Elsevier

Journal of Infection

Volume 60, Issue 1, January 2010, Pages 52-64
Journal of Infection

Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV

https://doi.org/10.1016/j.jinf.2009.10.047Get rights and content

Summary

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63 kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis.

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).

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