Abstract
Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K D, 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K D of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.
Highlights
Leptospirosis is a febrile disease caused by pathogenic spirochaetes of the genus Leptospira
Bioinformatic analysis The genes encoding LIC10258, LIC12880 and LIC12238 were identified by analysis of the genome sequences of the chromosome I of L. interrogans serovar Copenhageni and each one is present as a single copy [16]
Similar putative coding sequence of LIC12880 was found in L. interrogans serovar Lai (99% identity), in L. borgpetersenii serovar Hardjo-bovis (76% identity), but is absent in L. biflexa serovar Patoc
Summary
Leptospirosis is a febrile disease caused by pathogenic spirochaetes of the genus Leptospira. The disease became prevalent in cities with sanitation problems and large population of urban rodent reservoirs, which contaminate the environment through their urine [1,2]. Due to the broad spectrum of symptoms, such as, fever, chills, headache, and myalgias, and similarity with other tropical illness, leptospirosis often remains largely under diagnosed. Available vaccines, consisting of heat or chemically inactivated leptospires, named bacterins, provide serovar-specific protection against infection. The lack of serovar cross-protection in addition to the need for annual revaccination imposes a major limitation of whole-cell Leptospira vaccines [3]. The search for novel protein antigens that could elicit a broad protective and long-term immunity is currently under investigation
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