A comprehensive approach to identification of surface-exposed, outer membrane-spanning proteins of Leptospira interrogans

Abstract

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via fresh water and colonization of the renal tubules of their reservoir hosts or infection of accidental hosts, including humans. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in virulence mechanisms of pathogens and the adaptation to various environmental conditions, including those of the mammalian host. Little is known about the surface-exposed OMPs in Leptospira, particularly those with outer membrane-spanning domains. Herein, we describe a comprehensive strategy for identification and characterization of leptospiral transmembrane OMPs. The genomic sequence of L. interrogans serovar Copenhageni strain Fiocruz L1-130 allowed us to employ the beta-barrel prediction programs, PRED-TMBB and TMBETA-NET, to identify potential transmembrane OMPs. Several complementary methods were used to characterize four novel OMPs, designated OmpL36, OmpL37, OmpL47 and OmpL54. In addition to surface immunofluorescence and surface biotinylation, we describe surface proteolysis of intact leptospires as an improved method for determining the surface exposure of leptospiral proteins. Membrane integration was confirmed using techniques for removal of peripheral membrane proteins. We also demonstrate deficiencies in the Triton X-114 fractionation method for assessing the outer membrane localization of transmembrane OMPs. Our results establish a broadly applicable strategy for the elucidation of novel surface-exposed outer membrane-spanning proteins of Leptospira, an essential step in the discovery of potential virulence factors, diagnostic antigens and vaccine candidates.

Figure 1. Localization of OmpL36, OmpL37, OmpL47 and OmpL54 after detergent fractionation of L. interrogans Fiocruz L1–130.

Equivalents of 1×10⁸ of leptospires per lane or 0.5 µg of recombinant proteins per lane are separated on gel electrophoresis (Bis-Tris 4–12% NuPage gel, Novex), blotted to PVDF membrane and probed with rabbit immune sera. L. interrogans serovar Copenhageni strain Fiocruz L1–130 whole cell lysate (lane WC), the aqueous fraction (lane AQ), the protoplasmic cylinder fraction (lane PC) and the detergent fraction (lane DET). rOmpL36, rOmpL37, rOmpL47, and rOmpL54 denote the corresponding recombinant proteins. (A) Membranes probed with OmpL36, OmpL37, OmpL47 and OmpL54 antisera. (B) Membrane probed with ImpL63, FlaA1 and LipL32 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.

Figure 2. Surface localization of L. interrogans serovar Copenhageni strain Fiocruz L1–130 proteins by proteinase K treatment.

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.

Figure 3. Surface localization OmpL36, OmpL37, OmpL47, OmpL54, and OmpL1 by surface immunofluorescence assay (IFA).

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.

Figure 4. Analysis of biotinylated proteins from intact and lysed Leptospira.

Proteins of L. interrogans 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.

Figure 5. Membrane affinity analysis of OmpL36, OmpL37, OmpL47 and OmpL54.

Membrane fraction of L. interrogans 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.