Surface localization of <i>L. interrogans</i> serovar Copenhageni strain Fiocruz L1–130 proteins by proteinase K treatment.

<p>Whole intact spirochetes were incubated with different concentrations of proteinase K, equivalents of 1×10⁸ of leptospires per lane separated by gel electrophoresis (Bis-Tris 4–12% NuPage gel, Novex), transferred to a PVDF membrane, and probed with polyclonal rabbit antisera against: (A) OmpL37; (B) OmpL47; (C) OmpL54; (D) OmpL36; (E) FlaA1 and LipL31; (F) LipL46. The identities of individual proteins are indicated on the right, and the positions of molecular mass standard (in kilodaltons) are indicated on the left.</p

Membrane affinity analysis of LipL32, LipL41, LipL46 and OmpL1.

<p>The membrane fraction of <i>L. interrogans</i> was treated with lysis buffer as a control or 0.1 M Na₂CO₃ (pH 11), 1.6 M urea, or 0.6 M NaCl for 15 min at 4°C. Samples were pelleted by centrifugation to separate the membrane pellet (P) and soluble supernatant (S), followed by gel electrophoresis (Bis-Tris 4–12% NuPage gel, Novex), and immunoblotting with specific antisera. Lane WC contained the whole cell unfractionated lysate of <i>L. interrogans</i>. The location of individual proteins are indicated on the right, and the positions of molecular mass standard (in kilodaltons) are indicated on the left.</p

Primers for amplification of new <i>ompL</i> genes.

a<p>Restriction sites are indicated in bold type.</p

Membrane affinity analysis of OmpL36, OmpL37, OmpL47 and OmpL54.

<p>Membrane fraction of <i>L. interrogans</i> was treated with lysis buffer as a control or 0.1 M Na₂CO₃ (pH 11), 1.6 M urea, or 0.6 M NaCl for 15 min at 4°C. Samples were pelleted by centrifugation to separate the pellets (P) and supernatants (S), followed by gel electrophoresis (Bis-Tris 4–12% NuPage gel, Novex), and immunoblotting with specific antisera. The identities of individual proteins are indicated on the right, and the positions of molecular mass standard (in kilodaltons) are indicated on the left.</p

Confirmation of subsurface locale of LipL32 by surface IFA and various outer-membrane permeabilization methods.

<p>Intact spirochetes or cells disrupted by methanol, vortexing and high-speed centrifugation, 2 mM EDTA or shear force were probed with affinity purified LipL32 antibodies from leptospirosis patient sera or FlaA2 rabbit serum as a control. The data is representation of three experiments performed separately. Binding of antibodies to leptospires were detected either with Alexa Fluor 488 conjugated goat anti-human IgG fragments (for LipL32) or Alexa Fluor 488 conjugated goat anti-rabbit IgG fragments (for FlaA2). A DAPI counterstain was used to demonstrate the presence of spirochetes. The identities of individual proteins recognized by the particular antibody reagent are indicated on the top of each column.</p

Purification and specificity of LipL32 antibodies from leptospirosis patient sera.

<p>(A) Affinity purification of LipL32-specific antibodies. Recombinant LipL32 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051025#pone.0051025-Haake4" target="_blank">[17]</a> was coupled to an AminoLink Plus column. Pooled convalescent sera from 23 individuals with laboratory-confirmed leptospirosis was added to the LipL32-affinity column. The chromatography products were analyzed by gel electrophoresis (Bis-Tris 4–12% NuPage gel, Novex), and Coomassie G250 staining. Abbreviations: LeptoPS, leptospirosis patient sera (pooled); FT, flow-through fraction; W, fraction after washing with PBS; E1-E4, eluted IgG fractions. (B) Extract of 1×10⁸ leptospires (lane WC) or 0.5 µg of recombinant LipL32 (lane rLipL32) were separated by gel electrophoresis, blotted onto PVDF membrane, and probed with affinity purified LipL32 IgG fraction E2 (1∶200).</p

Surface localization of <i>L. interrogans</i> serovar Copenhageni strain Fiocruz L1-130 proteins by protease K treatment.

<p>(A) Whole intact spirochetes were incubated with different concentrations of Proteinase K. 1×10⁸ of leptospires per lane were separated by gel electrophoresis (Bis-Tris 4–12% NuPage gel, Novex), transferred to a PVDF membrane, and probed with polyclonal rabbit antisera against: LipL32, OmpL47, OmpL37, FlaA2 and LipL31. (B) Whole intact leptospires and cells lysed with 50 mM Tris-HCL (pH 8.0), 100 mM NaCl, 2 mM EDTA, 0.2% SDS and boiling for 5 min were treated as above and probed with rabbit serum recognizing LipL32. The data is representation of four experiments performed separately. The identities of individual proteins are indicated on the right, and the positions of molecular mass standard (in kilodaltons) are indicated on the left.</p

Surface localization OmpL36, OmpL37, OmpL47, OmpL54, and OmpL1 by surface immunofluorescence assay (IFA).

<p>Intact or membrane-permeabilized spirochetes were probed with immune and pre-immune sera (when utilized). Binding of rabbit sera to leptospires were detected with Alexa Fluor 488 conjugated goat anti-rabbit IgG fragments. A DAPI counterstain was used to monitor the presence of spirochetes. The identities of individual proteins recognized by the particular antiserum are indicated on the left. All images are taken after 4 sec long exposure.</p

Analysis of biotinylated proteins from intact and lysed <i>Leptospira.</i>

<p>Proteins of <i>L. interrogans</i> serovar Copenhageni strain Fiocruz L1–130 were treated with Sulfo-NHS-LC-Biotin (BT) and equivalents of 1×10⁸ of leptospires per lane were separated on gel electrophoresis (Bis-Tris 4–12% NuPage gel, Novex). A whole cell lysate without Sulfo-NHS-LC-Biotin (lane WC), a total protein of intact (INTACT) or lysed (LYSED) leptospires after biotinylation (lanes TP) and material captured from biotinylated leptospires by streptavidin affinity gel (lanes STR). (A) Streptavidin blot. Proteins were blotted to PVDF membrane and the biotin labeled proteins detected by streptavidin horseradish peroxide (HRP) conjugate. (B) A Coomassie G-250 stained gel of samples described above. (C) Immunoblots with specific rabbit sera. The identities of individual proteins are indicated on the right, and the positions of molecular mass standard (in kilodaltons) are indicated on the left.</p

Reused from:

<p><u>PLoS One. 2011; 6(7): e21962</u>. Confocal microscopy was performed with live <i>L. interrogans</i> using antisera specific for LIC10258, LIC12880, LIC12238, LipL32 (surface-exposed lipoprotein) and GroEL (protoplasmic cylinder marker). FITC-conjugated secondary antibodies were used to detect the surface-bound antibodies (B). Leptospires were identified by propidium iodide (A) staining of the DNA. Co-localization is shown in the merged images (C).</p