We now describe Lsa33 as a novel PLG – binding protein. Similar to the previously

reported proteins, bound PLG could be converted to plasmin by the addition of urokinase – type PLG activator (uPA), showing specific proteolytic activity. It is thus possible that the Lsa33 besides playing a role in the attachment to host and acting as PLG – receptor, may also help leptospires to surmount tissue barriers. The inhibitory effect exerted on the binding of leptospires to laminin and PLG by the recombinant proteins was statistically significant with both, in the case of Lsa33, and with laminin for the MCC950 Lsa25. The intensity of the interference upon the binding is expected given the presence of several ECM – or PLG-binding proteins in Leptospira. These data are comparable to the ones already reported in the literature [6, 7, 16–18, 21]. Partial inhibitory

effect was observed by EPZ5676 chemical structure laminin on the binding of Lsa33 to PLG, suggesting a competition for the same binding site. Conclusions We report in these studies a characterization of two leptospiral proteins, genome annotated as proteins of unknown function. The recombinant proteins Lsa33 and Lsa25 are laminin binding proteins that might be involved in the attachment to host. Moreover, both proteins showed the ability to bind C4bp, a feature suggesting their possible involvement in the immune evasion of leptospires. The recombinant Lsa33 is also PLG – binding protein that could help the bacteria during the infection process. Thus, it appears

that Lsa33 and to a lesser degree, Lsa25, are multifaceted proteins that might have multiple functions in the leptospiral pathogenesis. To date, Lsa33 is the first described laminin -, PLG – and C4bp – leptospiral binding protein. Rabusertib in vitro Methods Leptospira strains and sera The pathogenic Leptospira strains used were: L. interrogans serovar Canicola strain Hound Utrech IV, L. interrogans serovar Copenhageni strain M 20, L. interrogans serovar Hardjo strain Hardjoprajitno, L. interrogans serovar Icterohaemorrhagiae strain RGA, L. interrogans serovar Pomona strain Pomona, L. borgpetersenii serovar Whitcombi strain Whitcomb and serovar Grippothyphosa strain Moskva V, L. kirshneri serovar Cynoptery strain 3522 C, L. santarosai serovar Shermani strain 1342 K, L. noguchi serovar Panama strain CZ 214 and L. biflexa serovar Patoc strain Patoc, were PIK3C2G cultured at 28°C under aerobic conditions in liquid EMJH medium (Difco®) with 10% rabbit serum, enriched with L – asparagine (wt/vol: 0.015%), sodium pyruvate (wt/vol: 0.001%), calcium chloride (wt/vol: 0,001%), magnesium chloride (wt/vol: 0.001%), peptone (wt/vol:0.03%) and meat extract (wt/vol: 0.02%) (Turner LH. Leptospirosis. 3. Maintenance, isolation and demonstration of leptospires. Trans R Soc Trop Med Hyg 1970; 64: 623–646). Leptospira cultures are maintained in Faculdade de Medicina Veterinária e Zootecnia, USP, São Paulo, SP, Brazil.