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.
Highlights
The etiologic agents of leptospirosis, pathogenic Leptospira spp., have a significant impact on public health throughout the developing world [1,2,3]
Application of the bioinformatic criteria described in Material and Methods led to the selection of OmpL36 (LIC13166), OmpL37 (LIC12263), OmpL47 (LIC13050), and OmpL54 (LIC13491) for further study, designated according to their apparent molecular mass determined by gel electrophoresis
The expression of OmpL36, OmpL37, OmpL47 and OmpL54 in whole cell lysates of L. interrogans serovar Copenhageni strain Fiocruz L1–130 cultivated in vitro was confirmed by immunoblot analysis using antisera raised against the respective recombinant proteins (Fig. 1A)
Summary
The etiologic agents of leptospirosis, pathogenic Leptospira spp., have a significant impact on public health throughout the developing world [1,2,3]. Especially rodents, serve as reservoir hosts in the transmission of pathogenic Leptospira spp. to humans. Exposure of mucous membranes or broken skin to water or soil contaminated with leptospires shed in animal urine can lead to a potentially fatal infection, characterized by jaundice, renal failure, and/or pulmonary hemorrhage [1,3,4]. Large outbreaks of leptospirosis occur in tropical and subtropical regions after heavy rainfall and dispersal of leptospires in contaminated water [2,5]. One approach to infection control involves vaccines based on lipopolysaccharide (LPS), which dominates the leptospiral cell surface and can elicit protective immunity [6,7]. Leptospiral outer membrane proteins (OMPs) are generally well conserved [8,9] and would have the potential advantage of inducing comprehensive immunity [10]
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