The distinct transcriptional signatures of BMD-DLs housing live LV79 amastigotes.

<p>Authors' interpretation of key distinct early events is depicted. Are highlighted five immune processes including DL property to recruit leucocytes and initiate inflammatory processes DL invasiveness (1, 2) and migration (3, 4) DL property to communicate with T lymphocytes (5).</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

Monitoring of amastigotes-loaded C57BL/6 and DBA/2 DLs.

<p><b>A, B</b>) LV79 amastigotes were added to BMD-DL cultures (MOI of 5∶1) and sampled at 5, 24 and 48 hours. Fluorescence microscopy analyses were performed exclusively on MHC class II positive (High and Low) DLs. The intracellular amastigotes were evidenced by the 2A3-26 staining of fixed and permeabilized DLs from C57BL6 and DBA/2 mice (black and grey histograms, respectively). (<b>A</b>) Percentages of 2A3-26⁺ cells and (<b>B</b>) number of intracellular amastigotes in BMD-DLs from C57BL/6 or DBA/2 mice. Means and SDs are shown. Statistical differences obtained between mouse genotypes and time points are indicated.</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

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

Antibiotic tolerance profile of starved planktonic and biofilm bacteria.

<p>Static cultures were grown for 24 h in M63B1_Gluc for wild-type prototroph TG1 (WT) and with the addition of 25 µg ml⁻¹ of leucine, lysine, or cysteine for the corresponding auxotrophs. Planktonic bacteria from the 24-h static culture were removed from each well and therefore separated from the attached-biofilm cells. (A, C) Planktonic bacteria were then collected, spun, washed and treated in parallel with the biofilm bacteria (B, D) with ticarcillin (100 µg ml⁻¹, 100× MIC) or ofloxacin (5 µg ml⁻¹, 80× MIC) in the absence of glucose for WT prototroph TG1 (M63B1 medium) or amino acids for the different auxotrophic mutant strains (M63B1_Gluc medium) for 24 h. Survivor cells were quantified by viable cell counts. Percent survival represents the tolerant population after 24 h of treatment compared to the total number of CFU prior to addition of antibiotics. Equivalent CFU were present in all compared planktonic populations before antibiotic treatment. Data represented are means ± SEM of at least three replicates. Asterisks indicate values significantly different from no starvation condition by the two-tailed unpaired t test: * <i>P</i>≤0.01, ** <i>P</i>≤0.001, *** <i>P</i>≤0.0001 and n.s. (not significant). The genotypes of the three amino acid auxotrophic mutants constructed in the WT prototroph TG1 genetic background are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003144#pgen.1003144.s007" target="_blank">Table S1</a> or as follows: leucine (Leu: Δ<i>leuC</i>::GB), lysine (Lys; Δ<i>lysA</i>::KmFRT), and cysteine (Cys; Δ<i>cysD</i>::KmFRT).</p

Auxotrophic transposon mutants with increased antibiotic tolerance in biofilms.

*<p>Transposon inserted at different locations within <i>leuC</i> and <i>proA</i>.</p>**<p>Phenylalanine, tryptophan and tyrosine.</p><p>N.D. Not determined.</p

The high ofloxacin tolerance exhibited by starved biofilms is SOS-response-dependent.

<p>The impact of SOS response loss-of-function mutations Δ<i>recA</i> and <i>lexAind3</i> on the ofloxacin tolerance of starved biofilms for glucose (A), leucine (B), lysine and tryptophan (C). Briefly, all biofilms were grown for 24 h in M63B1_Gluc for all prototrophic strains and with the addition of 25 µg ml⁻¹ of leucine, lysine or tryptophan for corresponding auxotrophic strains. All biofilms were treated for 24 h in M63B1_Gluc containing ofloxacin (5 µg ml⁻¹). For the glucose starvation environment, M63B1 without glucose was used instead of M63B1_Gluc (Panel A - black bars). Ofloxacin hypertolerance of <i>ΔleuCΔrecA</i> was not restored when adding the vector control plasmid pAM34 (vector), but complete restoration of high ofloxacin tolerance upon leucine starvation was observed by <i>in trans</i> complementation using pAM34<i>recA</i> (p<i>recA</i>). Complementation of Δ<i>leuClexAind3</i> with pAM34<i>recA</i> (p<i>recA</i>) failed to fully restore ofloxacin hypertolerance back to leucine auxotroph levels. Both pAM34 and pAM34<i>recA</i> were maintained by the presence of 0.5 mM IPTG. (D) The SOS response mutation Δ<i>recA</i> (SOS-) did not affect ticarcillin hypertolerance of starved biofilms for leucine (Leu - <i>ΔleuCΔrecA</i>), lysine (Lys – <i>ΔlysAΔrecA</i>) or tryptophan (Trp - <i>ΔtrpAΔrecA</i>). Viable cells of the treated biofilm population were quantified by viable cell counts. Percent survival represents the tolerant population after 24 h of treatment compared to untreated biofilm prior to addition of antibiotics. All compared biofilms had similar numbers of CFUs prior to antibiotic treatment (data not shown). Data represented are means ± SEM of at least three replicates. Asterisks indicate values significantly different by the two-tailed unpaired t test: * <i>P</i>≤0.05, ** <i>P</i>≤0.01, *** <i>P</i>≤0.001, and n.s. (not significant). All strains used here were originally constructed in the WT TG1 genetic background and are detailed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003144#pgen.1003144.s007" target="_blank">Table S1</a>. The different SOS response mutant strains made in amino acid auxotrophic background are derivatives of Δ<i>leuC</i>::ΔFRT (Leu - leucine), Δ<i>lysA</i>::ΔFRT (Lys - lysine), and Δ<i>trpA</i>::ΔFRT (Trp – tryptophan).</p

Antibiotic-tolerant populations of <i>E. coli</i> K-12 TG1 biofilms.

<p>Twenty-four-hour biofilms of <i>E. coli</i> TG1 were preformed in M63B1_Gluc and treated for a period of 24 h with concentrations up to 100- and 80-times the MIC values for ticarcillin and ofloxacin, respectively. Untreated controls represent 48 h biofilms in which fresh M63B1_Gluc without antibiotic was added after 24 h, explaining their values above 100%, represented by a dotted line in each panel. Viable cells of the treated biofilm population were quantified by viable cell counts (A and B) and the XTT-reduction assay (C and D) and were compared to numbers obtained prior to antibiotic treatment. % survival (CFU) values are indicated on top of each bar and are means ± standard error of the means (SEM) of at least six replicates.</p