NixA and NiuBDE are necessary for resistance to extreme acidity in the presence of urea.

<p>Wild type strains and mutants were exposed during 40 min at pH 2, without or with 6 mM urea. <i>ΔniuB</i> corresponds to a <i>ΔniuB1-ΔniuB2</i> double mutant. Controls were performed at pH 7 without urea. Survival of the bacteria was assessed using an Alamar blue-based test, in which a pink staining reveals metabolically active cells, while a blue staining means that cells are metabolically inactive. The pink staining is proportional to the amount of metabolically active cells. The pH was evaluated for each strain and results were compared to control cells (exposed at pH 7) and to a pH-scale (right side of the figure). A reminder of the results obtained in other experiments for urease activity and ⁶³Ni(II) uptake is given at the bottom of the figure for comparison.</p

Role of NixA and NiuBDE in urease activity of <i>H</i>. <i>pylori</i> at pH 5 and 7.

<p>Urease activity was measured on whole cells of the different mutants strains by measuring ammonia production. For each condition, ammonia production is expressed as a percentage of the wild type strain put at 100%. These data indicate that NiuBDE functions at both pH 5 and 7, while NixA is mainly active at pH 5. <i>ΔniuB</i> corresponds to a <i>ΔniuB1-ΔniuB2</i> double mutant. The mutant strains were either complemented by c-<i>niuDE</i> (c- for chromosome) inserted at a neutral site on the chromosome under the control of the P_<i>ureI</i> promoter or by p-<i>niuB1</i> and p-<i>niuB2</i> (p- for plasmid) expressed from derivatives of plasmid pILL2157 under the control of an IPTG-inducible P_<i>ureI</i> promoter in the presence of IPTG. The data correspond to the mean value of three independent experiments and error bars represent the standard deviation. *** and <b>**</b> indicate that the mean value is significantly different from that of the wild type strain (<i>P</i> ≤ 0.001 and <i>P</i> ≤ 0.01, respectively). For the complemented strains, <i>ΔnixA</i> Δ<i>niuD</i> + c-<i>niuDE</i> or <i>ΔnixA</i> Δ<i>niuB</i> p-<i>niuB1/2</i>, the <i>P</i> values correspond to comparison with the corresponding parental <i>ΔnixA ΔniuD</i> and <i>ΔnixA</i> Δ<i>niuB</i> mutants, respectively.</p

NiuD and NiuB are required for mouse colonization by <i>H</i>. <i>pylori</i> strain SS1.

<p>Each point corresponds to the colonization load for one mouse one month after infection with the strain indicated below. Horizontal bars represent the geometric means of the colonization load for the wild type, each mutant and the chromosomally complemented mutants (designated c-). <i>ΔniuB</i> corresponds to a <i>ΔniuB1-ΔniuB2</i> double mutant. The results presented correspond to a representative experiment out of two. The detection limit is shown by a dashed horizontal line. <b>***</b> indicates that the geometric value is significantly different (<i>P</i> ≤ 0.001) from that of the wild type strain.</p

NiuD and NixA mediate <i>H</i>. <i>pylori</i> sensitivity to high nickel concentrations.

<p>Effect of 1.5 and 2 mM NiCl₂ on growth of <i>H</i>. <i>pylori</i> B128-S wild type strain, isogenic mutants and complemented strains. The results are presented as % of growth in the presence of nickel relative to growth without nickel after 24h incubation. The data correspond to the mean value of three independent experiments. Error bars represent the standard deviation. <b>***</b> indicates that the mean value is significantly different from that of the wild type strain (<i>P</i> ≤ 0.001).</p

NiuD and NixA control the intracellular nickel content and uptake in <i>H</i>. <i>pylori</i>.

<p>Panel A: Nickel amounts measured by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) and expressed as μg of nickel.g^-1 of protein. Strains were grown either without added nickel, with 10 μM or with 100 μM NiCl₂. Panel B: ß-galactosidase activity expressed by the P_<i>fecA3</i>::<i>lacZ</i> reporter fusion in wild type and mutant strains. The expression of the fusion decreases in a NikR-dependent manner with increasing intracellular nickel concentration. ß-galactosidase activities are presented as the ratio of activity measured in strains grown in the presence of 200 μM nickel versus without nickel, expressed in percentages. Panel C: Measurements of radioactive nickel uptake rates in wild type and mutant strains expressed in cpm/min. On the right side scale, the uptake rates were normalized with respect to the value measured for the wild type strain. Complementation with the <i>niuDE</i> operon inserted at a neutral locus on the chromosome is designated c-<i>niuDE</i> (c- stands for “chromosomally inserted”). In these different experiments, the data correspond to the mean value of three independent experiments and error bars represent the standard deviation. <b>***</b> indicates that the mean value is significantly different (<i>P</i> ≤ 0.001) from that of the wild type strain.</p

NiuB1 and NiuB2 control the nickel sensitivity and uptake in <i>H</i>. <i>pylori</i>.

<p>Panel A: Effect of nickel on growth of <i>H</i>. <i>pylori</i> B128-S wild type strain and isogenic mutants. <i>ΔniuB</i> corresponds to a <i>ΔniuB1-ΔniuB2</i> double mutant. Results are presented as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006018#ppat.1006018.g001" target="_blank">Fig 1</a>. Panel B: ß-galactosidase activity of the P_<i>fecA3</i>::<i>lacZ</i> reporter fusion in different backgrounds presented as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006018#ppat.1006018.g002" target="_blank">Fig 2</a>. Panel C: Measurements of radioactive nickel uptake rates in wild type and mutant strains. On the right scale, the uptake rates were normalized with respect to the value measured for the wild type strain. The mutant strains were either complemented by c-<i>niuB1</i> or c-<i>niuB2</i> (c- for chromosomally inserted) inserted at a neutral site on the chromosome under the control of the P_<i>ureI</i> promoter or by p-<i>niuB1</i> and p-<i>niuB2</i> (p- for inserted on a plasmid) expressed from derivatives of plasmid pILL2157 under the control of an IPTG-inducible P_<i>ureI</i> promoter in the presence of IPTG. In these different experiments, the data correspond to the mean value of three independent experiments and error bars represent the standard deviation. <b>***</b> indicates that the mean value is significantly different (<i>P</i> ≤ 0.001) from that of the wild type strain.</p

Model for nickel transport in <i>H</i>. <i>pylori</i>.

<p>In <i>H</i>. <i>pylori</i>, nickel (small blue circles) is transported across the outer membrane by FrpB4 (purple barrel), a TonB-dependent transporter. This uptake activity is most efficient at acidic pH. Once in the periplasm, uptake of nickel through the inner membrane can be performed by the NixA permease (green). Alternatively, nickel can form a Ni(II)-(L-His)₂ complex, that is specifically recognized by the periplasmic solute binding protein NiuB (orange). In <i>H</i>. <i>pylori</i> strains with two NiuB paralogs, NiuB1 (dark orange) seems to be the major contributor for nickel uptake, while NiuB2 (light orange) is less efficient. Then, NiuB docks onto the NiuD permease (orange), and nickel is transferred across the inner membrane upon ATP consumption by NiuE (light blue), and delivered to the cytoplasm. There, it is stored by histidine-rich proteins, such as HspA (GroES, purple), Hpn (blue) or Hpn-2 (green) and/or channeled by the UreEFGH machinery toward urease, or by the HypAB machinery towards hydrogenase. The figure presents urease activation through nickel delivery by UreEFGH (to which HypAB also contributes) in the cytoplasm. Ultimately, this pathway results in nickel-dependent urease activation, this enzyme catalyzing urea breakdown into ammonia and carbon dioxide, both contributing to maintaining the intracellular pH close to neutrality and allowing the bacteria to resist acidity.</p

Sensitivity to cobalt and bismuth of wild type strain and isogenic mutants.

<p>Effect of NiCl_2, CoCl₂ and bismuth subcitrate potassium on growth of <i>H</i>. <i>pylori</i> B128-S wild type strain, isogenic mutants and complemented strains. <i>ΔniuB</i> corresponds to a <i>ΔniuB1-ΔniuB2</i> double mutant. Strains were grown 24 h in the presence or absence of the metal examined. Results are presented as a Growth ratio G_Ratio = 100x[(OD_+metal/OD_-metal)_test−(OD_+metal/OD_-metal)_ref] / (OD_+metal/OD_-metal)_ref allowing to normalize the growth ratio of the test strain compared to the growth ratio of the reference strain. The reference is the wild type strain on panel A and the <i>ΔnixA ΔniuB</i> mutant on panel B. Bars correspond to the G_Ratio values. This representation allows to visualize the results in such a way that bars on the left relative to the vertical axis correspond to G_Ratio values of strains that are more sensitive to the metal than the wild type strain (negative values) and those on the right side to G_Ratio values of strains that are more tolerant (positive values) to the metal. The results are presented together with those with nickel for comparison. The data correspond to the mean value of three independent experiments and error bars represent the standard deviation.</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

Analysis of the protein-protein interaction network of Hpn and Hpn-2 by <i>in vivo</i> BACTH.

<p>(A-C) the tables represent the results of the β-galactosidase activity measurements on <i>E</i>. <i>coli</i> strains containing the pair-wise combinations of the different constructs. T18 and T25 correspond to the two fragments of the adenylate cyclase and the protein extremity at which the fusion was made (N_ter or C_ter) is shown. Panel A: interaction between Hpn, Hpn∆Cter, Hpn-2 and Hpn-2∆Cter. Panel B and C: interaction of Hpn, Hpn∆Cter, Hpn-2 and Hpn-2∆Cter with UreA, HypA and HypB. Red squares correspond to β-galactosidase activity that are > 3,000 U, orange squares between 2,000 and 3,000 U, yellow squares between 500 and 2,000 U and light yellow squares, between 240 and 500 U. None of the potential false positive interactions was presented (interactions of pUT18/pKNT25(Hpn) and pUT18(Hpn)/pKNT25 with ß-galactosidase activity below 600 units). Black squares represent combinations that lead to a lethal phenotype. The β-galactosidase activity values for each strain and the controls are indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005312#ppat.1005312.s006" target="_blank">S2 Table</a>. (D) Schematic representation of the protein-protein interaction network of Hpn and Hpn-2. Thick arrows correspond to strong interactions, thin arrows to moderately strong interactions, according to the ß-galactosidase. * Indicates that this interaction was established before [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005312#ppat.1005312.ref053" target="_blank">53</a>].</p