Additional file 1 of Ex-vivo RNA expression analysis of vaccine candidate genes in COPD sputum samples

Additional file 1: Table S1. PCR primer sequences for NTHi and Mcat genes. Figure S1. NTHi gene RNA concentrations in NTHi positive samples did not differ between stable visits (ST) and exacerbation visits (EX). Figure S2. Mcat gene RNA concentrations in Mcat positive samples did not differ between stable visits (ST) and exacerbation visits (EX). Additional methods

Transcriptome changes elicited by glucose in GBS.

<p>Comparison of gene expression changes (log2) between mid-log cultures of 2603 V/R wild-type strain and <i>CovRS</i> mutant following challenge with or without glucose. Data for the wild type and mutant strains are shown on the <i>x</i> axis and <i>y</i> axis, respectively.</p

CovR binds to <i>bibA</i> promoter <i>in vivo</i>. (A)

<p>Quantification by qRT-PCR of <i>bibA</i> promoter immunoprecipitated with CovR antiserum in 2603 V/R wild type strain grown in medium devoid of glucose or in the presence of 55mM glucose. <i>cfb</i> promoter and <i>cylX</i> promoter were used as a positive control while <i>sag0017</i> promoter was used as a negative control. The level of PCR products of eluate from the isogenic Δc<i>ovRS</i> deletion mutant grown with or without glucose was negligible. The data are representative of 3 independent experiments, each in triplicate. Error bars, SD. (<b>B)</b> Competitive EMSA experiment. Labelled <i>PbibA</i> fragment (3.3 nM) was incubated without <i>(lane1)</i> or with CovR (2 µM) <i>(lane2</i>–<i>6)</i>, in the presence of different amounts of unlabelled <i>PbibA (lane 3</i>–<i>4)</i>, as a specific competitor, and <i>Psag0017 (lane5</i>–<i>6)</i>, as a non-specific competitor. The labelled DNA was detected by chemioluminescence. <b>(C)</b> CovR phosphorylation increases its affinity for <i>bibA</i> promoter. Electrophoretic mobility shift assay using recombinant CovR (left) and chemically phosphorylated recombinant CovR (right). Labelled <i>PbibA</i> DNA fragment (3.3 nM) was incubated without or with the indicated amounts of CovR. The labelled DNA was detected by chemioluminescence.</p

Graphical representation summarizing adaptive regulation of GBS in high glucose conditions.

<p>Genes of interest are color-grouped according to main functional categories. Arrows indicate up- or down-regulation relative to time of 30′ in high glucose <i>vs.</i> no glucose.</p

Real-time RT-PCR evaluation of <i>bibA</i> expression in 2603V/R and Δc<i>ovRS</i> strains grown in medium containing 55 mM glucose or in sugar-free medium.

<p>Transcript levels were normalized to the expression level of <i>gyrA</i>. Syber green runs were performed with cDNAs from the same reverse transcription reaction from 1 µg of total RNA. The ΔΔCT method was applied as a comparative method of quantification, using strains grown in sugar free medium as reference. The data are representative of 2 independent experiments, each in triplicate. Error bars, SD.</p

List of genes highly regulated in GBS strain 2603V/R after incubation in high glucose medium.

<p>List of genes highly regulated in GBS strain 2603V/R after incubation in high glucose medium.</p

Differential regulation of gene expression in GBS strain 2603 V/R versus the isogenic Δ<i>CovRS</i> mutant strain after incubation in medium with 55 mM glucose versus a sugars-free complex medium.

<p>White bars indicate the number of glucose-regulated genes in the wild-type strain; black bars indicate the number of genes that are glucose- dependent and CovRS-dependent; grey bars indicate the number of genes that are glucose-dependent and CovRS-independent.</p

Anti-CnaBE3 domain antibodies recognize all the three Sdr full length proteins.

<p>Western blot analysis of bacterial cell wall extracts from NCTC8325, Newman, MSSA476, MW2, N315, Mu50, Mu3, USA300 FPR3757, MRSA252, TW20, and MN8 <i>S. aureus</i> strains blotted with anti-CnaBE3 domain antibodies. The Sdr proteins of each strain are highlighted.</p

Schematic representation of Sdr proteins and amino acid sequence of CnaBC2 CnaBD5 and CnaBE3 domains.

<p>A) Schematic representation of Sdr proteins. A putative leader peptide (LP) sequence and an LPXTG motif are depicted in black. The A domain is reported in light gray, whereas B repeats (two, three, or five, for SdrC, SdrE, and SdrD, respectively) are shown in white, and contain putative CnaB domains shown in dark gray. Finally, at the C-terminus, the SD repeat domain is depicted in dark gray. In addition, boundaries of CnaBC2, CnaBD5 and CnaBE3 domains are reported. B) CnaBC2, CnaBD5 and CnaBE3 domain amino acid sequences are aligned. Identical residues are highlighted, and putative CnaB domains are encompassed by a black box.</p

CnaBE3 domain sequence is highly conserved among phylogenetically distinct strains.

<p>A) The depicted phylogenetic tree was obtained using the Sequence Types (ST) of a panel of 59 epidemiologically relevant <i>S. aureus</i> strains. Clonal complexes 1, 5, 8 and 30 are highlighted. The eleven bacterial strains selected for conservation analysis are in bold. B) The percentages of amino acid sequence identity obtained from the comparison of Sdr proteins and the CnaBC2, D5 and E3 domain amino acid sequences of Newman strain to those of an epidemiologically relevant panel of <i>S. aureus</i> strains are reported. Dark gray color means that proteins are absent, whereas white color means present and conserved with an identity percentage ≥ 90, and light gray color means present but variable with an identity percentage ≥ 75 and ≤ 89, on at least 75% of the amino acid sequence.</p