No abstract

No abstract

Leptospirosis is the most widespread zoonosis and also a major cause of economic loss in animal production worldwide. It has been considered a major emerging infectious disease in the last ten years and it is included in the list of Neglected Tropical Diseases, according to the World Health Organization, with approximately 1 million cases per year worldwide. In urban areas, rodents are the main reservoirs of the disease; leptospires colonize the proximal renal tubules and are excreted live in urine. To date, there is no effective vaccine. The identification of outer membrane proteins, conserved among pathogenic strains, is the main focus of research to elucidate the mechanisms of pathogenicity. These proteins probably mediate the interaction of leptospires with the external environment, and can also serve as targets for the host immune system. The study of surface antigens, not yet described in Leptospira interrogans literature, is intriguing and may indicate an unprecedented knowledge in the initial pathogen-host interactions. It also represents new alternatives in the search for prophylactic or serodiagnosis candidates. The project proposed: cloning and expression of two predicted lipoproteins, encoding by the LIC13059 and LIC10879 genes, identified by bioinformatics the genome sequences of L. interrogans serovar Copenhageni; Structural characterization by circular dichroism; evaluation of the presence and conservation of the native proteins in different species and serovars of Leptospira spp.; determining the location of the native proteins in bacteria; characterization of the recombinant proteins interacting with tissue and plasma components of the host. The genes were amplified without the signal peptide and the recombinant proteins were expressed in Escherichia coli, in insoluble form, as inclusion bodies. After refolding and purification, circular dichroism analysis showed that the proteins have secondary structure. The proteins encoded by the genes are shown to be conserved in the pathogenic strains and absent in saprophytic one. The LIC13059 protein was assessed as surface exposed as well as the dimer form of LIC10879 in the assay of proteinase K. The two recombinant proteins were recognized by confirmed humam leptospirosis serum samples, suggesting that they are expressed during the infection. Recombinant proteins interact with plasminogen, fibrinogen and laminin. Because of the bind with laminin in a dose-dependent and saturable manner, the rLIC13059 and rLIC10879 were named Lsa25.6 and Lsa16 (Leptospiral Surface Adhesin, followed by their respective molecular masses), respectively. In addition, the protein Lsa16 interacted with e-cadherin, a mammalian cell receptor. These two recombinant proteins are able to recruit plasminogen from the normal human serum. The plasminogen captured by both recombinant proteins could be converted into plasmin, a mechanism that could help bacterial penetration in the host. Both proteins interacted with fibrinogen but only Lsa25.6 was capable of inhibiting the formation o...