Phenol-Soluble Modulin Toxins of Staphylococcus haemolyticus

Abstract

Coagulase-negative staphylococci (CoNS) are important nosocomial pathogens and the leading cause of sepsis. The second most frequently implicated species, after Staphylococcus epidermidis, is Staphylococcus haemolyticus. However, we have a significant lack of knowledge about what causes virulence of S. haemolyticus, as virulence factors of this pathogen have remained virtually unexplored. In contrast to the aggressive pathogen Staphylococcus aureus, toxin production has traditionally not been associated with CoNS. Recent findings have suggested that phenol-soluble modulins (PSMs), amphipathic peptide toxins with broad cytolytic activity, are widespread in staphylococci, but there has been no systematic assessment of PSM production in CoNS other than S. epidermidis. Here, we identified, purified, and characterized PSMs of S. haemolyticus. We found three PSMs of the β-type, which correspond to peptides that before were described to have anti-gonococcal activity. We also detected an α-type PSM that has not previously been described. Furthermore, we confirmed that S. haemolyticus does not produce a δ-toxin, as results from genome sequencing had indicated. All four S. haemolyticus PSMs had strong pro-inflammatory activity, promoting neutrophil chemotaxis. Notably, we identified in particular the novel α-type PSM, S. haemolyticus PSMα, as a potent hemolysin and leukocidin. For the first time, our study describes toxins of this important staphylococcal pathogen with the potential to have a significant impact on virulence during blood infection and sepsis.

Keywords: Staphylococcus haemolyticus; coagulase-negative staphylococci; hemolysin; leukocidin; phenol-soluble modulin; toxin.

Figure 1

RP-HPLC/MS of S. haemolyticus culture filtrate in comparison with S. aureus and S. epidermidis. (A) Stationary-phase culture filtrates of S. haemolyticus, S. epidermidis, and S. aureus were subjected to routine HPLC/MS detection of PSMs. (B) The averaged mass spectrum over that elution range showed the expected PSMs of S. epidermidis and S. aureus, among which the abundant m/z peaks belonging to δ-toxin are marked. S. haemolyticus showed a series of m/z peaks suggestive of three β-type PSMs and one α-type PSM.

Figure 2

Purification of S. haemolyticus PSMs. S. haemolyticus PSMs were purified from stationary-phase culture filtrate by initial chromatography on SOURCE PHE material and subsequent high-resolution HPLC on C18 material, shown in (A). Peaks in the elution range of PSMs were then further analyzed by RP-HPLC/MS (B).

Figure 3

Amino acid sequences and genes of S. haemolyticus PSMs and encoding loci in comparison with S. aureus and S. epidermidis. (A) Amino acid sequences of S. haemolyticus PSMs determined by N-terminal sequencing and/or comparison of molecular weights with annotated open reading frames in the S. haemolyticus genome. (B) Location of PSM-encoding genes in the genome of S. haemolyticus, in comparison with corresponding loci in S. aureus and S. epidermidis. Gene numbers for S. aureus and S. epidermidis are those from the USA300 FPR3757 or RP62A strain genomes, respectively. (C,D) Alignments and calculated phylogenetic trees of PSMα and PSMβ peptides of S. haemolyticus, S. aureus, and S. epidermidis. Sequences were analyzed using the online tool Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Next to the alignment in (C), the pairwise similarity matrix is shown.

Figure 4

S. haemolyticus PSMs form amphipathic α-helices. (A) Computation of α-helical wheels in the α-helical part of the peptides (using the program available at http://heliquest.ipmc.cnrs.fr). Note opposite arrangement of hydrophobic (yellow) vs. charged (red, blue) and hydroxyl (purple) amino acids. (B) Circular dichroism spectra of peptides in membrane environment conditions (50% trifluoroethanol).

Figure 5

Production of S. haemolyticus PSMs is conserved among different isolates and correlates with hemolytic capacity. Stationary-phase culture filtrates of 11 S. haemolyticus isolates were analyzed by RP-HPLC/MS. Below, hemolytic capacity of isolates is shown. Isolates were analyzed by spotting on human or sheep agar plates and growing overnight in a 37°C incubator.

Figure 6

Hemolytic activities of S. haemolyticus PSMs. Hemolytic activities of S. haemolyticus PSMs at different concentrations were measured using incubation with human erythrocytes and compared to that achieved with equal amounts of PSMα3 of S. aureus. Activities are compared as percentage compared to a control with total lysis. Data are from assays performed in quadruplicate.

Figure 7

Cytolytic activities of S. haemolyticus PSMs toward human neutrophils. Cytolytic activities of S. haemolyticus PSMs and S. aureus PSMα3 at 10 μg/ml were measured using incubation with human neutrophils by release of lactate dehydrogenase (LDH). Activities are compared as percentage compared to a control with total lysis. Triplicate measurements were performed each using neutrophils obtained from three donors.

Figure 8

Chemotactic activities of S. haemolyticus PSMs toward human neutrophils. Chemotactic activities of S. haemolyticus PSMs and S. aureus PSMα3 toward human neutrophils were determined using a transwell system. PSMs were applied at a concentration of 5 mM.