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

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

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

Muscle histology score.

<p>Legs and paravertebral regions fragments were collected from guinea pigs injected intraperitoneally with recombinant proteins or PBS (control) and stained with H&E and Gomori´s trichrome. The muscular lesions were graded as described in the Material and Methods. Data are means ± SEM.</p

Biochemical parameters of renal function in guinea pigs injected intraperitoneally with recombinant proteins or PBS (control).

<p>(A) GFR evaluated using creatinine clearance. (B) Urinary volume values. (C) Serum potassium values. Data are means ± SEM.</p

Expression in <i>Leptospira</i> species and surface localization of the Lp25 protein.

<p>(A) Whole-cell lysates were analyzed by immunoblotting using anti-LipL32 and anti-Lp25 antisera. Saprophytic <i>Leptospira biflexa</i> serovar Patoc strain Patoc I (Lane 1) and pathogenic species: <i>L</i>. <i>noguchii</i> serovar Panama strain CZ 214K (Lane 2), <i>L</i>. <i>borgpetersenii</i> serovar Javanica strain Veldrat Batavia 46 (Lane 3), <i>L</i>. <i>borgpetersenii</i> serovar Tarassovi strain 17 (Lane 4), <i>L</i>. <i>kirschneri</i> serovar Cynopteri strain 3522C (Lane 5), <i>L</i>. <i>interrogans</i> serovar Hardjo strain Hardjoprajitno (Lane 6), <i>L</i>. <i>interrogans</i> serovar Pomona strain 13A (Lane 7), and <i>L</i>. <i>interrogans</i> serovar Copenhageni strain L1-130 (Lane 8). (B) Triton X-114 fractions of <i>L</i>. <i>interrogans</i> serovar Copenhageni strain L1-130 were analyzed by immunoblotting with anti-Lp25 antiserum. As controls, membranes were probed with antisera against LigA (surface protein), LipL31 (cytoplasmatic protein) and LipL32 (subsurface protein). The fractions analyzed were whole lysate (W), Triton X-114-insoluble pellet (P), the aqueous phase (A), and the detergent phase (D). (C) Proteinase K accessibility assay. Whole intact leptospires were incubated with different concentrations of proteinase K and processed for immunoblot analyses using antibodies against Lp25 or LipL32.</p

Lp25 membrane protein from pathogenic <i>Leptospira</i> spp. is associated with rhabdomyolysis and oliguric acute kidney injury in a guinea pig model of leptospirosis

<div><p>Acute kidney injury (AKI) from leptospirosis is frequently nonoliguric with hypo- or normokalemia. Higher serum potassium levels are observed in non-survivor patients and may have been caused by more severe AKI, metabolic disarrangement, or rhabdomyolysis. An association between the creatine phosphokinase (CPK) level and maximum serum creatinine level has been observed in these patients, which suggests that rhabdomyolysis contributes to severe AKI and hyperkalemia. LipL32 and Lp25 are conserved proteins in pathogenic strains of <i>Leptospira</i> spp., but these proteins have no known function. This study evaluated the effect of these proteins on renal function in guinea pigs. Lp25 is an outer membrane protein that appears responsible for the development of oliguric AKI associated with hyperkalemia induced by rhabdomyolysis (e.g., elevated CPK, uric acid and serum phosphate). This study is the first characterization of a leptospiral outer membrane protein that is associated with severe manifestations of leptospirosis. Therapeutic methods to attenuate this protein and inhibit rhabdomyolysis-induced AKI could protect animals and patients from severe forms of this disease and decrease mortality.</p></div

Biochemical parameters of rhabdomyolysis in guinea pigs injected intraperitoneally with recombinant proteins or PBS (control).

<p>(A) Serum creatine phosphokinase values. (B) Serum phosphate values. (C) Uric acid values. Data are means ± SEM.</p