LigA and LigB Big domains are important for binding to C4BP.

<p>(<b>A</b>) The interaction of C4BP with the conserved N-terminus region of LigA and LigB proteins (LigBN), the C-terminus region of LigA (LigAC) and the C-terminus region of LigB (LigBC) was measured (<b>B-C</b>) The binding of two-domain serial constructs of LigAC (<b>B</b>) or LigBC (<b>C</b>) (described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004192#pntd.0004192.g001" target="_blank">Fig 1D</a>) to C4BP was studied using increasing concentrations of C4BP. BSA was used as negative control. Each value represents the mean ± SD of three independent experiments, each performed in triplicate and binding of C4BP to the recombinant Lig proteins was compared with the binding of these molecules to BSA by the ANOVA (*<i>p</i><0.05; **<i>p</i><0.01 ***<i>p</i><0.001) (<b>D</b>) Estimation of the dissociation constant (K_d) for the interactions of LigAC and LigBC with C4BP. K_d was calculated by fitting the data to the equation Y = Bmax*X/(K_d + X) using GraphPad Prism 5.0 (GraphPad Software, Inc.). Each data point represents the mean +/- SD of three trials in triplicate. C4BP binding to leptospiral proteins was detected using rabbit anti-C4BP as primary antibody and anti-rabbit IgG conjugated with peroxidase as secondary antibody.</p

Interaction of C4BP and recombinant proteins of <i>L</i>. <i>interrogans</i> are dependent on ionic strength.

<p>LigA, LigB or LcpA were immobilized on microtiter wells. C4BP WT (commercial) diluted in 10 mM Na₂HPO₄ and 1.8mM KH₂PO₄ was prepared in different concentrations of NaCl and then added to the wells. To detect the binding of C4BP to recombinant proteins of <i>L</i>. <i>interrogans</i>, polyclonal rabbit anti-C4BP antibodies were used, followed by peroxidase-conjugated anti-rabbit IgG. Each point represents the mean absorbance value at 492 nm +/- the SD of 3 independent experiments, each performed in triplicate. The binding of recombinant proteins of <i>L</i>. <i>interrogans</i> and C4BP WT in the absence of NaCl was considered 100%. Data were analyzed using ANOVA test; *<i>p</i><0.05; ***<i>p</i><0.0001.</p

LigAC and LigBC interact with heparin mainly via Big 7 and Big 8.

<p>(<b>A</b>) Using different LigA and LigB tandem domain constructs, we observed that Big7-8 are the major heparin binding sites on the C-terminus variable regions of LigB and LigA. (<b>B</b>) Binding of C4BP to different LigA and LigB tandem domain constructs is affected by heparin. Immobilized Lig protein constructs were incubated with C4BP mixed with different concentrations of heparin. C4BP bound to each Lig protein fragments in the absence of heparin was set as 100%. Each point represents the mean absorbance value at 492 nm or 630 nm +/- the SD of 3 independent experiments each performed in triplicate. Binding of heparin to LigA7-8, LigA8-13 and LigB 7–8, LigB7-12 were compared to LigA10-11 and LigB 9–10, respectively. ***p<0,001 (<b>C</b>) Estimation of the dissociation constant (K_d) for the interactions of LigAC and LigBC with heparin. K_d was calculated by fitting the data to the equation Y = Bmax*X/(K_d + X) using GraphPad Prism 5.0 (GraphPad Software, Inc.). Each data point represents the mean +/- SD of three trials in triplicate.</p

Binding of recombinant proteins LcpA, LigAC, LigBC and whole <i>L</i>. <i>interrogans</i> to recombinant mutant C4BP molecules.

<p>Microtiter plates were coated with LigAC (<b>A</b>), LigBC (<b>B</b>), LcpA (<b>C</b>) or LIC10301 (as negative control). After adding of each C4BP recombinant mutant or wild type (rec C4BP WT) protein (described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004192#pntd.0004192.g001" target="_blank">Fig 1B</a>), binding was measured using rabbit polyclonal anti-human C4BP and peroxidase-conjugated anti-rabbit IgG. Each point represents the mean absorbance value at 492 nm +/- the SD of 3 independent experiments each performed in triplicate. The interaction of recombinant proteins of <i>L</i>. <i>interrogans</i> with rec C4BP WT was set as 100% binding. (<b>D</b>) Binding of C4BP mutant proteins to whole <i>L</i>. <i>interrogans</i>. Leptospires (1x10⁸) were incubated with rec C4BP WT, C4BP mutants or PBS (negative control). To detect the C4BP binding to leptospires, polyclonal mouse anti-C4BP and FITC-conjugated anti-rabbit IgG were used. Each point represents the geometric mean fluorescence intensity (GMFI) +/- SE of 3 independent experiments each performed in triplicate. Data were analyzed using ANOVA test; *<i>p</i><0.05; ***<i>p</i><0.0001.</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

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

Image4.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

Image2.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