Missense non synonymous SNP mutations found by DNA sequencing of genome of M58 compared to the parental strain L495.

<p>Missense non synonymous SNP mutations found by DNA sequencing of genome of M58 compared to the parental strain L495.</p

NOD1 and NOD2 receptors do not affect <i>L</i>. <i>interrogans</i> dissemination.

<p>(A) Survival curves (left panel) and weight variation (right panel) of C57BL6/J (WT) and NOD1 and NOD2 deficient (NOD1/2DKO) mice after intraperitoneal infection with 4 different doses (from 5.10⁸ to 4.10⁶ leptospires/mouse) of MFLum1, a bioluminescent strain of <i>L</i>. <i>interrogans</i> serovar Manilae L495 (weekly passage 14). Survival curves and weight loss were established within the 5 days following the infection, corresponding to the acute phase of the disease. Percentage of weight loss is represented as the mean ± SEM of individual mice compared to their initial weights before infection at day 0 (D0). For each dose, n = 5 WT and n = 5 NOD1/2 DKO mice. (B) Bacterial loads in WT and NOD1/2DKO mice IP infected with a sub-lethal dose of 10⁷ bioluminescent <i>L</i>. <i>interrogans</i> (MFLum1). Bacterial loads were measured by qPCR in blood at 8, 24, 48 and 72 hours post infection, and in urine at 5 and 7 days post infection, and by live imaging (IVIS), 1 month post infection. The graphs represent a compilation of 3 independent experiments with n = 3 to 6 mice for the blood panel, 1 experiment for the urine panel and 1 experiment representative of 3 with n = 4 mice for the IVIS panel. (A),(B). Statistics are not indicated as no significant differences were found at any time points between WT and NOD1/2DKO mice.</p

Effect of <i>L</i>. <i>paracasei</i> derived-peptidoglycans on influenza_infected mice.

<p>(A) Effects of consumption of PG of <i>L</i>. <i>paracasei</i> by mice (n = 17 at D0) on body weight loss and temperature loss compared with PBS control group (n = 17 at D0). (B) Effect of consumption of PG of <i>L</i>. <i>paracasei</i> (n = 10) on the recruitment of immune cells in the lungs at day 0 compared with the control group (n = 10): aM: Alveolar Macrophage, iM: Interstitial Macrophage, DC: Dendritic cells, iM: Inflammatory Monocyte, pM: Patrolling monocytes, Eo: Eosinophils, N: Neutrophils, B: B cells, T: T cells. Results are expressed as mean ± SEM for each group. (C) Effect of consumption of PG of <i>L</i>. <i>paracasei</i> (n = 17 at D0) on viral load in IAV-infected BALB/c mice compared with the control group (n = 17 at D0), 7 and 10 days after viral infection. Data are means ± SEM of each group of mice (*p<0.05).</p

Expression of LipL21 impairs <i>E</i>. <i>coli</i> peptidoglycan digestion into muropeptides and recognition by NOD receptors.

<p>(A) Color phenotypes of strains expressing alkaline phosphatase (phoA) derivatives and grown as colonies on agar with chloramphenicol and 5-bromo-4-chloro-3-indoyl-phosphate (XP). FL-<i>phoA</i>, Δ(2–22)phoA, FL-<i>lipl21-phoA</i> and ΔN-<i>lipl21-phoA</i> correspond respectively to <i>E</i>. <i>coli</i> (BTH₁₀₁) expressing the full length phoA (positive control), <i>E</i>. <i>coli</i> expressing phoA without signal peptide (negative control), <i>E</i>. <i>coli</i> expressing the full length LipL21 fused with Δ(2–22)<i>phoA</i> and <i>E</i>. <i>coli</i> expressing the LipL21 without signal peptide fused with Δ(2–22)<i>phoA</i>. (B) Growth curves of BL-21 Rosetta-2 <i>E</i>. <i>coli</i> expressing the LipL21 lipoprotein (pLipL21) or not (pEmpty) in the pRSFDuet-1 vector, without IPTG induction. (C) Crude bacterial extracts of BL-21 Rosetta-2 <i>E</i>. <i>coli</i> with empty pASK-IBA6 vector (pEmpty) or LipL21 expressing vector (pLipL21), induced by anhydrous-Tetracyclin or not (NI) and migrated on 12% acrylamide gel. Stain free coloration (left) and immunodetection (right) using a LipL21 antiserum. The Rainbow marker ladder gives an indication for the LipL21size.(D) HPLC separation profiles of muropeptides after digestion with mutanolysin. Each peptidoglycan was extracted with both the 0.5 h and 4 h protocols from BL-21 Rosetta-2 <i>E</i>. <i>coli</i> expressing the LipL21 lipoprotein (pLipL21) or not (pEmpty), after induction with anhydrous-Tetracyclin. (E) and (F) <i>E</i>. <i>coli</i> PGs, extracted from the 0.5 h protocol, were used to stimulate HEK 293T cells expressing human NOD1 (E) or NOD2 (F). HEK 293T cells were co-transfected with 6 μg of PG or 100 nM of muropeptides controls (MTP for NOD1 and MDP for NOD2, along with the reporter constructions and NOD1 or NOD2 expression plasmids. Luciferase activity was measured 24 h after transfection. Cells left untreated with muropeptides were used as negative control (water). Data are expressed as the mean of triplicates ± SEM of relative light units representing luciferase activity normalized with respect to β-galactosidase activity. The graph shown is representative of 3 equivalent experiments. The unpaired <i>t</i> test was used to compare the recognition of PGs prepared from <i>E</i>. <i>coli</i> with the empty vector (pEmpty) to the PG prepared from <i>E</i>. <i>coli</i> expressing LipL21 (pLipL21). A <i>p</i> value < 0.05 was considered significant. <i>p</i> values: *** <i>p</i> < 0.001. For clarity, statistics comparing each PG or muropeptide control to the water treated cells have not been indicated but are all significant with at least p<0,05.</p

Effect of consumption of <i>L</i>. <i>paracasei</i> (N = 30) on the recruitment of immune cells in the lungs at different days.

<p>A: day 0, B: day 7, C: day 10 after influenza infection, compared with the control group (Mice gavaged with PBS and infected by influenzae virus) (N = 30). Or to mice that were only infected with influenzae virus but with no gavage (N = 5) aM: Alveolar Macrophage, iM: Interstitial Macrophage, DC: Dendritic cells, iM: Inflammatory Monocyte, pM: Patrolling monocytes, Eo: Eosinophils, N: Neutrophils, B: B cells, T: T cells. Results are expressed as mean ± SEM for each group. (*p<0.05, **P<0.01).</p

The human NOD1 receptor expressed in mice confers a slightly enhanced clearance of M58.

<p>(A) Transgenic C57BL6/J mice for the human NOD1 gene and deficient for NOD1 (hNOD1Tg/mNOD1KO) and mNOD1KO mice were IP infected with 2.10⁷ M58 <i>lipl21</i>- mutant. Bacterial loads were measured by qPCR in blood at 8, 24 and 48 hours post infection. This graph is a compilation of 2 independent experiments, representative of 3 equivalent experiments, with n = 3 to 5 mice in each experiment. (B) hNOD1Tg/mNOD1KO mice were IP infected with 2.10⁷ M58 <i>lipl21</i>- mutant or 2.10⁷ complemented C5M58 <i>lipl21</i>-/+ mutant. Bacterial loads were measured by qPCR in blood at 8, 24 and 48 hours post infection. The unpaired <i>t</i> test was used to compare at each time point the 2 strains. In grey, we indicated the p values that were close to, but did not reach 0.05, the <i>p</i> value considered as significant. This experiment is representative of 3 equivalent experiments with n = 3 to 5 in each group, suggesting the same trend.</p

Effects of consumption of <i>L</i>. <i>paracasei</i> on cytokine profiles, compared with the control group.

<p>A: day 0, B: day 7, C: day 10 after influenza infection. Results are expressed as mean ± SEM for each group (n = 8).</p

Impact of consumption of <i>L</i>. <i>paracasei</i> CNCM I-1518 on gut microbiota.

<p>(A) Alpha-diversity measured by number of OTUs before and after influenza infection. (B) Weighted Unifrac PcoA before and after infection infection (D0, D3 and D7). (C) Relative abundance (%) of <i>Allobaculum</i> through the experiment. (D) Relative abundance (%) of <i>Prevotella</i> through the experiment.</p

The LipL21 lipoprotein impairs leptospiral peptidoglycan digestion into muropeptides.

<p>(A) PG 0.5 h and PG 4 h from <i>L</i>. <i>interrogans</i> Manilae L495 and Fiocruz L1-130 strains, were loaded on coomassie-stained gels (upper panels; protein gels), revealing a 21-kDa protein corresponding to the LipL21. The protein specificity was checked by immunodetection using LipL21 antiserum (lower panels; western blot, (WB)). (B) LipL21 expression checked by immunodetection on bacterial extracts from <i>L</i>. <i>interrogans</i> Manilae L495 (<i>lipl21</i>⁺⁾, the (<i>lipl21</i>^-) M58 mutant and the complemented C5M58 strain (<i>lipl21</i>^-/+). (C) HPLC separation profiles of muropeptides after digestion by mutanolysin of <i>L</i>. <i>interrogans</i> Manilae <i>lipl21</i>⁺, <i>lipl21</i>^- and <i>lipl21</i>^-/+ peptidoglycans, extracted with the 0.5 h protocol. As positive control for the mutanolysin digestion, <i>E</i>. <i>coli</i> PG was extracted with the leptospiral 0.5 h protocol. D) Growth curves of <i>L</i>. <i>interrogans</i> Manilae <i>lipl21</i>⁺, <i>lipl21</i>^- and <i>lipl21</i>^-/+ strains in EMJH medium at 28°C, with agitation. (E) Muropeptides or 6 μg of PGs extracted from the parental Manilae strain (<i>lipl21</i>+), the LipL21 mutant (<i>lipl21</i>^-) and the complemented strain (<i>lipl21</i>^-/+) were co-transfected with the reporters and NOD1 plasmids in HEK293T cells. 24 h after transfection, luciferase activity was measured. MurTriDAP (MTP) (100 nM), the NOD1 agonist was used as positive control and water as negative control (water). Data are expressed as the mean ± SEM of relative light units representing luciferase activity normalized with respect to β-galactosidase activity. This graph is representative of 3 independent experiments. The unpaired <i>t</i> test was used to compare the recognition of each PG by hNOD1 transfected cells to water as a negative control (none). A <i>p</i> value < 0.05 was considered significant. <i>p</i> values: *** <i>p</i> < 0.001. Non significant differences for the + and -/+ PG are not indicated.</p

Effects of consumption of <i>L</i>. <i>paracasei</i> on ILCs and T effector cells.

<p>(A). gating strategy for FACS analysis of mouse lung. ILC1 were gated on CD45⁺CD3^-CD5^-RORγt^-NKp46⁺, ILC2 were gated on CD45⁺CD3^-CD5^-RORγt^-GATA-3^hi cells and ILC3 were gated on CD45⁺CD3^-CD5^-RORγt⁺ cells. The analysis of ILC3 subsets were supported by the expression of NKp46 and CCR6. Numbers adjacent to boxed areas indicate relative percentage of gated populations. Representative results from 4 independent experiments. (B-C) Representative histograms show the absolute numbers of ILC subsets (B) T cells (C) in lung. (D) Lung cells were stimulated for 4h with IL-12, IL-18, IL-33, IL-23 and IL-1β and IFN-γ, IL-5, IL-13 and IL-22 expression in ILCs and T cells was assessed by intracellular cytokine staining and flow cytometry. Graph represents the absolute numbers of cells. Representative results from 4 independent experiments (n = 9/group). *, P<0.05.</p