Phenol-soluble modulins--critical determinants of staphylococcal virulence

Abstract

Phenol-soluble modulins (PSMs) are a recently discovered family of amphipathic, alpha-helical peptides that have multiple roles in staphylococcal pathogenesis and contribute to a large extent to the pathogenic success of virulent staphylococci, such as Staphylococcus aureus. PSMs may cause lysis of many human cell types including leukocytes and erythrocytes, stimulate inflammatory responses, and contribute to biofilm development. PSMs appear to have an original role in the commensal lifestyle of staphylococci, where they facilitate growth and spreading on epithelial surfaces. Aggressive, cytolytic PSMs seem to have evolved from that original role and are mainly expressed in highly virulent S. aureus. Here, we will review the biochemistry, genetics, and role of PSMs in the commensal and pathogenic lifestyles of staphylococci, discuss how diversification of PSMs defines the aggressiveness of staphylococcal species, and evaluate potential avenues to target PSMs for drug development against staphylococcal infections.

Keywords: Staphylococcus aureus; Staphylococcus epidermidis; biofilm; phenol-soluble modulin; toxin; virulence.

Fig. 1. Structure of PSMs

Shown is a structural representation of PSMα3 modeled after NMR data available for δ-toxin (a) and an α-helical wheel representation of the same peptide (b) as examples. Hydrophobic amino acids are in yellow/brown, hydrophilic amino acids in blue (positive charge) or red (negative charge). The two C-terminal asparagine residues are in pink. Note that in the α-helix formed by PSMα3 hydrophilic and hydrophobic residues occupy opposite locations, making the α-helix distinctly amphipathic.

Fig. 2. Analysis of PSMs by RPLC/MS

Shown is RPLC chromatography routinely used for RPLC/MS analysis of PSMs. Culture supernatant of an S. aureus strain (USA300) was directly applied to a 2.3 × 30 mm C8 reversed phase column and a brief gradient from 10 to 50% followed by an extended gradient from 50 to 90% water/0.1% triflouroacetic acid (TFA) to acetonitrile/0.1% TFA as described in Wang et al. (Wang et al., 2007) was run. Note that PSMs are separated from other proteins, but not all PSMs completely separate from other PSMs, necessitating coupling to MS for measurement of single PSM quantities. WT, wild-type USA300 (LAC); Δpsmα, psmβ, hld, isogenic mutant not expressing any PSM.

Fig. 3. PSMs in S. aureus and S. epidermidis

Amino acid sequences are shown at the top. Numbers at the right show the net charge of the peptides at pH 7.0, rounded to whole numbers, and considering N-formylation. Genes are shown at the bottom. Gene numbering is according to strains S. aureus USA300 FPR3757 (Diep et al., 2006) and S. epidermidis RP62A (Gill et al., 2005), respectively.

Fig. 4. Regulation of PSMs by Agr

The Agr quorum-sensing circuit, as described in the text, is shown at the top. Direct regulation of the psmα and psmβ operons by AgrA is shown left (blue shadowing) and RNAIII-dependent Agr control of other target genes at the right (yellow shadowing). The likely evolution of the connection of quorum-sensing, RNAIII-dependent and –independent control is apparent in the encoding of the PSM δ-toxin (hld) by RNAIII. Agr also controls the psm-mec gene in an RNAIII-independent manner, but binding of AgrA to the psm-mec promoter has not yet been demonstrated.

Fig. 5. PSM-mec

The PSM-mec peptide and the psm-mec RNA surrounding the PSM-mec-encoding gene are encoded on SCCmec elements of types II, II, and VIII, of which SCCmec type III is shown here as example.

Fig. 6. PSM export

The genetic locus encoding the four Pmt ABC transporter components is shown at the top (a). Numbers refer to gene numbers in the USA300 FPR3757 genome (Diep et al., 2006). (b) Pmt functions. (c) Consequences of Pmt absence/blocking.

Fig. 7. PSM-mediated cytolysis

Likely mechanism of receptor-independent membrane attachment and disintegration, and subsequent pore formation by PSMs. This model is based on experimental evidence obtained with S. aureus δ-toxin, which forms receptor-independent short-lived pores in artificial membranes (Verdon et al., 2009). (b) Mechanism of intracellular neutrophil killing by PSMα peptides of S. aureus (Surewaard et al., 2013).

Fig. 8. PSM receptor interaction

In the nanomolar range, PSMs bind to and activate FPR receptors, with by far the strongest activation occurring with FPR2. Pro-inflammatory, FPR2-mediated activities of PSMs include neutrophil activation, chemotaxis, and release of specific cytokines such as IL-8. N-formylated and N-deformylated PSMs activate FPR2 receptors, with activation by formylated PSMs being somewhat stronger. The S. aureus virulence factors CHIPS and FLIPr block recognition by FPR1 and FPR2, respectively.

Fig. 9. PSMα peptides and skin infection

Representative pictures of abscesses formed by the USA300 CA-MRSA strain and its isogenic psmα deletion mutant in mice (Wang et al., 2007). (b) Comparative analysis of virulence determinants in a rabbit skin infection model (Kobayashi et al., 2011). The abscess volume is shown on the y-axis. Significantly smaller abscesses compared to the wild-type strain were found with the hla, psmα, and agr, but not lukSF mutants.

Fig. 10. PSMs and biofilm structuring

Confocal laser scanning microscopy pictures of biofilms formed by an S. epidermidis wild-type strain (strain 1457) and an isogenic deletion mutant of the psmβ operon (Wang et al., 2011). Note the thicker biofilm formed by the mutant and the absence of channels in the biofilm.

Fig. 11. PSM structure-function analysis

An alanine exchange library of the PSMα3 peptide was created and used to determine changes in PSM function caused by structural changes (Cheung, et al., 2013). Overall, large hydrophobic side chains impacted biofilm-structuring capacities (such as some phenylalanine residues shown in regular font) and predominantly cationic amino acids impacted cytolytic and pro-inflammatory functions. In addition, the C-terminus exclusively affected receptor-mediated but not cytolytic activities. Finally, removal of large hydrophobic side chains such as all phenylalanine residues led to derivatives that showed antimicrobial activities, indicating that these large hydrophobic side chains are involved in preventing cytolysis of bacterial versus eukaryotic membranes in PSMs.