Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV
Introduction
Leptospirosis is a life threatening disease caused by pathogenic spirochetes of the genus Leptospira. The transmission of leptospirosis has been associated with exposure of individuals in close proximity to wild or farm animals.1, 2 Lately, the disease became prevalent in cities with sanitation problems and large population of urban rodent reservoirs that contaminate the environment through their urine.3 Fever, chills, headache, and severe myalgias characterize the early phase of the disease. Progression to multi-organ system complications occurs in 5–15% of cases, with mortality rates of 5–40%.1, 4 Although leptospirosis can be treated, an early diagnosis is critical for an effective antibiotic therapy. The gold standard reference method for serological diagnosis of the disease is the microscopic agglutination test (MAT) in which sera from patients are reacted with live antigen suspensions of leptospiral serovars.1, 5 However, MAT serology has low sensitivity in the early phase of the disease since it relies on antibodies to leptospiral antigens not detected in the first days of post-exposure.6, 7 Leptospirosis also has a great economic impact in the agricultural industry since the disease affects livestock inducing abortions, stillbirths, infertility, reduced milk production and death.1, 4 Commercially available vaccines, consisting of heat or chemically inactivated leptospires provide serovar-specific protection against infection.1, 8 The lack of serovar cross-protection in addition to the need for annual revaccination has limited the usefulness of whole-cell Leptospira vaccines. The search for novel protein antigens that could promote cross-protective and long-term immunity has been pursued.5
Surface exposed proteins are potential targets for inducing immune responses during infection and may also mediate the initial adhesion process to host cells.9, 10, 11, 12, 13, 14, 15, 16 The genomes of pathogenic Leptospira have been sequenced17, 18, 19 and in silico analysis identified more than 200 predicted outer membrane proteins.18, 20 We have used proteomic studies in an attempt to narrow down our search for new surface antigens.21 Proteomic analysis using virulent, low-passage Leptospira interrogans serovar Pomona, allowed us to identify several proteins that were assigned as hypothetical in genome annotation and predicted to be outer membrane by bioinformatics analysis. One of these proteins encoded by the gene LIC10314 contains a p83/100 domain that is conserved in antigens broadly distributed in the spirochetes Borrelia and Treponema spp. Recombinant antigens containing p83/100 domain have been claimed to be useful for serodiagnosis of Lyme borreliosis.22, 23 Protein containing this domain was also identified in the genome sequences of L. interrogans serovar Lai17 and in both strains of Leptospira borgpertensenii.17 Thus, we decided to characterize the protein encoded by the LIC10314 gene. The gene was cloned and the protein expressed using E. coli as a heterologous host system. Assessment of 63 kDa purified recombinant protein against serum samples of confirmed-leptospirosis patients showed that this protein is recognized by antibodies in serum samples of individuals in convalescent phase of the disease. The ability of this protein to mediate attachment to various extracellular matrix (ECM) components was evaluated. We have found that this novel leptospiral protein binds strongly to laminin and collagen IV. The gene coding for Lsa63 (leptospiral surface adhesin of 63 kDa) is expressed on the surface of bacteria because it is detected by immunofluorescence assay with intact living leptospires. It is thus possible that this surface protein may participate in pathogenesis of Leptospira.
Section snippets
ECM components
All macromolecules, including the control protein fetuin, were purchased from Sigma Chemical Co. (St. Louis, Mo.). Laminin-1 and collagen Type IV were derived from the basement membrane of Engelbreth–Holm–Swarm mouse sarcoma, cellular fibronectin was derived from human foreskin fibroblasts, plasma fibronectin was isolated from human plasma and collagen Type I was isolated from rat tail.
Leptospira strains and sera
The pathogenic Leptospira strains used: L. interrogans serovar Canicola strain Hound Utrech IV, L. interrogans
Bioinformatic analysis
Several membrane proteins, genome annotated as hypothetical, were identified by proteomic studies.21 One protein encoded by the gene LIC10314 was identified in the chromosome I of the genome sequences of L. interrogans serovar Copenhageni18, 20 and was selected for further characterization because it contains a p83/100 conserved domain broadly distributed in protein antigens of Borrelia and Treponema spp.22, 37, 38 The p83/100 domains in LIC10314 CDS are depicted in Fig. 1A. Also shown in this
Discussion
In an effort to complement the data obtained by genome annotation of L. interrogans17, 18 and to narrow down our search for membrane proteins, we carried out proteomic studies with low passage, virulent strain of Leptospira.21 Among the proteins identified we opted for the one encoded by the gene LIC10314, annotated as hypothetical and presenting p83/100 conserved domain. The p83/100 sequence tag is broadly distributed in the spirochetes, particularly in Borrelia spp.22, 37 but also in Treponema
Acknowledgements
We are deeply indebted to Dr. Toshie Kawano andAlexsander Seixas de Souza (Departamento de Parasitologia, Instituto Butantan), São Paulo, Brazil) for use of Confocal facilities and helpful discussion. This work was supported by FAPESP, CNPq and Fundação Butantan, Brazil; MLV has a fellowship from FAPESP (Brazil).
References (56)
- et al.
Urban epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group
Lancet
(1999) - et al.
Overview of the epidemiology, microbiology, and pathogenesis of Leptospira spp. in humans
Microbes Infect
(2000) - et al.
Identification of a 36-kDa fibronectin-binding protein expressed by a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae
FEMS Microbiol Lett
(2000) Leptospirosis. 3. Maintenance, isolation and demonstration of leptospires
Trans R Soc Trop Med Hyg
(1970)- et al.
Molecular cloning and characterization of a novel leptospiral lipoprotein with OmpA domain
FEMS Microbiol Lett
(2003) - et al.
Leptospira and leptospirosis
(1999) Leptospirosis
Clin Microbiol Rev
(2001)Usefulness of serologic analysis as a predictor of the infecting serovar in patients with severe leptospirosis
Clin Infect Dis
(2003)- et al.
Assessment of the efficacy of an IgM-elisa and microscopic agglutination test (MAT) in the diagnosis of acute leptospirosis
Am J Trop Med Hyg
(1999) - et al.
Evaluation of four commercially available rapid serologic tests for diagnosis of leptospirosis
J Clin Microbiol
(2003)