The HVRs of M4.1 and M114 Bind C4BP

<div><p>Fusion proteins, derived from the HVR of M4.1 or M114 and the C-terminal part of M5, were expressed in the M-negative strain S. pyogenes ΔM5 using genes carried on plasmid pLZ12Spec. Controls included a strain expressing the C4BP-binding fusion protein M22⁵⁷–M5, a strain expressing the non–C4BP-binding M5 protein, and the M-negative strain ΔM5.</p><p>(A) Surface expression analyzed with rabbit antibodies directed against the C-repeat region of M5. Bound antibodies were detected with radiolabelled protein A. Binding of protein A to the M22⁵⁷–M5 strain, incubated with antiserum diluted ×10², was defined as 100%. The M5-negative strain ΔM5 served as negative control.</p><p>(B) Binding of radiolabelled C4BP. Binding at the highest bacterial concentration to the control expressing M22⁵⁷–M5 was defined as 100%. The non–C4BP-binding M5 strain served as negative control. All data in (A) and (B) are based on three separate experiments with duplicate samples, and are presented as means ± SD.</p></div

The Binding Site for Human C4BP in the Hypervariable Region (HVR) of M Protein

<div><p>(A) Schematic representation of C4BP bound to the HVR of an M protein, a dimeric coiled-coil. The most common form of C4BP has seven identical α-chains and one short β-chain. Both chains are composed of CCP modules, as indicated. The binding site for M protein in C4BP is located in the CCP1–2 region of the α-chain [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b017" target="_blank">17</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b024" target="_blank">24</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b047" target="_blank">47</a>].</p><p>(B) Multiple sequence alignment of HVRs that bind C4BP. The five upper sequences are from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b025" target="_blank">25</a>]. Three residues that are identical in these five sequences are boxed. PrtH is a second M protein expressed by certain M1 strains [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b035" target="_blank">35</a>]. The lower part of the alignment shows the HVRs of M4.1 and M114, characterized in this paper. The vertical hatched lines, corresponding to residues 1–39 in M22, indicate the region used to generate the logo in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-g005" target="_blank">Figure 5</a>A.</p><p>(C) Construction of fusion proteins derived from the M22 and M5 proteins. An N-terminal region derived from M22 was fused to the C-terminal part of M5 (residues 104–450 of M5). The fusion proteins contain the Fg-binding B-repeat region of M5.</p><p>(D) Schematic representation of the N-terminal region of different fusion proteins. The sequence of the N-terminal region of M22 is given at the top. Asterisks indicate the position of residues L28, E31, and D40 in M22 (corresponding to the three boxed residues in [B]). The ability of the fusion proteins to bind C4BP, indicated to the right, is based on the results shown in (E).</p><p>(E) Ability of fusion proteins to bind C4BP. The fusion proteins (D) are referred to as M22⁵⁷–M5, etc. Whole-cell lysates of E. coli strains, expressing the indicated proteins from genes carried on pBR322, were analyzed by Western blot using Fg or C4BP as the probe. The strain expressing M5 was used as a negative control. The control blot with Fg showed that the proteins were expressed in <i>E. coli.</i> The presence of double bands probably reflects incomplete processing of signal peptides in E. coli and/or intracellular degradation of M protein in this heterologous host.</p></div

Sequence Analysis of C4BP-Binding HVRs and Site-Specific Mutagenesis in M22

<div><p>(A) Sequence logo of C4BP-binding HVRs. The logo was generated from the seven C4BP-binding HVRs aligned in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-g001" target="_blank">Figure 1</a>B, using WebLogo (<a href="http://weblogo.berkeley.edu" target="_blank">http://weblogo.berkeley.edu</a>). Only regions in the HVRs corresponding to the shortest known C4BP-binding region in M22 (residues 1–39) were used to create the logo. These regions are demarcated by the vertical hatched lines in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-g001" target="_blank">Figure 1</a>B. In the logo, each column in the alignment is represented by a stack of letters, with the height of each letter proportional to the observed frequency of the corresponding residue at that position, while the overall height of each stack is proportional to the sequence conservation at that position [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b063" target="_blank">63</a>]. The sequence of the M4.1 HVR was included in the generation of the logo, although it is virtually identical to M4, because the single residue difference between these two HVRs was important for the conclusion that the different HVRs completely lack residue identities (see text). The numbering below the logo refers to residue numbers in the M22 protein and putative coiled-coil heptads (a–g) in M22 are indicated. Asterisks show the position of four M22 residues (L21, E24, L28, and E31) analyzed by site-specific mutagenesis.</p><p>(B) Helical wheel representation of a dimeric coiled-coil [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b042" target="_blank">42</a>]. The sequence of the L21–E31 region of M22 is included, with asterisks above residues L21, E24, L28, and E31, which were analyzed by site-specific mutagenesis and are located within the predicted coiled-coil region. The positions of residues within putative coiled-coil heptads (a–g) are indicated.</p><p>(C–E) The four mutant M22 proteins indicated, constructed by site-specific mutagenesis, and the wild-type (wt) M22 protein, were expressed in <i>S. pyogenes,</i> and the strains were analyzed for surface expression of the proteins and ability to bind C4BP. The genes encoding the proteins were present on plasmid pLZ12Spec, carried by an M-negative strain. This M-negative strain also served as negative control. (C) and (D) show that the different proteins were expressed normally on the bacterial surface (see text). (E) shows that mutants L21A and L28A had completely lost C4BP-binding ability, while mutants E24A and E31A were unaffected. The results shown in (C–E) are based on three separate experiments with duplicate samples and are presented as means ± SD.</p></div

Characterization of the C4BP-Binding HVR in M114

<div><p>(A) The HVR of M114 is a distinct protein domain that binds C4BP with high specificity. A dimerized synthetic peptide, derived from the 52 N-terminal residues in M114 and designated M114-N, was immobilized in a column. Whole human serum was passed through the column, which was washed and eluted. Control columns contained the C4BP-binding M22-N peptide or the nonbinding M5-N peptide. The eluates, and human serum, were analyzed by SDS-PAGE, as indicated. The ~70 kDa polypeptide present in the eluates from the M22-N and M114-N columns was identified as the C4BP α-chain by Western blot analysis with specific antiserum (not shown).</p><p>(B) The M114 and M22 proteins bind to the same region in C4BP. The peptides indicated were used to inhibit the binding of radiolabelled M22 protein to C4BP immobilized in microtiter wells. Data from three separate experiments with duplicate samples, presented as means ± SD.</p></div

Single Amino Acid Changes Not Affecting C4BP Binding Cause Major Antigenic Changes in the HVR of M22

<div><p>(A) Schematic representation of an inhibition test used to analyze the antigenic properties of mutant M22 proteins expressed on the surface of <i>S. pyogenes.</i> The test was based on the binding of mouse anti–M22-N to pure M22 protein immobilized in microtiter wells. This binding was inhibited with whole S. pyogenes bacteria expressing mutant M22 proteins.</p><p>(B) Ability of S. pyogenes strains expressing the M22 mutants E24A and E31A to cause inhibition. The positive control expressed the wild-type M22 protein and the negative control lacked M protein. As compared to the positive control, 50% inhibition (dashed line) required ~30-fold more bacteria expressing either of the mutant proteins. Results based on three separate experiments with duplicate samples, presented as means ± SD.</p></div

Binding of Human C4BP to S. pyogenes Strains of Different M Types

<p>Strains of the M types indicated were analyzed for ability to bind radiolabelled C4BP. Upper panel: OF⁺ strains. Lower panel: OF⁻ strains. Only strains that bound Fg, as determined in parallel tests, were used for the analysis because binding of Fg is a characteristic property of S. pyogenes isolates expressing members of the M protein family. Binding is expressed as percent of added radioactivity. The threshold for binding of C4BP was set at ≥10% binding (dashed line). Background binding to an M-negative strain (~3%) has been subtracted. All strains were tested at least twice, with duplicate samples, and the results were highly reproducible. For each strain, data from one experiment are shown. The data include binding data for one allelic variant per M type, except that data for both M4 and M4.1 are included. For strains of some M types, several allelic variants were tested and in most cases these strains did not differ in ability to bind C4BP (unpublished data).</p

Additional file 5: Figure S4. of HOXC8 regulates self-renewal, differentiation and transformation of breast cancer stem cells

Sequencing chromatograms of bisulfite converted HOXC8 CpG island PCR products. Bisulfite converted DNA extracted from CSC was amplified by PCR and then directly sequenced. Chromatograms show the average DNA methylation of the PCR products. CG residues are circled. A TG peak indicated unmethylated C, T/G peak indicates partially methylated C, CG peak indicates methylated C. (PDF 311 kb

Additional file 1: Table S1. of HOXC8 regulates self-renewal, differentiation and transformation of breast cancer stem cells

Primers and assays used in the study. (DOC 41 kb

Additional file 4: Figure S3. of HOXC8 regulates self-renewal, differentiation and transformation of breast cancer stem cells

HOXC8 copy number variation in the breast TCGA and METABRIC studies. Bar plots and scatter plots representing the copy number variation and correlation with gene expression in patient samples from the TCGA and METABRIC datasets [29, 30, 33, 34]. (PDF 271 kb

Additional file 6: Figure S5. of HOXC8 regulates self-renewal, differentiation and transformation of breast cancer stem cells

HOXC8 overexpression in TN and HER2-E cells. Overexpression of HOXC8 was induced by lentiviral transduction with pSIN-HOXC8 vector and gene expression measured by TaqMan® qRT-PCR. Results are presented as relative fold expression relative to RPLP0 and control (pSIN empty) vector used as calibrator (n = 3). Relative fold expression levels were analysed by Unpaired Student t-test. ***P < 0.001. Bottom panel represents Western Blotting of nuclear lysates from cells transduced with pSIN-HOXC8 overexpressing vector or control vector was conducted to detect the expression of HOXC8 (34 kDa) and LAMIN A/C (41-50 kDa) as loading control. (PDF 92 kb