Molecular determinants of staphylococcal biofilm dispersal and structuring

Abstract

Staphylococci are frequently implicated in human infections, and continue to pose a therapeutic dilemma due to their ability to form deeply seated microbial communities, known as biofilms, on the surfaces of implanted medical devices and host tissues. Biofilm development has been proposed to occur in three stages: (1) attachment, (2) proliferation/structuring, and (3) detachment/dispersal. Although research within the last several decades has implicated multiple molecules in the roles as effectors of staphylococcal biofilm proliferation/structuring and detachment/dispersal, to date, only phenol soluble modulins (PSMs) have been consistently demonstrated to serve in this role under both in vitro and in vivo settings. PSMs are regulated directly through a density-dependent manner by the accessory gene regulator (Agr) system. They disrupt the non-covalent forces holding the biofilm extracellular matrix together, which is necessary for the formation of channels, a process essential for the delivery of nutrients to deeper biofilm layers, and for dispersal/dissemination of clusters of biofilm to distal organs in acute infection. Given their relevance in both acute and chronic biofilm-associated infections, the Agr system and the psm genes hold promise as potential therapeutic targets.

Keywords: Staphylococcus aureus; Staphylococcus epidermidis; biofilm; medical devices; phenol-soluble modulins.

Figure 1

Stages of biofilm development. (A) During the attachment phase, planktonic bacteria adhere to a biotic surface, such as human tissue or a human matrix-covered indwelling device, by non-covalent interactions between human matrix proteins and dedicated bacterial surface binding proteins (mostly, MSCRAMMs). (B) After attachment is accomplished, biofilm cells multiply producing an extracellular biofilm matrix that is composed of a variety of macromolecules, including specific exopolysaccharides (in many staphylococci, PIA), eDNA, teichoic acids, and a series of proteins such as the fibril-forming accumulation-associated protein, Aap. Furthermore, the biofilm develops a structured form with channels and mushroom-like towers, which is dependent on the disruptive forces of the PSM structuring molecules discussed in this review. (C) In the last phase of biofilm development, clusters of bacteria or single bacteria may detach from the biofilm in a process also called dispersal or sloughing. This process is stimulated by mechanic forces (such as under flow), the PSM surfactants, and by enzymes that degrade biofilm matrix molecules such as nucleases and proteases. The relevance of the latter mechanism for infection is unclear.

Figure 2

Surfactant properties of PSMs. (A) All PSMs form amphipathic α-helices. In the α-type PSMs, the helix stretches over virtually the whole peptide, while the longer β-type PSMs contain an α-helical part at their C-terminus. The graph shows an α-helical wheel presentation of PSMα3. Hydrophilic and hydrophobic amino acids occupy opposite sides of the helix, giving the helix strongly amphipathic character. (B) Model of PSMα3 structure (modeled after the known structure of δ-toxin that was determined by NMR studies). The hydrophobic side is shown. Replacement of amino acids on the hydrophobic side, mainly of large hydrophobic residues such as phenylalanine, leads to impaired biofilm structuring capacity.

Figure 3

PSM genes in S. aureus and S. epidermidis. PSMs are known to occur in a variety of staphylococci, but only in S. aureus and S. epidermidis were psm genes identified and gene products analyzed in a systematic manner. The graph shows the genetic arrangement of psm genes in S. aureus and S. epidermidis. The hld/RNAIII, psmβ, and psmα/psmδ loci show strong similarity between S. aureus and S. epidermidis, suggesting that they are evolutionarily related. The psm-mec gene is encoded on SCCmec mobile genetic elements present in similar form in S. aureus and S. epidermidis.

Figure 4

The Agr quorum-sensing system. The Agr system is an auto-regulatory system controlling gene expression in response to increasing cell density. It consists of the structural gene coding for the extracellular signal (AgrD), which is post-translationally modified and exported via AgrB. Upon reaching a certain threshold concentration, the AgrD AIP triggers auto-phosphorylation of the histidine kinase AgrC, which in turn leads to phosphorylation and activation of the DNA-binding response regulator AgrA. AgrA binding activates transcription from the AgrP2, AgrP3, psmα, and psmβ promoters. Agr targets other than PSMs are regulated by RNAIII, the regulatory RNA surrounding the hld (δ-toxin) gene.