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A Comprehensive Approach to Identification of Surface- Exposed, Outer Membrane-Spanning Proteins of Leptospira interrogans Marija Pinne1,3*, David A. Haake2,3 1 Research Service, 151, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, United States of America, 2 Division of Infectious Diseases, 111F, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, United States of America, 3 Department of Medicine, The David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, California, United States of America 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 b-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 leptosp