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 β-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.

Characterization of Three Novel Adhesins of Leptospira interrogans

“…We performed a proteinase K accessibility assay by using a described assay 14,39 with some modifications. Live L. interrogans serovar Copenhageni strain M-20 was treated with 25 μg/mL of proteinase K, and aliquots of the bacterial suspensions were obtained at 0, 30, 60, 120, and 240 minutes.…”

Leptospira interrogans Catalase Is Required for Resistance to H 2 O 2 and for Virulence

“…Bioinformatic analysis to predict the cellular location of KatE was performed by using pSORTb v3.0 (66). The experimental subcellular localization of KatE in Leptospira was determined by Triton X-114 solubilization of the outer membrane and subsequent fractionation into detergent (DET) and aqueous (AQ) phases, as described previously (21,48,68), except that fractionation was performed on approximately 4 ϫ 10 10 cells at a concentration of 5 ϫ 10 9 bacteria/ml. Immunoblot analyses.…”

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

“…Such a result indicates that either the protein is in a sub-surface location (with some non-specific background staining of intact cells), the antibodies are binding preferentially to non-native epitopes that are exposed after methanol permeabilization or a protein might have multiple localization sites as described for Erp and Elp proteins of another spirochete, Borrelia burgdorferi 14 . In either case, an ambiguous IFA result such as this requires alternative approaches such as surface biotinylation or surface proteolysis to determine whether the protein of interest is surface-exposed or not 10 . …”

“…A green fluorescence filter was used to detect Alexa Fluor 488 labeled secondary antibody binding to surface exposed proteins and blue/cyan fluorescence filter to detect DAPI binding to cell DNA. A) Leptospires probed with antibodies recognizing a surface exposed protein, OmpL54 10 . B) Cells probed with antibodies against a periplasmic subsurface protein, FlaA1 (negative control).…”