Staphylococcus quorum sensing in biofilm formation and infection

Abstract

Cell population density-dependent regulation of gene expression is an important determinant of bacterial pathogenesis. Staphylococci have two quorum-sensing (QS) systems. The accessory gene regulator (agr) is genus specific and uses a post-translationally modified peptide as an autoinducing signal. In the pathogens Staphylococcus aureus and Staphylococcus epidermidis, agr controls the expression of a series of toxins and virulence factors and the interaction with the innate immune system. However, the role of agr during infection is controversial. A possible second QS system of staphylococci, luxS, is found in a variety of Gram-positive and Gram-negative bacteria. Importantly, unlike many QS systems described in Gram-negative bacteria, agr and luxS of staphylococci reduce rather than induce biofilm formation and virulence during biofilm-associated infection. agr enhances biofilm detachment by up-regulation of the expression of detergent-like peptides, whereas luxS reduces cell-to-cell adhesion by down-regulating expression of biofilm exopolysaccharide. Significant QS activity in staphylococci is observed for actively growing cells at a high cell density, such as during the initial stages of an infection and under optimal environmental conditions. In contrast, the metabolically quiescent biofilm mode of growth appears to be characterized by an overall low activity of the staphylococcal QS systems. It remains to be shown whether QS control in staphylococci represents a promising target for the development of novel antibacterial agents.

Introduction

To establish an infection, bacteria have to orchestrate the expression of a group of molecules that determine pathogenicity, which are collectively known as the virulence factors. A delicate coordination of these usually species-specific virulence factors is crucial for the survival of the pathogen and the successful invasion of the host. Hence, pathogens have developed sophisticated regulatory systems to adapt virulence gene expression to the changing environmental conditions during infection.

Among bacterial global regulatory systems, cell–cell communication or quorum-sensing (QS) systems have gained broad attention in the scientific community. The signals of QS systems are small molecules called autoinducers (AIs). At low cell population density, AIs are low in concentration. When the cells reach a certain population density and the AIs accumulate to a threshold concentration, a transcriptional regulator is activated (Fuqua et al., 1994). This transcriptional factor in turn regulates the expression of various genes, which often includes a series of virulence factors.

Gram-negative microorganisms have a QS system that consists of homologues of the LuxI and LuxR proteins and an N-acyl homoserine lactone (AHL) signal molecule. The LuxI protein functions as an AHL synthase. Upon binding of the AHL molecule to the LuxR sensor, the AHL–LuxR complex activates the expression of multiple genes (Fuqua et al., 1994). In addition, a different QS system has been described in the marine bacterium Vibrio harveyi (Bassler, 1999). This signaling system uses a novel furanosyl borate diester, called AI-2, which is synthesized by the product of the luxS gene (Surette et al., 1999; Chen et al., 2002). The luxS gene encoding the AI-2 synthase shows no significant sequence similarity to other AI synthase genes (Surette et al., 1999). Interestingly, luxS homologues are found in many Gram-positive and Gram-negative bacteria, including Staphylococcus aureus and Staphylococcus epidermidis, suggesting that the luxS system might function as a widespread bacterial signaling system. In Gram-positive bacteria, several QS systems have been characterized with AIs that vary in structure, but are frequently peptide based. In contrast to the diffusible AIs of Gram-negative systems, the AIs of Gram-positive QS systems usually bind to a membrane-located protein that forms part of a two-component system (Lyon and Novick, 2004).

Staphylococci have one well-characterized QS system called agr for accessory gene regulator (Fig. 1). The agr system is composed of two divergently transcribed units, RNAII and RNAIII, the transcription of which is driven by the P2 and P3 promoters, respectively (Morfeldt et al., 1995). The RNAII contains four genes, agrB, agrD, agrC, and agrA (Novick et al., 1995). The agrB and agrD gene products are engaged in the production of the AI (Ji et al., 1995). The AI is an autoinducing peptide of ∼8 amino acids in length, which is encoded within the agrD gene. It is synthesized as a larger polypeptide, and then presumably trimmed by the agrB gene product (Saenz et al., 2000; Zhang et al., 2004) to form a thiolactone-containing ring structure (Otto et al., 1998; Mayville et al., 1999). AIs from different staphylococcal strains and species have a divergent primary amino acid sequence, but conserve the typical ring structure. They show the unique phenomenon of cross inhibition (Otto et al., 1999). As agr controls a series of virulence factors, it has been proposed to exploit agr antagonism by cross-inhibiting AIs as a means to control staphylococcal infection (Mayville et al., 1999).

The staphylococcal AI binds to a transmembrane protein, AgrC, which acts as the sensor kinase of the bacterial two-component regulatory system (Ji et al., 1995). Upon binding to the AI, AgrC activates the response regulator, AgrA, which in turn induces the transcription of RNAII and RNAIII (Ji et al., 1995). AgrA is a DNA-binding protein that recognizes a pair of direct repeats with a consensus sequence 5′-ACAGTTAAG-3′ separated by a 12-bp spacer region (Koenig et al., 2004). It is generally believed that RNAIII is the effector molecule of the accessory gene regulation (Novick et al., 1995). This 514-nt RNA acts as both a regulatory RNA and the messenger RNA for the hld gene, which encodes the delta toxin (Novick et al., 1995). RNAIII folds into a 14stem-loop structure with two long helices (Benito et al., 2000). It has been shown that the 5′-end of RNAIII positively regulates the translation of the alpha hemolysin (Morfeldt et al., 1995), whereas the 3′ domain is required for the repression of protein A synthesis (Novick et al., 1993; Huntzinger et al., 2005). However, it remains unknown how other virulence factor genes are regulated by RNAIII.

Since the identification of agr, it quickly became apparent that the agr system plays a central role in staphylococcal pathogenesis. However, recent data suggest that in vivo the role of agr may be subtle. In this review, we focus on the role of agr in biofilm formation and infection.