Circular dichroism of VapB and VapC confirmed predicted secondary structure.

<p>(<b>A</b>) The VapB and VapC CD spectra were recorded in the wavelength range of 195–255 nm as average of five scans at 20°C. Measured ellipticities, <i>θ</i> (mdegree), were converted to molar mean residue ellipticities, [<i>θ</i>] (degree.cm².dmol⁻¹). The assays were reproduced with at least 2 samples of each protein. (<b>B</b>) Prediction of secondary structure by the PSIPRED algorithm using the primary sequence of the proteins. The experimental data confirmed the secondary structure predicted by computational analysis.</p

3D model of leptospiral VapC closely matches the experimental X-ray structure of <i>Shigella</i>'s VapC.

<p>Alignment of the sequences of VapCs from <i>S. flexneri</i> and <i>L. interrogans</i> is shown. On the left panel, the structure model (ribbon) of VapC from <i>L. interrogans</i> (green) was superimposed to the VapC template from <i>S. flexneri</i> (white) (PdB: 3TND-C). The green regions in the target appear almost identical to the template, while the red region does not correspond. The amino acids composing the red region are written in the same color in the sequence alignment. On the right panel, superimposition shows the perfect matching of the conserved threonine and the four acidic residues responsible for coordinating metal ions in the catalytic site, and the cysteine involved in dimerization, positioned in the neighborhood of the catalytic site. These six residues are numbered in the structure according to leptospiral VapC sequence and colored as highlighted in the alignment.</p

Primers used in the amplification of leptospiral genes and expression plasmids.

<p>* The restriction sites for the enzymes are underlined in the primers.</p

Analysis of purified recombinant proteins by SDS-PAGE revealed VapC dimerization.

<p>(<b>A</b>) VapB purified from the soluble fraction of <i>E. coli</i> extracts (<b>B</b>) VapC expressed as inclusion bodies, before [NP] or after [P] pressurization in buffer containing 0.5 M L-arginine; (<b>C</b>) VapB and VapC purified from the soluble fraction of <i>E. coli</i> co-expressing both proteins. Samples were prepared with or without β-mercaptoethanol ([Reduced] or [NReduced], respectively). M - Molecular Marker (kDa). The arrows indicate purified VapB and monomeric (15.1 kDa) or dimeric (30.2 kDa) forms of VapC. Remarkably, VapC dimers were observed in non-reduced samples of the pressurized protein (B) and in the co-purification with VapB (C). This analysis was made with 3 VapC preparations.</p

VapB and VapC interact <i>in vivo</i> and <i>in vitro</i>.

<p>(<b>A</b>) Pull-down assay. The soluble fraction of <i>E. coli</i> pAE<i>vapBC</i> extract was applied to a Ni⁺²-Sepharose column. Samples were analyzed by SDS-PAGE. Lane 1: initial sample; lane 2: washing; lanes 3–4: elution with 250 mM imidazole. It is important to observe that no VapC was released during the washing step, being co-purified with VapB-His, denoting the <i>in vivo</i> interaction. The arrows indicate the VapB and VapC bands. M - Molecular Weight Marker (kDa). Pull-down assay was perfomed more than 5 times. (<b>B</b>) Ligand affinity blotting. To analyze specific binding between VapB and VapC, the VapC and LipL32 proteins (negative control) were subjected to 15% SDS-PAGE (left panel) and transferred to nitrocellulose membrane (right panel). After blocking, the membrane was incubated with a VapB solution (3 µg ml⁻¹). Following extensive washing, the membrane was incubated with anti-VapB antibodies. M - Prestained Molecular Weight Marker (kDa). VapB in solution bound to both monomeric (15.1 kDa) and dimeric (30.2 kDa) forms of VapC immobilized in the membrane, denoting <i>in vitro</i> interaction. VapB did not bound the negative control, protein LipL32.(<b>C</b>) Western blot control showing that anti-VapB antibodies recognizes specifically VapB, and not VapC.</p

Evaluation of VapC models.

<p>VapC structural models were evaluated by QMEAN4 and Z score in order to compare and rank alternative models of the same target. QMAN4 is a reliability score consisting of a combination of four structural descriptors which ranges between 0 and 1 with higher values for better models. Z-score provides an estimate of the “degree of nativeness” of the structural features observed in a model; ‘good-quality’ models reach a mean <i>Z</i>-score of −0.65, ‘medium-quality’ -1.75, and the ‘low-quality’ −3.85. The analysis of the model quality scores showed that <i>Shigella</i> VapC is the only template to render a “good” model for VapC from <i>Leptospira</i> (*), as “good” as the one created for <i>Salmonella</i> VapC (**), which shares 89% identity with the temp<b>l</b>ate. In opposition, <i>Mycobacterium</i> VapC20 model (^#) displayed low scores.</p

Solubility and availability of VapC influences the growth rate of <i>E. coli</i>.

<p>(<b>A</b>) The growth of <i>E. coli</i> transformed with control pAEØ (◊), pAE-<i>vapB</i> (▪), pAE-<i>vapC</i> (•) or pAE-<i>vapBC</i> (▴) was followed after induction, for 5 h to 8 h. <i>E. coli</i> harboring the empty vector pAE was used as control. The data represents one experiment that was reproduced at least three times in the laboratory. (<b>B</b>) Samples (10 µl) were applied to SDS-PAGE to analyze the expression of soluble [S] or insoluble [InS] proteins from induced [I] and not induced [NI] cultures. The arrows indicate the recombinant proteins. The relative amount of soluble and insoluble VapB and VapC proteins were estimated by densitometry of the bands in the gel, considering that the insoluble fraction was resuspended 5× concentrated in relation to the soluble fraction. VapB was expressed predominantly (∼80%) in soluble form (▪). <i>E. coli</i> growth was arrested by the expression of VapC (•), probably due to the toxic effect of the protein in soluble and active form (∼10%). Co-expression of the VapB antitoxin (▴) restored the bacterial growth and increased the expression of the soluble form of VapC (∼89%).</p

Leptospiral VapC toxin cleaves tRNA^fMet and is inhibited by the VapB antitoxin.

<p>Unless specified, the reactions were carried out under the following general condition: 5 pmol VapC was incubated with 3 ρmol of tRNA^fMet in 10 mM Hepes pH 7.5, 15 mM KCl, 1 mM DTT, 10 mM MgCl and 10% glycerol at 37°C for 30 min and analyzed by denaturing 8% PAGE 6 M urea, stained with ethidium bromide. Digestion of tRNA^fMet resulted in only one band indicating that it contains two fragments of same size. (<b>A</b>) VapC showed no activity over <i>E. coli</i> rRNA. VapC (2.5 and 5 pmol) was incubated with 1 µg of rRNA at 37°C for 10 min and analyzed by 1% Tris-Acetic-EDTA ethidium bromide agarose gel electrophoresis. (<b>B</b>) VapC activity is dose dependent. The initiator tRNA^fMet was incubated with increasing amounts of VapC (0 to 5 pmol). Incubation with 10 mM EDTA abrogates RNAse activity. (<b>C</b>) Rnase activity is time dependent. VapC was incubated with the substrate for 0.5 to 30 min. (<b>D</b>) VapC activity is inhibited by VapB. VapC was pre-incubated for 15 min with increasing amounts of VapB (0 to 6.8 pmol), before the addition of tRNA^fMet. (<b>E</b>) VapC cleaves tRNA^fMet more effectively using Mg⁺² than Mn⁺². VapC aliquots were incubated with Mg⁺² (0.01 to 100 mM) or Mn⁺² (0.01 to 10 mM). Each assay was performed at least twice.</p

Image5.TIF

<p>Leptospirosis is considered one of the most important zoonosis worldwide. The activation of the Complement System is important to control dissemination of several pathogens in the host. Only a few studies have employed murine models to investigate leptospiral infection and our aim in this work was to investigate the role of murine C5 during in vivo infection, comparing wild type C57BL/6 (B6 C5^+/+) and congenic C57BL/6 (B6 C5^−/−, C5 deficient) mice during the first days of infection. All animals from both groups survived for at least 8 days post-infection with pathogenic Leptospira interrogans serovar Kennewicki strain Fromm (LPF). At the third day of infection, we observed greater numbers of LPF in the liver of B6 C5^−/− mice when compared to B6 C5^+/+ mice. Later, on the sixth day of infection, the LPF population fell to undetectable levels in the livers of both groups of mice. On the third day, the inflammatory score was higher in the liver of B6 C5^+/+ mice than in B6 C5^−/− mice, and returned to normal on the sixth day of infection in both groups. No significant histopathological differences were observed in the lung, kidney and spleen from both infected B6 C5^+/+ than B6 C5^−/− mice. Likewise, the total number of circulating leukocytes was not affected by the absence of C5. The liver levels of IL-10 on the sixth day of infection was lower in the absence of C5 when compared to wild type mice. No significant differences were observed in the levels of several inflammatory cytokines when B6 C5^+/+ and B6 C5^−/− were compared. In conclusion, C5 may contribute to the direct killing of LPF in the first days of infection in C57BL/6 mice. On the other hand, other effector immune mechanisms probably compensate Complement impairment since the mice survival was not affected by the absence of C5 and its activated fragments, at least in the early stage of this infection.</p

Image3.tif

<p>Leptospirosis is considered one of the most important zoonosis worldwide. The activation of the Complement System is important to control dissemination of several pathogens in the host. Only a few studies have employed murine models to investigate leptospiral infection and our aim in this work was to investigate the role of murine C5 during in vivo infection, comparing wild type C57BL/6 (B6 C5^+/+) and congenic C57BL/6 (B6 C5^−/−, C5 deficient) mice during the first days of infection. All animals from both groups survived for at least 8 days post-infection with pathogenic Leptospira interrogans serovar Kennewicki strain Fromm (LPF). At the third day of infection, we observed greater numbers of LPF in the liver of B6 C5^−/− mice when compared to B6 C5^+/+ mice. Later, on the sixth day of infection, the LPF population fell to undetectable levels in the livers of both groups of mice. On the third day, the inflammatory score was higher in the liver of B6 C5^+/+ mice than in B6 C5^−/− mice, and returned to normal on the sixth day of infection in both groups. No significant histopathological differences were observed in the lung, kidney and spleen from both infected B6 C5^+/+ than B6 C5^−/− mice. Likewise, the total number of circulating leukocytes was not affected by the absence of C5. The liver levels of IL-10 on the sixth day of infection was lower in the absence of C5 when compared to wild type mice. No significant differences were observed in the levels of several inflammatory cytokines when B6 C5^+/+ and B6 C5^−/− were compared. In conclusion, C5 may contribute to the direct killing of LPF in the first days of infection in C57BL/6 mice. On the other hand, other effector immune mechanisms probably compensate Complement impairment since the mice survival was not affected by the absence of C5 and its activated fragments, at least in the early stage of this infection.</p