Abstract
The agents of leptospirosis, a zoonosis with worldwide distribution, are 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. Infection of accidental hosts, including humans, may result in life-threatening sequelae. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen virulence mechanisms and adaptation to environmental conditions, including those found in the mammalian host. Therefore, elucidation and characterization of the surface-exposed OMPs of Leptospira spp. is of great interest in the leptospirosis field. A thorough, multi-pronged approach for assessing surface exposure of leptospiral OMPs is essential. Herein, we present evidence for a sub-surface location for most or all of the major leptospiral lipoprotein, LipL32, based on surface immunofluorescence utilizing three different types of antibodies and four different permeabilization methods, as well as surface proteolysis of intact and lysed leptospires. We reevaluate prior evidence presented in support of LipL32 surface-exposure and present a novel perspective on a protein whose location has been misleading researchers, due in large part to its extraordinary abundance in leptospiral cells.
Highlights
Leptospirosis, a zoonosis caused by pathogenic Leptospira spp. transmitted from rodents and other reservoir hosts to humans via contaminated water, has a significant public health impact in tropical and sub-tropical regions [1,2,3,4,5]
Surface immunofluorescence assays utilizing these three different types of antibodies revealed that LipL32 was readily recognized by anti-LipL32 rabbit serum, monoclonal antibodies or affinity-purified antibodies from leptospirosis patient sera only after the OM was permeabilized by methanol (Fig. 3)
These data clearly demonstrate that LipL32 is not detected on the surface of intact L. interrogans by IFA (Fig. 3)
Summary
Leptospirosis, a zoonosis caused by pathogenic Leptospira spp. transmitted from rodents and other reservoir hosts to humans via contaminated water, has a significant public health impact in tropical and sub-tropical regions [1,2,3,4,5]. The two major types of leptospiral OMPs, outer membrane lipoproteins and transmembrane OMPs, differ significantly in their structure and how they are associated with the outer membrane. Lipoproteins become associated with membranes via a hydrophobic interaction between the N-terminal acyl moieties and the phospholipids of the lipid bilayer [9,10]. The bioinformatic algorithm, SpLip, is suitable for prediction of spirochetal protein lipidation but does not address the cellular destination of lipoproteins [11]
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