacts with oligopeptide permease and is transported into the cell where it inhibits the activity of TprA leading to de-repression of phrA, tprA, and a change in transcription of lantibiotic genes (wide arrows). Only three of the eight lantibiotic biosynthesis cluster genes are shown here for simplicity (black outlined, wide arrows with one not shown to scale (angled lines)). Bent arrows indicate the location of data-supported (black) or predicted (gray) promoters, all of which are negatively regulated by TprA (denoted by lines that end with a horizontal line).