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

Distribution of NHBA peptides in the 300 isolates panel.

<p>Others^a: NHBA peptides not present in more than one isolate (peptides 8, 9, 13, 25, 47, 53, 115, 160, 187, 237, 304, 307, 308, 309, 310, 355, 367, 368, 370, 385, 460, 461, 462, 463, 464, 465, 468, 469, 470, 471). Others cc^b: Others clonal complexes. NA^c: Clonal complexes non assigned.</p

CC156 strain panel used in this study.

<p>For each strain name, ST, serotype/serogroup, country of isolation, number of MLST alleles in common with ST156 and ST4945, data source, strain source and lineage (as identified by 96-MLST hierarchical clustering, <i>see</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061003#pone-0061003-g002" target="_blank">Figure 2</a>) are indicated.</p

Sequence Analysis of 96 Genomic Regions Identifies Distinct Evolutionary Lineages within CC156, the Largest <i>Streptococcus pneumoniae</i> Clonal Complex in the MLST Database

<div><p>Multi-Locus Sequence Typing (MLST) of <i>Streptococcus pneumoniae</i> is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize <i>S. pneumoniae</i> strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.</p> </div

Distribution of MATS strains predicted to be covered by cc and specific vaccine antigen combinations.

<p>Strains were defined as covered if they possessed PorA P1.4 or had relative potency greater than the positive bactericidal threshold for fHbp, NHBA or NadA. *NA: Clonal complex non assigned.</p

Distribution of fHbp variant families and peptides in the 300 isolates panel.

<p>Others¹: FHbp variant family 1 peptides not present in more than three isolates (peptides 4, 15, 35, 37, 54, 65, 90, 87, 108, 110, 144, 213, 218, 236, 252,275, 322, 357, 358, 359, 360, 361, 362, 363, 365, 373, 374, 403, 456, 480, 544, 545). Others²: FHbp variant family 2 peptides not present in more than three isolates (peptides 18, 24, 34, 104, 367, 548, 551). Others³: FHbp variant family 3 peptides not present in more than three isolates (peptides 29, 31, 174, 188, 294, 364, 366, 368, 398, 399, 400,401,402, 485, 494, 532, 536, 549, 550, 552, 555). Others cc*: Others clonal complexes, included clonal complexes non assigned.</p

Distribution of clonal complexes and main PorA genotypes in the 300 isolates panel.

<p>*NA: Clonal complex non assigned.</p

Distribution of NadA variants and peptides in the 300 isolates panel.

<p>Frameshift¹: The <i>nadA</i> nucleotide allele presents a frameshift mutation resulting in a premature stop codon (variant 1 allele 44; variant 4/5 alleles 12, 34, 38, 39, 40 and 46; variant 6 alleles 37, 43 and 94). IS1301²: The <i>nadA</i> gene (allele 50) is disrupted by an insertion sequence IS1301. NA³: Clonal complex non assigned.</p

Minimum Spanning Tree analysis based on 96-MLST allelic profiles identifies seven distinct lineages by imposing a maximum threshold of 75 different loci.

<p>The Minimum Spanning Tree analysis was performed by using PHYLOVIZ on the 96-MLST alleles of the 66 strains considered in this study. The lineages identified by applying the threshold of 75/96 different loci are highlighted with shadowed shapes and named according to the lineage identification of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061003#pone-0061003-g002" target="_blank">Figure 2</a>.</p

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