The overall goal of this proposal is to define the leptospiral surface proteome and the relevance of post-translational modifications to immunity. We have identified a number of surface-exposed lipoproteins that are expressed during infection of the mammalian host. However, many leptospiral surface lipoproteins remain to be identified and those that are known appear to undergo extensive post-translational modifications that likely affect recognition by the host immune system. Lipoproteins are dominant leptospiral surface antigens. The genome of Leptospira interrogans serovar Copenhageni encodes approximately 168 lipoproteins. We have described a number of these lipoproteins, localized them to either the inner or outer membrane, and determined whether they are surface exposed. L. interrogans has genes encoding two possible lipoprotein export pathways: The LOL pathway and Type II secretion. Methods for leptospiral genetic manipulation are now available to determine the signals required to target lipoproteins to the outer membrane and leptospiral surface, as has recently been achieved for the lipoproteins of Borrelia burgdorferi by Wolfram Z?ckert, who is an export on spirochetal surface lipoprotein export pathways and a co-investigator on this proposal. Recent proteomic studies, including those performed in collaboration with co-investigator Caroline Cameron, reveal that many leptospiral surface proteins undergo post-translational modification, particularly by methylases. We now have evidence that the major outer membrane lipoprotein, LipL32, undergoes extensive differential methylation during infection. This would explain why recombinant LipL32 produced in E. coli is ineffective as a vaccine, even though it is an abundant surface lipoprotein. Understanding the nature of surface lipoprotein methylation provides an opportunity to create effective methylated peptide vaccines that target lipoprotein surface epitopes expressed during infection. The Research Plan has the following three Specific Aims: #1. What is the leptospiral surface lipoprotein export pathway? Our hypothesis is that, as in B. burgdorferi, leptospiral lipoproteins are exported to the leptospiral surface via the LOL export pathway. We will test this hypothesis by transforming L. interrogans with genes encoding lipoprotein-GFP fusions and test their susceptibility to surface proteolysis. We will determine the length of the tether needed for targeting lipoproteins to the surface and the role of negative-charged amino acids in preventing surface localization. #2. How does in vivo LipL32 methylation alter its surface epitopes? Our hypothesis is that increased methylation during infection alters the antigenic character of LipL32. We will isolate organisms from infected tissues and further define LipL32 sites that become methylated during infection. Those sites that are predicted to be surface-exposed based on the LipL32 crystal structure will be tested for recognition by infection-derived antibodies and T-cells. #3. Which methylated peptides are most effective at inducing protective immunity? Methylated peptides that are highly recognized by infection-derived antibodies and T-cells will be examined as immunoprotective antigens in the hamster model of leptospirosis. In vitro assays examining adherence inhibition, growth inhibition, bactericidal activity, and opsonophagocytosis will be performed to determine mechanisms of protective immunity.

Public Health Relevance

Leptospirosis is a widespread and important infection affecting homeless veterans in the United States and military personnel deployed to parts of the world with substandard housing. Leptospirosis can cause kidney failure and is potentially fatal. We will study how Leptospira interrogans, the causative agent of leptospirosis, alters its lipoproteins to escape the immune response during infection and exports these lipoproteins to the bacterial surface. We will use this information to develop safe and effective subunit protein-based vaccines that enable the immune system to recognize and kill leptospires at an early stage of the infection.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Infectious Diseases B (INFB)
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VA Greater Los Angels Healthcare System
Los Angeles
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Wunder Jr, Elsio A; Figueira, Claudio P; Santos, Gisele R et al. (2016) Real-Time PCR Reveals Rapid Dissemination of Leptospira interrogans after Intraperitoneal and Conjunctival Inoculation of Hamsters. Infect Immun 84:2105-2115
Lourdault, Kristel; Matsunaga, James; Haake, David A (2016) High-Throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants during Acute Infection. PLoS Negl Trop Dis 10:e0005117
Fouts, Derrick E; Matthias, Michael A; Adhikarla, Haritha et al. (2016) What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira. PLoS Negl Trop Dis 10:e0004403
Haake, David A; Levett, Paul N (2015) Leptospirosis in humans. Curr Top Microbiol Immunol 387:65-97
Haake, David A; Z├╝ckert, Wolfram R (2015) The leptospiral outer membrane. Curr Top Microbiol Immunol 387:187-221
Narayanavari, Suneel A; Lourdault, Kristel; Sritharan, Manjula et al. (2015) Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans. PLoS Negl Trop Dis 9:e0003952
Evangelista, Karen V; Hahn, Beth; Wunder Jr, Elsio A et al. (2014) Identification of cell-binding adhesins of Leptospira interrogans. PLoS Negl Trop Dis 8:e3215
Lourdault, Kristel; Wang, Long-Chieh; Vieira, Ana et al. (2014) Oral immunization with Escherichia coli expressing a lipidated form of LigA protects hamsters against challenge with Leptospira interrogans serovar Copenhageni. Infect Immun 82:893-902
Coutinho, Mariana L; Matsunaga, James; Wang, Long-Chieh et al. (2014) Kinetics of Leptospira interrogans infection in hamsters after intradermal and subcutaneous challenge. PLoS Negl Trop Dis 8:e3307
Witchell, Timothy D; Eshghi, Azad; Nally, Jarlath E et al. (2014) Post-translational modification of LipL32 during Leptospira interrogans infection. PLoS Negl Trop Dis 8:e3280

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