Quorum sensing (QS)
For many years, bacteria were thought to be isolated single-celled organisms lacking social abilities. Secreted signaling molecules were identified (Tomasz & Hotchkiss, 1964), although infrequently, and the full extent of bacterial communication was not appreciated until much later. Only recently have researchers come to understand bacterial populations as communities of organisms in which signals serve as communication devices, sharing information within and between bacterial populations. The study of this process, termed QS, has provided important insight into how bacteria regulate behaviors in synchrony with members of their community. QS-mediated processes include biofilm formation and dispersal (Davies et al., 1998; Hancock & Perego, 2004; Parsek & Greenberg, 2005; Rickard et al., 2006; Waters et al., 2008; Ueda et al., 2009; Chang et al., 2011), virulence factor regulation (Winzer & Williams, 2001; Zhu et al., 2002; Rutherford & Bassler, 2012; Subramoni & Sokol, 2012), competence development (Håvarstein et al., 1995; Fontaine et al., 2010; Mashburn-Warren et al., 2010), sporulation (Perego & Hoch, 1996; Steiner et al., 2012), and many others.
In Gram-negative bacteria, the QS signals are often acyl-homoserine lactone molecules that have been extensively discussed and reviewed elsewhere (Fuqua et al., 2001; Schauder et al., 2001; Fuqua & Greenberg, 2002; Waters & Bassler, 2005; Ng & Bassler, 2009). Gram-positive bacteria, on the other hand, are found to use small peptides, commonly termed pheromones, as signals to mediate QS behaviors. In this review, we use the terms QS signal and pheromone interchangeably, recognizing these compounds serve a variety of purposes that may provide a means to measure population density or as a mechanism to signal from one individual to another. These signaling peptides regulate a wide array of processes, including many related to host-microbe interactions, and thus may provide novel targets for therapies that interfere with communication to disrupt bacterial infection. Disrupting virulence without directly killing or inhibiting growth of bacterial pathogens is expected to place lower selective pressure on bacteria to evolve mechanisms that would overcome such treatments. Quorum-quenching strategies may therefore provide an alternative method of treatment against antibiotic-resistant pathogens by means that are less likely to perpetuate resistance. To advance development of such therapeutic methodologies, a deeper understanding of the mechanisms by which bacteria regulate behaviors by intercellular signaling should be pursued. This review focuses on the current understanding of pheromone pathways used by Gram-positive bacteria of the genera Streptococcus and Enterococcus.
Gram-positive pheromone systems
Recent years have seen a dramatic increase in studies revealing new pheromone pathways among Gram-positive bacteria that expand upon the understanding of peptide signaling described for model organisms like Bacillus subtilis, Staphylococcus aureus, and Streptococcus pneumoniae. It is our aim to categorize fundamental attributes of these pheromone signaling pathways. Although all of the QS peptides described to date among Firmicutes are ribosomally synthesized, their processing, secretion, and signaling abilities differ widely (Fig. 1). Gram-positive QS pathways fall into four general groups based on features of the pheromones and their receptors: (1) members of the RNPP (Rap, NprR, PlcR, and PrgX) family of regulators; (2) Agr-type cyclical pheromones; (3) peptides with double-glycine (Gly-Gly) processing motifs; and (4) regulators of the Rgg family. It is becoming evident that Gram-positive bacteria often utilize multiple types of QS pathways within a species to control a wide variety of processes.
Pheromones that bind to RNPP regulators are transported to the cytoplasm where they directly interact with an RNPP family member to modulate gene expression. The RNPP family was named for the Rap auxiliary regulatory proteins of B. subtilis, the neutral protease regulator, NprR, found in Bacillus species, the phospholipase C regulator, PlcR, of the B. cereus family, and the pheromone responsive gene regulator, PrgX, of Enterococcus faecalis (Declerck et al., 2007). The group has grown to contain many orthologs of these regulators as well. Often the RNPP family member and its cognate peptide are encoded adjacent to one another. Following transcription and translation, pre-peptides are secreted and processed into mature pheromones where they can encounter other cells in the population. The pheromones then are taken into the cell by transporters of the oligopeptide permease (Opp) family and subsequently bind to the RNPP cytoplasmic regulators. In the case of Rap proteins, pheromone binding disrupts the interaction of Rap with response regulator proteins that control gene expression (Core & Perego, 2003; Baker & Neiditch, 2011; Parashar et al., 2013). NprR, on the other hand, contains a helix-turn-helix (HTH) DNA binding domain. Binding of the NprX peptide activates interaction of the NprR HTH domain with DNA, thus activating transcriptional activity (Zouhir et al., 2013). Similarly, PrgX, a repressor of gene transcription, and PlcR, an activator, are bound by their cognate peptide (or multiple peptides in the case of PrgX) to exert a conformational change in the proteins thus modulating DNA binding and transcriptional regulation of target genes (Bae et al., 2002; Declerck et al., 2007). The RNPP family of QS systems have been recently reviewed (Rocha-Estrada et al., 2010).
Agr-type peptides are named for accessory gene regulator, consisting of the genes agrABCD, that stands as a well-studied QS circuit controlling virulence factor expression in S. aureus. A hallmark feature of this pathway is the utilization of a cyclical peptide pheromone encoded by agrD in S. aureus. The peptide is proposed to be exported and processed via a dedicated transport protein termed AgrB (Saenz et al., 2000; Nakayama et al., 2001). AgrB contains a putative cysteine endopeptidase domain (Qiu et al., 2005) and cyclization of the peptide is thought to be assisted by the transporter. Pheromone detection occurs by a two-component signal transduction system (TCSTS) at the cellular surface whereby the peptide binds to a dedicated histidine kinase, AgrC, that transmits the signal via phosphorylation of the cytoplasmic response regulator AgrA (Sturme et al., 2002). Orthologous signaling pathways have been identified in Enterococcus (Fsr, discussed below), Clostridium, and Listeria (Agr).
Competence-stimulating peptides (CSPs) of streptococci and class II bacteriocins belong to the Gly-Gly-type peptide family. As their name suggests, these peptides contain a double glycine motif in their conserved leader sequence (LSX2ELX2IXGG) (Havarstein et al., 1994). Gly-Gly peptides are secreted via a transporter containing an accessory domain that proteolytically processes the leader sequence at a site in the polypeptide immediately following the conserved Gly-Gly motif (Havarstein et al., 1995). As seen in Agr-type systems, peptides of the double glycine family are sensed via a TCSTS that transmits a signal internally via phosphorylation of cognate response regulators.
Like the RNPP family, regulators of the Rgg family directly bind to pheromones that are internalized subsequent to their export and maturation. Although secretion and processing of these peptides is not fully understood, several reports have found a role for the Eep (enhanced expression of pheromone) metalloprotease, which also cleaves the signal sequence of the enterococcal sex pheromones (An et al., 1999; Chang et al., 2011). These peptides are potentially processed further upon reaching the extracellular milieu, and mature peptides are internalized via Opp or Ami peptide uptake systems prior to interaction with Rggs. Not every Rgg-type regulator has been shown to interact with a peptide pheromone, although as this family of proteins has continued to receive attention, more peptide interactions have been found or are hypothesized to be present (Fleuchot et al., 2011; Shelburne et al., 2011; Cook et al., 2013). Structural information on Rgg proteins remains elusive; however, structure prediction algorithms suggest that Rgg proteins contain similar tricopeptide repeat (TPR)-like domains responsible for peptide interactions in the RNPP family leading some to propose that Rgg proteins should be included in the RNPP family (Mashburn-Warren et al., 2010; Fleuchot et al., 2011). For the purposes of this review, we will consider them as separate groups while still highlighting similarities between the two.