SARTools workflow.

<p>Left part (black): successive steps of the analysis; Right part (blue): SARTools functions associated with each step when running either DESeq2 or edgeR.</p

Example of target file for a KO vs. WT experiment including 3 replicates in each biological condition.

<p>Example of target file for a KO vs. WT experiment including 3 replicates in each biological condition.</p

Several ways to detect an outlier (d3-WT).

<p>(a) Low total number of reads for d3-WT, (b) High proportion of null counts for d3-WT, (c) Most expressed sequence different for d3-WT, (d) Different distribution of counts for d3-WT, (e-f) d3-WT falls far from the other samples on the first component of the PCA and on the dendrogram.</p

Transcriptional analysis of DLs hosting LV79 amastigotes.

<p>C57BL/6 and DBA/2 BMD-DL cultures were incubated or not with <i>Ds</i>Red2-LV79 amastigotes (MOI of 5∶1) for 24 hours. <b>A</b>) Gating strategy for sorting the DLs that were hosting live amastigotes. The double labelling of i) surface MHC class II molecules-detected by an antibody conjugated to PE-CY5- and ii) intracellular transgenic LV79 expressing the fluorescent <i>Ds</i>Red2 allowed the sorting of DLs hosting live amastigotes (black gate). <b>B, C</b>) Global analysis of modulated transcripts in C57BL/6 and DBA/2 DLs hosting live LV79 amastigotes. Once control DLs and amastigote-hosting DLs were sorted their respective total RNA were prepared and further processed for Affymetrix-based analyses. (<b>B</b>) Representation of the number of specific and common transcripts modulated in live amastigotes-hosting DLs sorted from C57BL/6 and DBA/2 bone marrow cultures. (<b>C</b>) Histogram showing the number of modulated transcripts according to their fold change values.</p

Monitoring of transcript abundance in the C57BL/6 and DBA/2 ear pinnas collected at day 4 and 15 post inoculation/pi of <i>L. amazonensis</i>.

<p>Ten thousand metacyclic promastigotes of <i>L. amazonensis</i> were inoculated into ear pinna dermis of C57BL/6 and DBA/2 mice. At days 7 and 15 pi, the ear pinnas were removed and processed for RNA isolation. Transcript abundance was determined by real-time RT-qPCR. Results are indicated as Fold Changes (FC) unexposed ears being used for calibrator for the calculation of relative expressions. One representative out of 3 independent experiments with three ears that were individually processed the means and SD being displayed.</p

Half of the <i>L</i>. <i>pneumophila</i> proteins are differentially expressed upon CsrA deletion.

<p>Protein intensities in the wt and <i>csrA</i>^<i>-</i> strains (three biological replicates, <i>csrA</i>^<i>-</i> = Mut1-3; wt = WT1-3) were measured by differential shotgun proteomics and visualized in a heat map (left) and a profile plot (right) after non-supervised hierarchical clustering. Every row represents a quantified protein (n = 1448) for which the normalized (LFQ) intensity in each biological replicate is color indicated in the columns.</p

CsrA interacts directly with <i>lqsR</i> mRNA <i>in vitro</i>.

<p><b>A)</b> Electromobility shift assay (EMSA) with 200nM of biotinylated <i>lqsR</i> mRNA combined with varying concentrations of purified CsrA-His <i>lqsR</i> mRNA and recombinant CsrA in 6% Native Tris-PAGE. Lane 1: no CsrA, lane 2: 0.2 μM CsrA, lane 3: 0.5 μM CsrA, lane 4: 1.0 μM CsrA, lane 5: 2.0 μM CsrA, lane 6: 5.0 μM CsrA, lane 7: 5.0 μM CsrA + 2.0 μM unlabled RsmZ. <b>B)</b> Mfold secondary structure prediction of the <i>lqsR</i> mRNA fragment used for the EMSA. Red, two potential CsrA-binding sites, which are mutated in C. Blue, transcriptional start codon. <b>C</b>) EMSA with recombinant CsrA and 200 nM of non-mutated RNA (pLqsR) or mutated in the indicated regions (mLqsR). AGGA motifs were replaced by an AAAA sequence using PCR mutagenesis. Lane 1: no CsrA + non-mutated <i>lqsR</i> mRNA, lane 2: 5.0 μM CsrA + non-mutated <i>lqsR</i> mRNA, lane 2: 5.0 μM CsrA + m<i>lqsR</i> mRNA mutated in region 1, lane 3: 5.0 μM CsrA + m<i>lqsR</i> mRNA mutated in region 2, lane 4: 5.0 μM CsrA + m<i>lqsR</i> mRNA mutated in both region 1 and 2.</p

Evaluation of the maturation features of DLs loaded with LV79 amastigotes.

<p>C57Bl/6 and DBA/2 BMD-DL cultures were incubated or not with LV79 amastigotes (MOI of 5∶1) or BCG (MOI of 10∶1) for 24 hours. Control cultures without exposition to microorganisms (unexposed DLs), LV79-exposed DL cultures and BCG-exposed DL cultures were analyzed by FCM. Histograms of gated C57BL/6 (A) or DBA/2 (B) BMD-DLs expressing surface MHC class II molecules are shown. The percentage of DLs expressing high cell surface levels of CD80, CD86, CD54, MHC class II are shown as means and standard deviations (4≤n≤7 independent experiments). In LV79-exposed DL cultures, the distinction between parasite-free (2A3-26- DL) and amastigote-hosting DLs (2A3-26⁺ DLs) was based on the recourse to the 2A3-26 mAb which binds exclusively to <i>L. amazonensis</i> amastigotes. * indicates a significant difference between control cultures and microorganisms-harbouring BMD-DLs (p<0.001).</p

Distinct ear pinna features and amastigote population size values in C57BL6 and DBA/2 mice.

<p>Ten thousand LV79 metacyclic promastigotes were inoculated into the ear dermis of C57BL/6 (n = 41) and DBA/2 (n = 65) mice. <b>A–C</b>) Ear thickness was monitored post LV79 inoculation (black dots) or not (white dots) in ears up to 80 days for C57BL/6 mice and up to 100 days for DBA/2 mice. Results are expressed as means and standard deviations. * indicates significant p values between inoculated and un-exposed/control ears. Phase 1 (day 0 to day 22 PI) and phase 2 (post day 22) are depicted. <b>B, C</b>) Mean delta of ear thickness and parasite load in C57BL/6 and DBA/2 mice are displayed over the phase 1 (day 4 to day 22). (B) – The delta of ear thickness was obtained from inoculated ear thickness measures of all mice from day 4 to day 22 minus ear thickness values of unexposed/control mice - (<b>C</b>) Mean amastigote load quantification in ears and ears-DLNs of C57BL/6 and DBA/2 mice by RT-qPCR (n = 12 mice) over the phase 1. Means and SDs are shown. * p<0.002.</p

CsrA modulates the expression of a thiamine pyrophosphate (TPP) riboswitch element.

<p><b>A</b>) Schematic representation of the <i>thi</i>-operon in <i>L</i>. <i>pneumophila</i> including the transcriptional start site (TSS), the CsrA-binding region, the thi element of the predicted TPP riboswitch and a predicted transcription termination site upstream of the start codon (AUG). The CsrA-binding site is overlapping the <i>thi</i> element of the TPP riboswitch. This organization suggests that CsrA is implicated in the fine-tuning of the expression of the downstream <i>thi</i>-operon most probably due to conformational changes in the secondary RNA structure. <b>B</b>) EMSA with 200nM of biotinylated thi-element (TPP) RNA and purified CsrA: Lane 1: no CsrA, lane 2: 1.0 μM CsrA, lane 3: 2.0 μM CsrA, lane 4: 5.0 μM CsrA, lane 5: 5.0 μM CsrA + 2.0 μM unlabeled RsmZ. <b>C)</b> Beta-lactamase (BlaM) assay in minimal medium grown <i>Legionella</i> without, with 1 mM and with 2 mM of TPP. BlaM activity in 10μg total protein of wt and <i>csrA</i>^<i>-</i> strain containing the 5'UTR of the <i>thi</i>-operon in a pXDC61 plasmid was measured. Each value represents the mean +/- SD of three independent experiments. BlaM activity is significantly decreased in the mutant at the different conditions indicating a positive effect of CsrA on the <i>thi</i>-operon expression in <i>L</i>. <i>pneumophila</i>. <b>D</b>) Model of the TPP riboswitch modulated by CsrA. Mfold prediction of the secondary structure of the 5'UTR <i>thi</i>-region: When TPP is bound, the OFF state of the riboswitch is favored in which the expression of the operon is inhibited (most likely due to premature termination at the predicted termination site). The presence of CsrA in contrast might stabilize the ON state where the structure of the thi-element is dispersed, hence, higher amounts of TPP would be necessary to shift the element back to the OFF state leading to the down-regulation of the <i>thi</i>-genes expression.</p