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

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

Digested <i>L</i>. <i>interrogans</i> PG is recognized by human NOD1 and NOD2 but barely by murine NOD1.

<p>(A) HPLC separation profiles of leptospiral muropeptides. Peptidoglycan (PG) extraction protocol influences the digestion of leptospiral PG into muropeptides. PG obtained from <i>L</i>. <i>interrogans</i> Manilae L495 and Fiocruz L1-130, extracted with 2 different protocols, called respectively 0.5 h and 4 h were digested by mutanolysin before HPLC. Commercial <i>E</i>. <i>coli</i> PG (Sigma) was used as the positive control for the mutanolysin digestion. Mutanolysin without PG (mutanolysin alone) was included as a control. (B) Leptospiral muropeptides signal through human NOD1 (hNOD1) and NOD2 (hNOD2). Six μg of PG 0.5 h and 4 h of <i>L</i>. <i>interrogans</i> Manilae (left panel) and Fiocruz (right panel), the NF-κB-luciferase reporter and NOD1 or NOD2 plasmids were co-transfected in HEK 293T cells. MurTriDAP (MTP) and MDP (100 nM) were used as positive controls (agonists) for NOD1 and NOD2 activation, respectively. Luciferase activity was measured 24 h after transfection. Data are expressed as the mean ± SEM of triplicates of relative light units, representing luciferase activity normalized with respect to β-galactosidase activity and are representative of 5 experiments. For each transfection, the unpaired <i>t</i> test was used to compare each condition to the negative control (water). A <i>p</i> value < 0.05 was considered significant. <i>p</i> values: *<i>p</i> < 0.05, ***<i>p</i> < 0.001. NS: non significant. (C) PG of pathogenic strains (Manilae L495 / Fiocruz L1-130) does not signal through murine NOD1. HEK293T cells were co-transfected with 10 μg of PG from Manilae L495, Fiocruz L1-130 strains or <i>L</i>. <i>biflexa</i> strain Patoc, prepared with the 4h SDS boiling protocol, along with the NF-κB-luciferase reporter (None) and human (h)NOD1, hNOD2 or murine NOD1 (mNOD1) plasmids. For each transfection, cells were stimulated with water as negative control, or with 100 nM of MurTriDAP, FK156, or MDP (agonists) as positive controls for hNOD1, mNOD1 and hNOD2 activation, respectively. Luciferase activity was measured 24 h after transfecting the cells. Data are expressed as the mean ± SEM of triplicates of relative light units representing luciferase activity normalized with respect to β-galactosidase activity and are representative of 3 experiments. The unpaired <i>t</i> test was used to compare the recognition of each PG by mNOD1 transfected cells (in red) to the negative control (water). A <i>p</i> value < 0.05 was considered significant. <i>p</i> values: ***<i>p</i> < 0.001. NS: non significant. For clarity, statistics relative to hNOD1 and hNOD2, already showed in panel (A), are not indicated. (D) HPLC analysis of the PG of <i>L</i>. <i>interrogans</i> strain Manilae prepared with the 4 h protocol and treated with chemotrypsin before digestion with mutanolysin. The numbered peaks were collected, and analyzed by mass spectrometry. The corresponding composition is in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006725#ppat.1006725.t001" target="_blank">Table 1</a>. The peak numbered 3 in red corresponds to GM4, the murine NOD1 agonist.</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

M58 is not virulent and the complementation does not restore virulence.

<p>(A)(B) M58 is not virulent in mice and the loss of virulence is not due to PG sensing. (A) C57BL6/J (WT) mice and mice deficient for both NOD1 and NOD2 receptors (NOD1/2DKO) and (B) FVB (WT) mice and mice deficient for lysozyme expressed in macrophages (LysMKO) were IP infected with 2.10⁷ M58 <i>lipl21</i>- mutant. Bacterial loads were measured by qPCR in blood at 8, 24 and 72 hours post infection. These experiments are representative of 2 equivalent experiments with n = 4 to 7 mice in each group. Statistics are not indicated as no significant differences were found at any time points between WT and NOD1/2DKO mice (A), or WT and LysMKO mice (B). (C) <i>L</i>. <i>interrogans</i> are not susceptible to lysozyme <i>in vitro</i>; leptospires were incubated at 28°C in presence of lysozyme and EDTA for the indicated times, then washed and further cultured for 5 days in EMJH medium. The Alamar blue dye was added and further incubated for 2 days. A pink color means that the bacteria are viable whereas the blue color indicates dead bacteria, reflecting the killing by lysozyme in presence of EDTA. Each plate included a positive control (bacteria with EDTA (+) and without lysozyme (-)), and a negative control (medium only). (D) The LipL21 complementation does not restore the virulence of M58 (<i>lipl21</i>-) in mice (left panel) nor in gerbils (right panel). C57BL6/J (WT) mice were IP infected with a sub-lethal dose of 10⁷ L495 strain or with 2.10⁷ of M58, the <i>lipl21</i>- mutant or C5M58, the complemented strain of M58 expressing LipL21 (<i>lipl21-/+</i>). Bacterial loads were measured by qPCR in blood at 8, 24, 48 and 72 hours post infection. This experiment is representative of 2 equivalent experiments with n = 4 to 7 mice in each group. Statistics are not indicated as no significant differences were found at any time points between M58 and C5M58 complemented strain. Survival curves of gerbils IP infected with 10⁶ bacteria/ ml in EMJH. n = 4 gerbils /group.</p

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

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