Phagocytosis and survival of GAS clinical isolates in BMDMs.

<p>Cells were infected with GAS as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101464#s2" target="_blank">Material and Methods</a>. (<b>A</b>) Percentage of phagocytosis of all (black bars), invasive (black symbols) and non-invasive (white symbols) GAS strains of different <i>emm</i> types. The results are expressed as the percentage of bacterial CFUs recovered after 30 min post-antibiotics treatment relative to the initial inoculum. (<b>B</b>) Bacterial survival experiments were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101464#s2" target="_blank">Material and Methods</a> and expressed as the percentage of phagocytosed bacteria that survived. (<b>C</b>) Intracellular bacteria were not killed by extracellular antibiotics. Cells were infected with GAS as described and after washing, either medium with antibiotics (ATB) (black) or medium alone (dash) was added to cells. The results represent the mean ± SD of three independent experiments, with significance levels indicated between given isolates from the same <i>emm</i> type or in between <i>emm</i> types (*p≤0.05; **p≤0.01; ***p≤0.005).</p

GAS clinical isolates induced pro-inflammatory mediator and IFN-β secretion by infected BMDMs.

<p>Graphics represent IL-6, TNF-α and IFN-β quantification in the cell culture supernatant at T2h (<b>A</b>), T4h (<b>B</b>) and T6h (<b>C</b>) after infection by the different <i>emm</i> type isolates. The mean values immune mediator productions induced by all (black bars), invasive (black symbols) and non-invasive (white symbols) of isolates of each <i>emm</i> type are shown. To note, the scales are different, within the IL-6 data in panel A and, within the IFN-β data in panel C. The results represent the mean ± SD of 3 independent experiments; with significance levels indicated between <i>emm1</i> and other <i>emm</i> types by stars above the corresponding black bar and within <i>emm</i> types by stars above a line overlapping the corresponding the black and white symbols (*p≤0.05; **p≤0.01; ***p≤0.005).</p

Reassociation capacity of rGAPDH.

<p>A) ELISA plates were coated with 6.5×10⁷ CFU/well of heat inactivated NEM316 overnight at 4°C. They were incubated with 100 µL/well of different amounts of rGAPDH (0, 10 or 50 µg/mL) or rGAPDH 50 µg/mL with 75 µg of rabbit anti-rGAPDH IgG pAb (50+αGAPDH). rGAPDH was detected using a HRP coupled mouse pentaHis antibody. After revelation, the OD₄₅₀ was registered using a microplate reader. B) 200 µg of rGAPDH (+rGAPDH) or PBS (−rGAPDH) was added to an exponentially grown culture of several GBS strains belonging to different serotypes (Ia, Ib, II, III, IV, V, VI, and VII), <i>S. pyogenes</i>, <i>L. lactis</i>, <i>S. aureus</i>, and <i>E. coli</i>. After incubation, total proteins extracts were subjected to SDS-PAGE followed by transfer to nitrocellulose membrane (rGAPDH: 10 ng; Total proteins +/− rGAPDH: 8 µg). This membrane was incubated with an HRP-coupled mouse pentaHis antibody or rabbit anti-SodA antibody (loading control) followed by HRP-conjugated goat anti-rabbit secondary antibody. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029963#s2" target="_blank">Results</a> shown are representative of two independent experiments. **, P<0.01; ***, P<0.001.</p

Comparative expression of the gene (<i>spb1</i>) encoding the major PI-2b pilin in <i>S</i>. <i>agalactiae</i> strains BM110 (ST17) and A909 (non ST17).

<p>(A) Schematic representation of the PI-2b pilus operon. Arrows represent coding sequence and the direction of transcription. Genes encoding pilus structural proteins are shown in purple, those encoding sortases are in brown, <i>lep</i> encodes a putative signal peptidase and <i>orf</i> codes for a conserved hypothetical protein. Gene nomenclature is according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169840#pone.0169840.ref011" target="_blank">11</a>]. (B) Immunofluorescence microscopy of <i>S</i>. <i>agalactiae</i> strains using anti-Spb1 antibody. (C) Flow-cytometry analysis of Spb1 expression in WT and isogenic <i>Δbp</i> strains. (D) Transcriptional analysis of <i>spb1</i> gene by quantitative RT-PCR in exponentially growing <i>S</i>. <i>agalactiae</i> cells using <i>gyrA</i> as an internal standard. Results are expressed as the n-fold change with respect to the WT strain BM110 whose value has been set arbitrarily to 1. Results are means +/- SD from at least two independent cultures in triplicates. Asterisks represent P values (*P<0.05) evaluated using a Student's <i>t</i> test.</p

Induction of apoptosis mediated by (r)GAPDH.

<p>A) 5×10⁵ RAW264.7 murine macrophages were left untreated (Medium) or treated for 24 h with 1 µM staurosporin or 50 µg/mL rGAPDH, or 200 µL of culture supernatant from GBS, <i>S. pyogenes</i>, <i>L. lactis</i> or <i>S. aureus</i> left untreated (unt) or immunoprecipitated with rabbit anti-GAPDH IgG pAb (ipGAPDH), with rabbit anti-SodA IgG pAb (ipSodA) or with a normal rabbit IgG (control IgG). B) 5×10⁵ Bone marrow-derived macrophages from C57BL/6 mice were left untreated (Medium) or treated for 24 h with 1 µM staurosporin, or rGAPDH at the indicated concentrations. After treatment, the macrophages were fixed and stained for TUNEL using the Promega DeadEnd™ Fluorometric TUNEL kit. Samples were acquired and the % of cells that incorporated fluorescein-dUTP (TUNEL positive cells) was quantified. C) For IF, 1×10⁵ C57BL/6 bone marrow-derived macrophages were left untreated or treated for 24 h with 50 µg/mL rGAPDH. After that period, cells were fixed and stained as mentioned in B) and visualized in a fluorescence microscope. Scale bars, 15 µm. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029963#s2" target="_blank">Results</a> shown are representative of two independent experiments. ns, non-significant; *, P<0.05; **, P<0.01.</p

Transcriptional start site of the PI-2b operon.

<p>(A) Characterization of <i>PI-2b</i> transcription start site in <i>S</i>. <i>agalactiae</i> strains A909 and BM110 by dRNA-seq. The sequence reads corresponding to transcript 5' ends generated after (TAP+) or without (TAP-) TAP treatment or obtained from strand-specific RNA-seq (RNA-seq) were aligned to the genomes of strains A909 and BM110. A significantly higher number of reads under TAP+ conditions as compared with TAP- is indicative of 5' -triphosphate ends of transcripts characteristic of transcription start sites (TSS). Identical TSS upstream PI-2b locus are detected in strains A909 and BM110. In addition, coverage of the intergenic regions between <i>sak1445 (or san1522)</i> and <i>orf</i> under conditions of RNA-seq experiments reveals a transcriptional read-through originating from <i>sak1445</i>, in A909 only, that could participate to the global level of <i>PI-2b</i> transcription in A909. (B) Primer extension analysis of the PI-2b mRNA. Primer elongation product obtained with oligonucleotide E3 and 15 μg of total RNA from A909 (lane1) or BM110 (lane 2). Lanes T, G, C, A, results of sequencing reactions performed with primer E3. Arrow indicated the transcriptional start site. (C) Schematic representation and genomic DNA sequence, from nucleotide positions -281 to +3 (numbering from the A of the ATG start codon of <i>orf</i>, negative in the -3'-to-5' direction and positive in the 5' to-3' direction) of the region upstream from the PI-2b locus. Transcription start site is indicated in boldface and arrow. Consensus -10 and -35 sequences are indicated by grey boxes. The 43-bp sequence, present in BM110 and absent in A909, is underlined. The ATG start codon of <i>orf</i> is indicated in boldface.</p

Release of cytoplasmic proteins upon induced bacterial lysis.

<p>A, B) NEM316 WT or SodA mutant cultures were treated (+) with 15 U/mL mutanolysin and 0.1% Triton X-100 or left untreated (−). The amount of surface GAPDH or SodA was determined by FACS with a rabbit IgG anti-rGAPDH pAb or anti-SodA followed by AlexaFluor 488-conjugated anti–rabbit antibody (A). The graphic expresses the fold change in MFI after treatment. B) Western Blot analysis of GAPDH and SodA proteins from NEM316 WT or SodA mutant detected using a rabbit anti-rGAPDH IgG pAb or rabbit anti-SodA followed by HRP-conjugated goat anti-rabbit antibody. rGAPDH: 10 ng; supernatants from untreated bacteria: 25 µL; supernatants from Triton-treated bacteria: 1 µL. Numbers in the western blot represent the fold increase of GAPDH or SodA amount after Triton treatment, as determined densitometrically and corrected for sample amount. C, D) 5×10⁶ CFU/mL of NEM316 were treated for 12 h with Penicillin G (+PenG) at 100× MIC (6.4 µg/mL) or left untreated. The bacteria were then subjected to FACS analysis as previously described (C). The numbers in the histogram represent the fold change in MFI after the treatment with PenG. D) Western blot analysis of GAPDH protein from NEM316 WT detected using a rabbit anti-GAPDH IgG pAb followed by HRP-conjugated goat anti-rabbit antibody. rGAPDH: 10 ng; supernatants: 25 µL. Numbers in the western blot represent the fold increase of GAPDH amount after PenG treatment, as determined densitometrically. E,F) GBS WT and PilB⁻ strains containing the inducible vector pMSP3545 empty (pMSP Ø) or encoding <i>gbs0093</i> (pMSPΩ<i>gbs0093</i>) were subjected to overnight induction with 20 ng/µL nisin after reaching an OD₆₀₀ = 0.3. After this period, the amount of surface exposed GAPDH was quantified by FACS using anti-GAPDH antibodies (E) and 15 µL of supernatant extracts were analyzed for the presence of GAPDH, SodA, and CAMP by western blot (F). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029963#s2" target="_blank">Results</a> shown are representative of at least three independent experiments. **, P<0.01.</p

Quantification of bacterial lysis.

<p>A) 10 µL of bacterial culture of GBS WT, PilB⁻ and PilA/C⁻ were stained with Syto9 and propidium iodide (PI). The bacterial suspensions were then subjected to FACS and counted using the LIVE/DEAD® <i>Bac</i>Light™ kit. B) OD_{600 nm} measurement of GBS or <i>S. aureus</i> suspensions in PBS at 37°C over 5 h. The graphic expresses the % in OD in different time points relative to the time 0. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029963#s2" target="_blank">Results</a> shown are representative of two independent experiments. *, P<0.05.</p

Evaluation of surface-bound and extracellular GAPDH levels in GBS strain NEM316 (WT) and pilus mutant strains.

<p>A) Immunoelectron microscopy (IEM) picture of the surface-bound GAPDH in NEM316. Scale bar, 100 nm. B) Fluorescence-activated cell sorter analysis (FACS) of WT, PilB⁻ and PilA/C⁻ incubated with a polyclonal rabbit anti-GAPDH IgG (grey filled histogram for WT or black line histogram for PilB⁻ and PilA/C⁻ strains) or normal rabbit IgG (control IgG-grey dotted histogram). Mean Fluorescence Intensity (MFI) in lower panel is reported relative to control IgG. C) Western Blot detection of GAPDH in culture supernatants (SN) of exponentially growing WT, PilB⁻ and PilA/C⁻ strains. 25 µL of each concentrated supernatant, 10 ng of rGAPDH or 2 µg of WT total proteins were loaded in the gel. After transfer to a membrane, proteins were detected using a polyclonal rabbit anti-rGAPDH IgG antibody, rabbit anti-SodA antibody and rabbit anti-CAMP antibody followed by HRP-conjugated goat anti-rabbit antibody. D) Quantifications of GAPDH relative to the loading control protein (CAMP) in the supernatant of WT, PilB⁻ and PilA/C⁻ strains, were performed with ImageJ software. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029963#s2" target="_blank">Results</a> shown are representative of three independent experiments. *, P<0.05.</p

Characterization of PI-2b promoter region using transcriptional fusions with <i>gfp</i> as a reporter gene.

<p>(A) Schematic representation of the region upstream from the PI-2b locus. The putative +1 transcriptional start site detected by primer extension is indicated. Length of the intergenic region is indicated at the bottom. The four pTCV plasmids (pTCV1 to pTCV4) are drawn schematically. The size of the PI-2b fragment inserted upstream of the reporter gene is indicated on the left. (B) Flow-cytometry analyses of GFP expression in <i>S</i>. <i>agalactiae</i> NEM316 (ST-23) (left part) or in the heterologous <i>Lactococcus lactis</i> strain NZ9000 harboring the various pTCV1-4 plasmids (right part). Left, Black NEM316 + pTCV4; blue, NEM316 + pTCV2; green, NEM316 + pTCV1; orange, NEM316 + pTCV3; Right, Black NZ9000 + pTCV4; blue, NZ9000 + pTCV2; green, NZ9000 + pTCV1; orange, NZ9000 + pTCV3. (C) Role of CovR on PI-2b pilus expression in <i>S</i>. <i>agalactiae</i>. Left panel, Flow-cytometry analysis of GFP expression in NEM316 WT (black) and isogenic <i>ΔcovR</i> (orange) and CovRD53A (blue) strains harboring pTCV3. NEM316 carrying pTCV2 (grey) was used as a negative control. Right panel, transcriptional analysis of <i>san1522</i> (AgB locus, according to COH1 annotation) and PI2b-<i>orf</i> by quantitative RT-PCR in exponentially growing <i>S</i>. <i>agalactiae</i> cells using <i>gyrA</i> as an internal standard. Results are expressed as the n-fold change with respect to the WT strain BM110 whose value has been set arbitrarily to 1. Results are means +/- SD from at least two independent cultures in triplicates. Asterisks represent P values (****P ≤ 0.001, ns for non- significative) evaluated using a Student's <i>t</i> test.</p