Microcolony and MAM diameter of <i>S. aureus</i> Newman strains after growth in 3D-CoG/Fib.

<p>The average diameter of microcolonies and MAM was determined after 16 h of growth in 3D-CoG/Fib (A). Wildtype (wt), <i>coa</i> mutant and <i>eap</i> mutant formed microcolonies and MAM of comparable size. The <i>vWbp emp</i> double mutant did not form any MAM, despite being unaffected in pseudocapsule formation. A <i>sae</i> mutant (Newman-29) neither formed pseudocapsules nor MAM but grew in clusters significantly larger than wt colonies. USA300 microcolonies were comparable in size to wt, pseudocapsule formation was present, but the diameter of the MAM was significantly smaller compared to wt. Data are averaged from at least three independent experiments. Addition of plasmin (8 µg/ml) led to rapid degradation of both pseudocapsule and MAM surrounding <i>S. aureus</i> Newman colonies grown in 3D-CoG/Fib for 17h (B1–B4). The time stamp in the single panels is relative to plasmin addition, scale bar 25 µm.</p

Localization of Coa and Emp to <i>S. aureus</i> pseudocapsules.

<p>Coa and Emp were detected in pseudocapsules by immunocytochemistry using rabbit antibodies specific for coagulase (anti-Coa, A) or Emp (anti-Emp, B and C). The pseudocapsule of the <i>coa</i> mutant is more irregularly shaped than its wildtype counterpart, illustrated by anti-Emp staining (C). Staphylococci were grown in 3D-CoG/Fib for 16 h and then fixed with 4% paraformaldehyde. Primary antibodies were detected by Alexa Fluor 555 anti-rabbit antibodies. Staphylococci were detected by staining DNA with DAPI (A1, B1, C1) and N-acetyl-glucosamine with FITC-Lectin (<i>T. vulgaris</i>) (A2, B2, B3). Staining of Coa and Emp was specific as the <i>coa</i> and <i>vwbp emp</i> mutant were not stained with the respective antisera (data not shown). Scale bar 7.5 µm. The images are representative of three independent experiments.</p

Proposed model for the differential actions of Coa and vWbp.

<p><i>S. aureus</i> Newman forms two concentric barriers in the presence of fibrinogen in the growth medium: the outer MAM and the inner pseudocapsule; both contain fibrin. The MAM is dependent on vWbp and inhibits neutrophil immigration into the vicinity of the microcolony. The pseudocapsule is partially dependent on Coa and acts as a second barrier preventing neutrophil invasion of the microcolony.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Presence of pseudocapsule and MAM structures in various clinical isolates.

<p>Clinical <i>S. aureus</i> isolates were cultivated as described for strain Newman and evaluated after 16 h of growth. MSSA strain MP9-11 (A). MSSA strain MP3-11 (B). MSSA strain MP6-11 (C). MRSA strain MP10-11 (D). CA-MRSA strain USA300 (E). MSSA strain MP2-11 (F). MRSA strain ST239-CC8 (G). MSSA strain MP1-11 (H). Pseudocapsules (yellow arrowheads) and MAM-like structures (blue arrowheads) are indicated. A–C: scale bar 20 µm. D–H: scale bar 75 µm.</p

The thrombin inhibitor argatroban antagonizes staphylococcal barrier activity.

<p><i>S. aureus</i> Newman was grown in 3D-CoG/Fib in the presence of different argatroban concentrations. After 16 h of growth, the growth phenotypes were analyzed: at 10 nM argatroban, the MAM was diminished and at higher concentrations also pseudocapsule formation was absent (A). Challenging the system with neutrophils after 16–17 h revealed loss of both barrier functions in a concentration-dependent manner (B). Scale bar 25 µm. The images are representative of three independent experiments. Data are averaged from three independent experiments.</p

Strains used in this study.

<p>*Formerly denoted as Newman <i>emp</i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002434#ppat.1002434-Johnson1" target="_blank">[47]</a>. Sequencing analysis revealed that assumedly by phage transduction not only the <i>emp</i> gene but also the neighboring functional <i>vWbp</i> gene of strain Newman has been replaced by a truncated version of <i>vWbp</i> from strain RN4220. This was further confirmed by the fact that vWbp could be detected in the supernatants of 3 h Newman wildtype cultures but not of this <i>emp</i> mutant strain (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002434#ppat.1002434.s001" target="_blank">Fig. S1</a>).</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

EGFP expression in different bacterial hosts was determined by FACS analysis.

<p>Streptococcal, enterococcal strains and <i>S. aureus</i> were grown overnight in THY broth as described in Material and Methods. Depicted are the values obtained for the ß-hemolytic species <i>S. agalactiae</i>, <i>S. pyogenes</i>, <i>S. dysgalactiae</i> subsp. <i>equsimilis</i> (SDSE) serogroup C, SDSE serogroup G, <i>Staphylococcus aureus</i>, the enterococcal species <i>E. faecalis</i> and <i>E. faecium</i> and the viridans group streptococci <i>S. mutans</i> and <i>S. anginosus</i>. Shown are mean values and standard deviations of five independent experiments.</p

Methionine metabolism and biosynthesis control among <i>Bacillales</i> and <i>Lactobacillales</i>.

<p>Presence of genes of the methionine salvage pathway, polyamine synthesis, SAM recycling and control mechanisms of methionine biosynthesis in genomes of bacteria of the <i>Bacillales</i> and <i>Lactobacillales</i> orders as well as number of methionine-specific T-box riboswitches in these genomes. Data were obtained by querying the KEGG <a href="http://www.genome.jp/kegg/" target="_blank">http://www.genome.jp/kegg/</a> and RegPrecise <a href="http://regprecise.lbl.gov/RegPrecise/index.jsp" target="_blank">http://regprecise.lbl.gov/RegPrecise/index.jsp</a> databases <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003606#ppat.1003606-Novichkov1" target="_blank">[15]</a> and from references <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003606#ppat.1003606-GutierrezPreciado1" target="_blank">[6]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003606#ppat.1003606-Joshi1" target="_blank">[20]</a>.</p