Distinct roles of phenol-soluble modulins in spreading of Staphylococcus aureus on wet surfaces

Abstract

The human pathogen Staphylococcus aureus is renowned for the rapid colonization of contaminated wounds, medical implants, and food products. Nevertheless, little is known about the mechanisms that allow S. aureus to colonize the respective wet surfaces. The present studies were therefore aimed at identifying factors used by S. aureus cells to spread over wet surfaces, starting either from planktonic or biofilm-associated states. Through proteomics analyses we pinpoint phenol-soluble modulins (PSMs) as prime facilitators of the spreading process. To dissect the roles of the eight PSMs produced by S. aureus, these peptides were chemically synthesized and tested in spreading assays with different psm mutant strains. The results show that PSMα3 and PSMγ are the strongest facilitators of spreading both for planktonic cells and cells in catheter-associated biofilms. Compared to the six other PSMs of S. aureus, PSMα3 and PSMγ combine strong surfactant activities with a relatively low overall hydropathicity. Importantly, we show that PSM-mediated motility of S. aureus facilitates the rapid colonization of wet surfaces next to catheters and the colonization of fresh meat.

Fig 1

Characteristic features of S. aureus spreading motility. (A) A transparent “halo” is produced by cells of spreading colonies. A similar halo is also generated by surfactants that are spotted on soft agar plates (not shown). (B) Spreading motility involves a rapid phase of spreading by the cells spotted on a plate. Subsequently, waves of cells emerge from the cells in the initial spreading zone. The boxes mark the rapid spreading zones that emerged from the sites of inoculation of strains SH1000 and Newman on soft agar plates.

Fig 2

Colony spreading depends on the growth phase of inoculated S. aureus cells and on secreted factors produced by agr⁺ strains. (A) Colony spreading by planktonic cells of S. aureus strains SH1000 or Newman collected from cultures in different growth stages; t3 corresponds to the early exponential growth phase (OD₆₀₀ = 1.5), t5 to the late exponential phase (OD₆₀₀ = 6.8), and t7 to early stationary phase (OD₆₀₀ = 9.0). (B) Filter-sterilized culture medium of agr⁺ cells of S. aureus strain Newman promotes rapid spreading of Δagr cells of strains SH1000, NCTC8325, and Newman. As shown for Δagr cells of strain SH1000, no spreading is observed when fresh medium is used (left plate labeled “agr−”). (C) Culture supernatants of agr⁺ cells need to be applied on top of soft agar plates to promote spreading. Fresh soft agar plates were prepared and inoculated as follows: plate 1, regular soft agar with the wild-type strain Newman; plate 2, regular soft agar with the wild-type strain Newman resuspended in 2 μl of filter-sterilized supernatant of the wild-type strain Newman; plate 3, 200 μl of filter-sterilized supernatant of the wild-type strain Newman included within the soft agar prior to inoculation with the wild-type strain Newman; and plate 4, 200 μl of filter-sterilized supernatant of the wild-type strain Newman included in the soft agar prior to inoculation with Δagr cells of strain Newman. (D) Δagr cells carrying plasmid GFPopt-pRIT5H for expression of GFP were coinoculated with agr⁺ cells carrying plasmid pAH9 for expression of mCherry. GFP fluorescence of the Δagr cells is detectable at the edges of the spreading zone (green color), whereas mCherry fluorescence of the agr⁺ cells (red color) is detectable in the entire area covered by spreading. All spreading assays were repeated at least five times.

Fig 3

Particular synthetic PSM peptides facilitate spreading of Δagr cells. To identify factors that facilitate spreading of Δagr cells (labeled “agr−” in the figure), chemically synthesized PSMs from S. aureus (plate rows 2 to 5) were spotted in the center of soft agar plates prior to the inoculation with S. aureus SH1000 Δagr cells. Both N-terminally formylated PSMs (marked with an “f” prefix) and nonformylated peptides were used in the assay. In addition, the PSM-mec peptides were tested for a potentially inhibitory role in spreading by cells of the S. aureus SH1000 agr⁺ strain (bottom row). All spreading assays were repeated at least five times.

Fig 4

Additive effects of psmα and psmβ gene deletions on the spreading of S. aureus cells. The psmα and/or psmβ loci of the S. aureus strains Newman, SH1000, or HG001 were deleted, and the effects on colony spreading were compared with the effects of an agr mutation. Subsequently, the spreading areas of the investigated mutant and parental strains were determined by ImageJ, and statistical analyses were performed based on triplicate measurements for each individual strain. The graphs show the areas covered in arbitrary units (AU).

Fig 5

The spread of S. aureus cells from catheter-associated biofilms is facilitated by particular PSM peptides. (A) To investigate whether the spreading of cells from a catheter-associated biofilm is facilitated by PSMs, biofilms of Δagr cells of S. aureus Newman were grown on ∼1-cm-long strips of catheter material. These strips were then incubated on soft agar plates under differing conditions. Plate 1, 1egative control plate, showing that the used S. aureus Newman Δagr strain is unable to spread; plate 2, biofilms of Δagr cells were grown on catheter strips placed on a soft agar plate as shown with plate 2; plate 3, catheter strip with a biofilm (as on plate 2) transferred to a fresh soft TSA plate without further additions. Some “outgrowth” of the cells is observed but no spreading. Plate 4, catheter strip with a biofilm (as on plate 2) transferred to a soft TSA plate to which the PSMα3 peptide was added prior to the positioning of the catheter strip; plate 5, catheter strip with a biofilm transferred to a plate with the PSMγ peptide (as in plate 4); plate 6, catheter strip with a biofilm transferred to a plate with the PSMβ1 peptide (as in plate 4). (B) Control experiments with agr⁺ cells of S. aureus Newman. Plate 7, positive control plate showing colony spreading of agr⁺ cells; plate 8, colony spreading of Newman agr⁺ cells from a catheter strip; plate 9, catheter strip from plate 8 transferred to a fresh soft TSA plate without added PSMs. All spreading assays were repeated at least five times.

Fig 6

Spreading of S. aureus on meat. (A) Overnight grown S. aureus SH1000 agr⁺ or Δagr (labeled “agr−” in the figure) cells were spotted on pork meat, which was subsequently incubated 48 h at 37°C. SH1000 agr⁺ cells covered a 2.5-fold larger area than SH1000 Δagr cells. The spreading areas are marked with dashed lines. (B) Spreading areas of agr⁺ and Δagr S. aureus SH1000 cells, and single or double psmα and/or psmβ mutant strains upon spotting onto fresh pork meat. All spreading assays were repeated at least three times.