36 research outputs found

    Uptake of <i>S. gallolyticus</i> UCN34 WT, Δ<i>pil1</i>, and Δ<i>term</i> (Pil1+<sub>locked</sub>) strains by human THP-1 macrophages.

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    <p>(A) Indirect immunofluorescence microscopy showing phagocytosis of Δ<i>pil1</i> or Δ<i>term</i> (Pil1+<sub>locked</sub>) mutants by THP-1 macrophages in the presence of anti-Pil1 antibody (opsonophagocytic assay). Actin is depicted in grey-purple, Hoeschst 33342 in blue, intracellular bacteria in green, and extracellular bacteria in yellow (green+red). (B) Percentage of phagocytosis by THP-1 human macrophages after 1 h of infection with WT, Δ<i>pil1</i>, and Δ<i>term</i> (Pil1+<sub>locked</sub>) strains, with or without an anti-Pil1 antibody. Values are representative of at least 4 independant experiments. (C) Flow-cytometry analysis of Pil1 expression performed on the UCN34 WT from the inoculum (grey) or from intracellular UCN34 bacteria recovered from THP-1 macrophages in an opsonophagocytic assay with an anti-PilB pAb (black lines are 3 replicates from one experiment).</p

    Whole blood survival of <i>S. gallolyticus</i> UCN34 WT, Δ<i>pil1</i>, and Δ<i>term</i> (Pil1+<sub>locked</sub>) strains.

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    <p>Exponentially growing bacteria were added to whole blood from three different human donors (10<sup>4</sup> bacteria in 100 µl PBS were added to 400 µl of human blood drawn fresh in eppendorf tube, incubated on a wheel at 37°C and plated for enumeration at 1 h, 2 h and 3 h time points. (A) Plating on TH agar plates of Δ<i>pil1</i> and Δ<i>term</i> (Pil1+<sub>locked</sub>) strains after 3 h of incubation in whole blood at 37°C. The petri plate sectors represent three replicates of a bacterial survival assay with the blood of one donor at 2 h. (B) Percentage of whole blood survival for the three strains WT, Δ<i>pil1</i>, and Δ<i>term</i> (Pil1+<sub>locked</sub>) after 3 h of incubation. Values are represented as means +/− standard deviations of three independent experiments. Each experiment was performed in triplicate. Asterisks represent P values (** <i>P</i><0. 05) evaluated using a Mann-Whitney test.</p

    The <i>pil1</i> locus of <i>S. gallolyticus</i> strain UCN34.

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    <p>(A) Schematic representation of the <i>pil1</i> operon constituted of three genes: <i>pilA</i>, encoding the pilus adhesin, <i>pilB</i> encoding the major pilin and <i>srtC</i>, encoding the sortase required for pilus polymerization. Relevant domains are: COL, collagen-binding domain (Pfam 05737); and LPXTG, cell wall-anchoring domain. The P<i>pil1</i> promoter, the premature terminator upstream <i>pilA</i> and the putative terminator (hairpin structures) downstream <i>srtC</i> delineating the <i>pil1</i> operon are indicated. (B) Schematic representation of the intergenic region <i>gallo2180-pilA</i> with the 22 GCAGA repeats striped in gray and white and the stem-loop structure. (C) Determination of transcription start sites (TSS) of the <i>pil1</i> operon by primer extension analysis. The band indicated as “+1” corresponds to the location upstream the leader peptide gene is the likely TSS of the <i>pil1</i> operon; the band marked as “*” is located within one inverted repeat of the stem-loop structure and is likely generated by a pause of the RNA polymerase during elongation. (D) Nucleotide sequence of the intergenic region <i>gallo2180-pilA</i> showing the -35 and -10 promoter boxes and the +1 site, the leader peptide coding sequence with the 22 GCAGA repeats, the downstream stem-loop structure, the <i>pilA</i> initiator codon and the predicted ribosome binding site (RBS). (E) The predicted secondary structure of the stem-loop transcription terminator identified upstream ATG<i><sub>pilA</sub></i> (according to The mfold WebServer, <a href="http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form" target="_blank">http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form</a>).</p

    Uncoupling transcription and translation in the <i>pil1</i> operon.

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    <p>Quantitative RT-PCR were performed on RNAs extracted from UCN34 WT and Pil1+<sub>var23</sub> variant grown without or with chloramphenicol (8 µg/mL) to induce ribosome stalling in leader peptide mRNA. The expression levels were normalized using 16S rRNA as an internal standard and are indicated as the n-fold change with respect to untreated WT strain UCN34, expressed as means and standard deviations of at least three separate experiments. The gene <i>tanA</i> was used as a reference gene whose transcription is not affected by addition of chloramphenicol. Asterisks represent P values (*<i>P</i><0.05) evaluated using a Student <i>t</i> test. The location in the <i>pil1</i> operon of the four primer pairs used is indicated at the bottom.</p

    Model of pilus regulation by an attenuation-like mechanism.

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    <p>(A) <i>S. gallolyticus</i> UCN34 WT displays a heterogeneous <i>pil1</i> expression and consists of two main subpopulations, a majority of low piliated cells (Pil1<sub>low</sub>, 70%) and a minority of hyper piliated cells (Pil1<sub>high</sub>, 30%). The Pil1<sub>low</sub> cells, characterized by a basal expression of <i>pil1</i>, possess a regulatory leader peptide-encoding gene with 22 GCAGA repeats that ends 39 bp upstream the hairpin transcription terminator. In-frame addition/deletion of GCAGA did not modify the distance between the leader peptide stop codon and the terminator. In this case, most transcripts initiated at P<i>pil1</i> promoter end at this premature terminator and the observed low expression of <i>pil1</i> probably occurs by readthrough transcription. The Pil1<sub>high</sub> cells, characterized by a strong expression of <i>pil1</i>, displayed out-of-frame addition/deletion of repeats resulting in the synthesis of longer regulatory peptides whose stop codon are located within or dowstream of the hairpin terminator. Translation of these long regulatory leader peptides enhances <i>pil1</i> genes transcription by preventing the formation of the transcription terminator. (B) Flow cytometry analysis of UCN34 WT and isogenic mutant derivatives (Δ<i>term</i>, ATG*, and 3 STOPs) labeled with anti-PilB pAb. The WT and mutant profiles are depicted by gray area and black lines, respectively. Note that, as expected, the UCN34 and “back to the WT” profiles, depicted by dotted lines, are almost entirely superimposable. (C) Immunolabeling screening of the Pil1+<sub>var23</sub> and Δ<i>term</i> strains with anti-PilB pAb. As opposed to the Pil1+<sub>var23</sub> variant, the Δ<i>term</i> mutant is homogeneous and only generates Pil1+ colonies. This is the consequence of the deletion of the transcription terminator that blocks this strain in the Pil1<sub>high</sub> configuration. (D) Quantitative RT-PCR were performed on RNAs extracted from WT UCN34 and mutant derivatives using pilB primers. The expression levels were normalized using 16S rRNA as an internal standard and are indicated as the n-fold change with respect to WT UCN34, expressed as means and standard deviations of at least three independent experiments with four technical replicates. Asterisks represent P values (** <i>P</i><0.05) evaluated using a Student <i>t</i> test.</p

    Isolation, phenotypic analyses and sequencing of Pil1+ variants.

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    <p>(A) Detection of Pil1+ variants by colony blot using anti-PilB pAb. Isolated bacteria on a TH agar plate (left) and corresponding replicate hybridized with anti-PilB pAb (right). Arrowheads indicate the few variants displaying a Pil1+ phenotype. (B) Serial analysis of Pil1 expression in WT and Pil1+<sub>var</sub> populations. Individual colonies of WT UCN34 strain isolated on TH agar plates were grown in 96 well plates, transferred to a Nylon membrane, and probed with anti-PilB. Analysis of one Pil1+ variant revealed that it subsequently generates a majority of Pil1+ colonies (90–95%) and a minority of Pil1<sub>low</sub> cells (5–10%) thus displaying phenotypic pilus heterogeneity as the WT UCN34. (C–F) Phenotypic comparison of WT UCN34 and Pil1+<sub>var23</sub> variant. Scanning immunogold electron microscopy (C) and flow cytometry (D) analyses of bacteria labeled with anti-PilB pAb. Western blot analysis (E) of cell wall protein extracts revealed with anti-PilB pAb. Quantitative RT-PCR analysis (F) of RNAs extracted from exponentially growing <i>S. gallolyticus</i> cells. The expression levels were normalized using 16S rRNA as an internal standard and are indicated as the n-fold change with respect to the WT strain UCN34, expressed as means and standard deviations of at least three independent experiments with four technical replicates. Asterisks represent P values (*<i>P</i><0.05) evaluated using a Student <i>t</i> test. The location in the <i>pil1</i> operon of the four primer pairs used is indicated at the bottom. (G) Sequence and flow cytometry analyses of WT UCN34 and Pil1+<sub>var</sub> strains. Frameshift addition/deletion GCAGA repeats in the leader peptide encoding gene did not modify the Pil1 expression profile of UCN34 WT whereas out-of-frame addition were always associated with a Pil1+ phenotype.</p

    Heterogeneous expression of Pil1 pilus at the <i>Streptococcus gallolyticus</i> UCN34 surface.

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    <p>(A–B) Scanning immunogold electron microscopy and immunofluorescence of <i>S. gallolyticus</i> UCN34 strain and <i>L. lactis</i> NZ9000 pOri23Ω<i>pil1</i> strain expressing <i>pil1</i>. Pili were revealed with an anti-PilB polyclonal antibody (pAb) coupled to 10 nm gold beads (A) or the DyLight 488 rabbit secondary antibody (B). (C) Flow cytometry analysis of <i>S. gallolyticus</i> UCN34 cell populations labeled with anti-PilB pAb and the DyLight 488 rabbit secondary antibody. Results are represented as dot plots (left) or as a graph (right). The two groups (left) or peaks (right) in black correspond to the heterogeneous UCN34 WT strain constituted of a weakly piliated subpopulation (Pil1<sub>low</sub> cells) and of a hyper piliated subpopulation (Pil1<sub>high</sub> cells) representing 67% and 28% of the total population respectively. The negative population in gray corresponds to the isogenic Δ<i>pil1</i> mutant (Pil1<sub>neg</sub>).</p

    Heterogeneous expression of Pil1 depends on its 5′ upstream region.

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    <p>(A–D) Flow cytometry profiles of the WT UCN34 strain, the isogenic Δ<i>pil1</i> mutant, and the Δ<i>pil1</i> complemented with pTCV<i>erm</i>-P<i>tet</i>-<i>pil1</i> or pTCV<i>erm</i>-P<i>pil1</i>-<i>pil1</i>. (A) The WT strain displays two cell subpopulations, a majority of Pil1<sub>low</sub> (66%) and a minority of Pil1<sub>high</sub> (27%). (B) The Δ<i>pil1</i> mutant that does not express the Pil1 pilus. (C) The Δ<i>pil1</i> mutant complemented with the plasmid pTCV<i>erm</i>-P<i>tet</i>-<i>pil1</i>, P<i>tet</i> being a constitutive promoter, displays a single population of highly piliated cells. (D) The Δ<i>pil1</i> mutant complemented with the plasmid pTCV<i>erm</i>-P<i>pil1</i>-<i>pil1</i>, P<i>pil1</i> being the entire <i>gallo2180-pilA</i> intergenic region, displays two subpopulations. (C–D) Immunofluorescence staining with anti-PilB pAb (green) confirms that only the P<i>pil1</i> promoter and downstream sequences restored the heterogeneous expression of the Pil1 pilus (Pil1<sub>low</sub>, 76%; Pil1<sub>high</sub>, 16%). Bacteria were stained with DAPI (blue).</p

    SpdC impacts cell wall homeostasis.

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    <p>A. The Δ<i>spdC</i> mutant displays increased resistance to lysostaphin-induced lysis. Cells were grown in TSB until mid-exponential phase, harvested and incubated in PBS with lysostaphin (200 ng/ml) with aeration at 37°C. Bacterial lysis was measured by monitoring OD<sub>600nm</sub> over time. Results are shown as the mean and standard deviation of three independent assays. HG001 parental strain (); Δ<i>spdC</i> mutant (■); Δ<i>spdC</i>/pMK4Pprot-<i>spdC</i> complemented strain (▲). B. The absence of SpdC leads to sensitivity to oxacillin and tunicamycin. Dilution series of the HG001, Δ<i>spdC</i> and Δ<i>spdC</i>/ pMK4Pprot-<i>spdC</i> strains on TSA plates with or without antibiotics. Oxacillin: 0.1 μg/ml; fosfomycin: 4 μg/ml; tunicamycin: 1 μg/ml.</p
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