Additional file 2: Table S2. of Genome-wide identification of transcriptional start sites in the haloarchaeon Haloferax volcanii based on differential RNA-Seq (dRNA-Seq)

Containing the dRNA-Seq results. Summary of all of the TSSs identified by the dRNA-Seq approach and the additional selected parameters, e.g., replicon, localization, strand, identification in the three datasets, TSS class, associated gene, and promoter score. The first Excel sheet contains the results, and the second Excel sheet contains a list of columns and definitions. (XLSX 572 kb

<i>S</i>. <i>pneumoniae</i> serotypes differentially induce PLY- and caspase-1-dependent production of IL-1β by PBMCs as well as cell death.

<p>(A-C, H-L) PBMCs were infected with <i>S</i>. <i>pneumoniae</i> strains (MOI = 1) as indicated or (D, E) were infected with <i>S</i>. <i>pneumoniae</i> D39 (MOI = 0.01) and treated with 10 μM Z-YVAD. Production of IL-1β (A, D, H) and IL-8 (C, E, J) was quantified by ELISA after 16 h. (D, E) 100% were defined as the amount of IL-1β or IL-8 released by <i>S</i>. <i>pneumoniae</i>-infected PBMCs. (B, I) Relative expression of <i>Il1b</i> was determined by quantitative RT-PCR after 5 h. (F) Human blood was incubated with pneumococcal lysates of <i>S</i>. <i>pneumoniae</i> serotypes 3, 6B, 7F, 9N, 1 and 8, and haemolytic activity was assessed. (G) Comparative protein model of allele 5 PLY. PLY domains are colour coded and mutations are marked in rose. (K) PBMCs were infected with <i>S</i>. <i>pneumoniae</i> D39 and D39Δ<i>ply</i>. (L) PBMCs were infected with serotypes 3, 6B, 7F, 9N, 1 and 8 pneumococci. (M) PBMCs were stimulated with allele 1 and allele 5 PLY for 16 h. (K-M) LDH release was quantified by cytotoxicity assay. Data are shown as mean ± SEM of three (D, E, M), five (B, I) or seven (A, C, H, J, K, L) independent experiments carried out in duplicates or triplicates. Significance is indicated by asterisks * = p<0.05, ** = p<0.01, *** = p<0.001.</p

The production of IL-1β in <i>S</i>. <i>pneumoniae</i>-infected human lung tissue is dependent on PLY and caspase-1.

<p>(A-E) Human lung tissue was infected with 1x10⁶ CFU/mL <i>S</i>. <i>pneumoniae</i> serotype 2 D39 or D39Δ<i>ply</i> and treated with 100 ng/ml Z-YVAD 1 hour before infection where indicated. After 24 h, production of IL-1β (A, B, E) was quantified by ELISA, relative expression of <i>Il1b</i> was determined by qRT-PCR (C), or expression of pro-IL-1β was assessed by immunoblotting (D). Data are shown as mean ± SEM of three (A), five (E) or six (B, C) independent experiments carried out in duplicates. (D) One representative of four independent experiments is shown. Significance is indicated by asterisks ** = p<0.01, *** = p<0.001.</p

Pneumolysin allele determination by sequencing of <i>ply</i> genes was performed from the 6 isolates used in this study.

<p>The first row refers to amino acid positions. The second row shows allele 1 PLY expressed in <i>S</i>. <i>pneumoniae</i> D39 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137108#pone.0137108.ref021" target="_blank">21</a>]. The amino acid polymorphisms in the clinical isolates are highlighted. Deletion of an amino acid is represented by the abbreviation DEL. Serotype 1 and 8 pneumococci express an allele 5 PLY as previously shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137108#pone.0137108.ref021" target="_blank">21</a>].</p

Pneumococci expressing haemolytic but not non-haemolytic PLY stimulate IL-1β production in human lung tissue.

<p>(A) Human lung tissue was infected with 1x10⁶ CFU/mL <i>S</i>. <i>pneumoniae</i> serotypes 3, 6B, 9N, 1 and 8, or (B) were stimulated with 1μg/ml allele 1 and allele 5 PLY for 24 h. IL-1β secretion was quantified by ELISA. Data are shown as mean ± SEM of eight (A) or six (B) independent experiments carried out in duplicates. Significance is indicated by asterisks *** = p<0.001.</p

NLRP3 expression is up-regulated in human lung tissue during <i>S</i>. <i>pneumoniae</i> infection.

<p>(A, B) Human lung tissue was infected with 1x10⁶ CFU/mL <i>S</i>. <i>pneumoniae</i> serotype 2 D39 or D39Δ<i>ply</i> and expression of NLRP3 was assessed by immunoblotting. (C) NLRP3 inhibitor glybenclamide efficiently reduced IL-1β secretion in human lung tissue during <i>S</i>. <i>pneumoniae</i> infection. Human lung tissue was preincubated with the inhibitor for 2 h and infected with 1x10⁶ CFU/mL <i>S</i>. <i>pneumoniae</i> D39 for 16 h. IL-1β production was quantified by ELISA. Data are shown as mean ± SEM of three (A, B) or six (C) independent experiments carried out in duplicates. Significance is indicated by asterisks *** = p<0.001.</p