Gel filtration column chromatography of calf serum.

<p>(A) Elution profile of gel filtration column chromatography using a Superdex 200. Open circles indicate diameters of colonies. Filled circles indicate absorbance at 280 nm. Molecular weight markers were eluted in the fraction described below. Catalase (250-kDa) was eluted in fraction 21, bovine serum albumin (66-kDa) in fractions 25–26, and cyanocobalamin (1.3-kDa) in fraction 38. 250-µl aliquots of each sample were applied to soft agar medium and their colony-spreading stimulatory activity was measured. (B) SDS-PAGE analysis of gel filtration fractions. The gel was stained with Coomassie Brilliant Blue. A 66-kDa protein coincided with the colony-spreading stimulatory activity in fractions 24–29.</p

Mammalian serum stimulates <i>S. aureus</i> colony-spreading activity.

<p>(A) Stimulation of colony-spreading activity by calf serum. Overnight culture of <i>S. aureus</i> MRSA NI-15 was spotted on soft agar supplemented with serially diluted calf serum and incubated for 8 h at 37°C. Each plate contained 20 ml soft agar medium. (B) Stimulation of colony-spreading by porcine serum. Porcine serum or calf serum was serially diluted 2-fold and applied to 20 ml soft agar medium and its colony-spreading stimulatory activity was measured. Open circles indicate the halo diameters of giant colonies supplemented with porcine serum and filled circles indicate those supplemented with calf serum. Horizontal axis represents the volume of serum added to 20 ml soft agar medium in a plate. (C) Stimulation of colony-spreading activity by silkworm hemolymph. Hemolymph was collected from fifth instar larvae of silkworms and applied to the soft agar plates in 2-fold serial dilutions and its colony-spreading stimulatory activity was measured. Open circles indicate the halo diameters of giant colonies supplemented with silkworm hemolymph and filled circles indicate those supplemented with calf serum. (D) Growth curves of MRSA NI-15, MW2, or FRP3757 in tryptic soy broth supplemented with or without 1.25% (v/v) calf serum.</p

Stimulation of colony-spreading by bovine serum albumin.

<p>(A) Colony-spreading stimulation by purified bovine serum albumin. Purified bovine serum albumin of 96% grade (open triangles, Nacalai, cat. no. 01861-26) or 99% grade (open circles, Sigma, cat. no. A0281) was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities against MRSA NI-15 strain were measured. (B) Elution profile of DEAE-cellulose column chromatography using bovine serum albumin (96% grade, Nacalai). Open circles indicate the colony-spreading stimulatory activity. Filled circles indicate absorbance at 280 nm. (C) SDS-PAGE analysis of DEAE-cellulose column chromatography fractions. The gel was stained with Coomassie Brilliant Blue. The 66-kDa protein coincided with colony-spreading stimulatory activity in fractions 10–20. (D) Bovine serum albumin (Nacalai), fatty acid-free albumin from bovine serum (Wako Chemicals, cat. no. 017-15146), casein from bovine milk (Sigma, cat. no. C4032), or fetuin from calf serum (Sigma, cat. no. F2379) was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured.</p

Purification of colony-spreading stimulatory factor in calf serum.

<p>Calf serum (19 ml) was used as the starting medium. To measure the colony-spreading stimulatory activity, samples were serially diluted 2-fold and applied to 20 ml of soft agar and incubated for 8 h at 37°C. The diameters of giant colonies were measured from each dish, and stimulatory activities were calculated.</p

Diverse colony-spreading response to calf serum among HA-MRSA and CA-MRSA strains.

<p>(A) Representative images of the colony-spreading activity of MRSA NI strains stimulated by calf serum. Overnight cultures of MRSA NI strains were spotted onto soft agar plates supplemented with or without calf serum (250 µl/plate) and incubated for 8 h. (B) Colony-spreading response of CA-MRSA strains to calf serum. Overnight cultures of MW2 (USA400, open circles) or FRP3757 (USA300, filled circles) were spotted onto soft agar plates supplemented with 2-fold serially diluted calf serum and incubated for 8 h. The halo diameter was measured. (C) Amount of PSMα3 in MRSA strains cultured in the presence or absence of calf serum. MRSA strains with high colony-spreading response against calf serum (NI-15, MW2, FRP3757, NI-5, NI-7, NI-27, NI-29, NI-36, and NI-38) were cultured in the presence or absence of 1.25% (v/v) calf serum and the culture supernatants were analyzed by HPLC as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097670#pone.0097670-Kaito4" target="_blank">[6]</a>. Asterisks means not detected.</p

Lipoprotein and albumin-depleted serum decreases colony-spreading stimulatory activity.

<p>(A) Stimulatory activities of calf serum, lipoprotein-depleted serum, lipoprotein- and albumin-depleted serum were examined. Fractionation was performed from a 50-ml volume of calf serum containing 3850 units of stimulatory activity. Each fraction was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured. Relative total activities of each fraction compared with that of calf serum are presented in graph. (B) SDS-PAGE analysis of the lipoprotein-depleted serum and Affi-Gel blue gel column effluent. 10 µg protein of the lipoprotein-depleted serum and the Affi-Gel blue gel column effluent was electrophoresed in 12.5% SDS-polyacrylamide gel.</p

Identification of lipoprotein particles as a colony-spreading stimulator.

<p>(A) Protein from each purification step was electrophoresed by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (B) The final fraction (Fraction IV) was serially diluted 2-fold, and the colony-spreading stimulatory activity was measured. (C) Elution profile of DEAE-cellulose column chromatography using Fraction III. <i>Open circles</i> indicate diameters of colony-spreading. <i>Filled circles</i> indicate absorbance at 280 nm. 500-µl aliquots of each sample were applied to the soft agar medium and their colony-spreading stimulatory activity was measured. (D) SDS-PAGE analysis of DEAE-cellulose column chromatography fractions. The 25-kDa protein coincided with colony-spreading stimulatory activity in fractions 46-58. The 25-kDa protein was identified as apolipoprotein A1 by peptide-mass fingerprinting. (E) Colony-spreading stimulation by HDL particles. Purified HDL, delipidated HDL, or recombinant human apolipoprotein A1 (Wako Chemicals, cat. no. 019-20731) was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured. Human apolipoprotein A1 shares 78% amino acids identity with calf apolipoprotein A1. (F) Agarose gel electrophoresis analysis of calf serum, HDL, LDL, and lipoprotein-depleted serum. Agarose gel electrophoresis was performed according to a previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097670#pone.0097670-Noble1" target="_blank">[23]</a>. Each fraction (4 µg phospholipid) was electrophoresed in agarose 0.5%, and then lipoprotein bands were detected by lipid-specific staining using Oil Red O. The electrophoresed protein amount of calf serum, HDL, LDL, and lipoprotein-depleted serum was 500 µg, 5 µg, 5 µg, and 1800 µg, respectively. (G) Colony-spreading stimulation by phosphatidylcholine. Purified HDL, lipid extract from HDL, phosphatidylcholine from egg yolk (99% purity, Sigma, cat. no. P3556), or cholesterol (99% purity, Sigma, cat. no. C8667) was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured. (H) Colony-spreading stimulation by HDL, LDL, and VLDL. Fractionated HDL, LDL, or VLDL was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured.</p

Presence of phenol soluble modulins on the <i>S</i>. <i>aureus</i> cell surface.

<p>A. S. <i>aureus</i> Newman overnight cultured cells were washed in water, 5 M NaCl, 2% CHAPS, 8 M urea, 6 M guanidine HCl, or 3 M LiCl. In another sample, <i>S</i>. <i>aureus</i> cells were digested with lysostaphin and treated with 2% CHAPS. Samples were centrifuged and the amount of PSMα3 or PSMα1+δ-toxin in the supernatant was measured by HPLC. Vertical axis represents the amounts of PSM recovered from <i>S</i>. <i>aureus</i> cells (1.33 ml bacterial culture). Data are means±standard errors from three independent experiments. B. The centrifuged supernatants obtained in <i>A</i> were analyzed by SDS-PAGE. Proteins in the supernatants were precipitated with 10% trichloroacetic acid and electrophoresed on a 12.5% SDS polyacrylamide gel. The gel was stained by Coomassie brilliant blue. Each lane contains proteins from the same number of <i>S</i>. <i>aureus</i> cells (0.09 ml bacterial culture). C. <i>S</i>. <i>aureus</i> Newman overnight cultured cells were washed in 6 M guanidine HCl or 2% SDS. Samples were centrifuged and the amount of PSMα3 in the supernatant was measured by HPLC. Vertical axis represents the amount of PSMα3 recovered from <i>S</i>. <i>aureus</i> cells (1.33 ml bacterial culture). Data are means±standard errors from triplicate experiments.</p

Summary of the cell surface PSMα1–4 and the colony spreading in <i>S</i>. <i>aureus</i> gene knockout strains.

<p>PSMα1–4 and δ-toxin are presented as orange and blue dots, respectively. Knockout of δ-toxin increases the amount of cell surface PSMα1–4. In contrast, knockout of PSMα1–4 does not affect the amount of cell surface δ-toxin. The amount of cell surface PSMα1–4 and the colony-spreading activity in the wild-type strain, the δ-toxin knockout strain, the PSMα1–4 knockout strain, and the PSMα1-4/δ-toxin knockout strain is summarized in the lower part of this figure. The amount of cell surface PSMα1–4 is a determinant of colony-spreading activity.</p

Competitive binding assay of PSMs against <i>S</i>. <i>aureus</i> cell surface.

<p><b>A.</b> Inhibitory activity of δ-toxin against PSMα2 binding to the <i>S</i>. <i>aureus</i> cell surface of the PSMα1-4/δ-toxin knockout strain was measured. Binding assay of PSMα2 (10 nmol) to the cell surface of the PSMα1-4/δ-toxin knockout strain was performed in the absence or presence of δ-toxin (0, 10, 20, and 30 nmol) and the amount of PSMα2 bound to the cell surface was measured (left graph). In the competition assay, the binding of δ-toxin to the <i>S</i>. <i>aureus</i> cell surface was also measured (center graph) and the binding of total PSM (PSMα2 and δ-toxin) is presented (right graph). In all graphs, horizontal axis represents the amount of PSM added to <i>S</i>. <i>aureus</i> cells and vertical axis represents the amount of PSM bound to <i>S</i>. <i>aureus</i> cells (3 x 10⁸ CFU). B. Inhibitory activity of PSMα2 against δ-toxin binding to the <i>S</i>. <i>aureus</i> cell surface was measured. Binding assay of δ-toxin (10 nmol) to the cell surface of the PSMα1-4/δ-toxin knockout strain was performed in the absence or presence of PSMα2 (0, 10, 20, and 30 nmol) and the amount of δ-toxin bound to the cell surface was measured (left graph). In the competition assay, binding of PSMα2 to the <i>S</i>. <i>aureus</i> cell surface was also measured (center graph) and the binding of total PSM (δ-toxin and PSMα2) is presented (right graph). C. Inhibitory activity of δ-toxin against PSMα3 binding to the <i>S</i>. <i>aureus</i> cell surface was measured. Binding assay of PSMα3 (10 nmol) to the cell surface of the PSMα1-4/δ-toxin knockout strain was performed in the absence or presence of δ-toxin (0, 10, 20, and 30 nmol) and the amount of PSMα3 bound to the cell surface was measured (left graph). In the competition assay, binding of δ-toxin to the <i>S</i>. <i>aureus</i> cell surface was also measured (center graph) and the binding of total PSM (PSMα3 and δ-toxin) is presented (right graph). D. Inhibitory activity of PSMα3 against δ-toxin binding to <i>S</i>. <i>aureus</i> cell surface was measured. Binding assay of δ-toxin (10 nmol) to the cell surface of the PSMα1-4/δ-toxin knockout strain was performed in the absence or presence of PSMα3 (0, 10, 20, and 30 nmol) and the amount of δ-toxin bound to the cell surface was measured (left graph). In the competition assay, binding of PSMα3 to the <i>S</i>. <i>aureus</i> cell surface was also measured (center graph) and the binding of total PSM (δ-toxin and PSMα3) is presented (right graph).</p