Binding of leptospiral EF-Tu to human plasminogen.

<p>(<b>A</b>) Binding of EF-Tu to plasminogen as a function of protein concentration by ELISA. EF-Tu and the positive and negative control proteins LipL32 and LIC10301 (0 - 2 μM) were allowed to interact with immobilized plasminogen (10 μg/mL), and were detected using specific antisera, followed by peroxidase-conjugated secondary antibodies. (<b>B</b>) Role of lysines in EF-Tu/plasminogen interaction. Plasminogen (10 μg/mL) was added to EF-Tu-coated wells in the presence (0.1 - 10 mM) or absence of ε-aminocaproic acid. Bound plasminogen was detected with a specific monoclonal antibody followed by peroxidase-conjugated anti-mouse IgG. In (<b>A</b>) and (<b>B</b>) each point represents the mean absorbance value at 492 nm ± the standard deviation of three independent experiments, each performed in duplicate. (* <i>p</i> < 0.05). .</p

Leptospiral EF-Tu interacts with the complement regulator FH and bound-FH remains functionally active.

<p>(<b>A</b>) Purified recombinant proteins were subjected to SDS–PAGE, and transferred to a nitrocellulose membrane. The membrane was incubated with 7% NHS as a source of FH, and probed with polyclonal goat antibodies recognizing human FH, followed by secondary HRP-conjugated antibodies. LigBC (56 kDa) and LIC10301 (13 kDa) were included as positive and negative controls, respectively. (<b>B</b>) <b>Cofactor activity of FH bound to EF-Tu</b>. The recombinant proteins EF-Tu, LigBC, and LIC10301 (10 μg/mL) were immobilized on microtiter plates and incubated with purified FH (2 μg). Control reactions in which we omitted FH were also included. After washing, C3b and FI were added. The reactions were incubated for 1, 2, and 4 h at 37°C. The products were analyzed by SDS-PAGE and the cleavage fragments of C3b were detected by Western blotting with anti-human C3 polyclonal. The presence of bands of 43 and 68 kDa indicates that acquired FH was able to promote FI-mediated cleavage of C3b. LIC10301 was used as a negative control since this protein does not bind FH and LigBC was included as a positive control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081818#B18" target="_blank">18</a>].</p

Cellular localization of EF-Tu on <i>Leptospira interrogans</i>.

<p>(<b>A</b>) Surface immunofluorescence assay was performed using confocal microscopy (Zeiss LSM-510 Meta). Intact leptospires were probed with anti-EF-Tu, anti-LigA/B or preimmune mouse serum. Alexa Fluor 488 conjugated goat anti-mouse IgG was used to detect bound antibodies. A iodide propidium counterstain (low panels) was used to demonstrate the presence of leptospires. (<b>B</b>) Immunoelectron microscopy analysis. Leptospires were incubated with anti-EF-Tu or pre-immune mouse serum, followed by treatment with colloidal-gold-conjugated anti-mouse IgG. Analysis was performed using an electron transmission microscope (LEO 906E - Leica Microsystems BmgH, Germany).</p

Immunoblot analysis with EF-Tu antiserum to different <i>Leptospira</i> species.

<p>Lanes contain whole-cell lysates of <i>L. biflexa</i> serovar Patoc (lane 1), <i>L. noguchii</i> serovar Panama (lane 2), <i>L. borgpetersenii</i> serovar Javanica (lane 3), <i>L. borgpetersenii</i> serovar Tarassovi (lane 4), <i>L. kirschneri</i> serovar Cynopteri (lane 5), <i>L. interrogans</i> serovar Copenhageni (lane 6), <i>L. interrogans</i> serovar Pomona (lane 7), <i>L. santarosai</i> serovar Shermani (lane 8), and recombinant EF-Tu (lane 9). </p

Binding of EF-Tu to ECM components.

<p>Wells were coated with 10 μg/mL of collagen type I (CI), collagen type IV (CIV), cellular fibronectin (FC), plasma fibronectin (FP), laminin (LAM), elastin (ELA), fibrinogen (FIB), plasminogen (PLG) and the control protein fetuin (FET). Recombinant protein attachment to those ECM macromolecules was assessed by ELISA. One microgram of recombinant EF-Tu protein was added per well. LigBC and LIC10301 were included as positive and negative controls, respectively. LipL32 was included as a positive control for plasminogen [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081818#B34" target="_blank">34</a>]. Bound proteins were detected using mouse specific antisera to the recombinant proteins, followed by peroxidase-conjugated secondary antibodies. Each point represents the mean absorbance value at 492 nm ± the standard deviation of three independent experiments, each performed in duplicate. Binding of EF-Tu to each ECM component was compared to the binding of LIC10301 to these molecules by the two-tailed t test (* <i>p</i> < 0.05). </p

Table4.XLSX

<p>Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.</p

Table5.DOCX

<p>Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.</p

Table1.XLSX

<p>Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.</p

Table3.XLSX

<p>Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.</p

Table2.XLSX

<p>Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.</p