In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

Abstract

BackgroundLeptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin.Methodology/Principal FindingsWe have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin.Conclusions/SignificancePLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.

Characterization of Three Novel Adhesins of Leptospira interrogans

“…Previous work by our group has shown that several proteins may probably act as PLG receptors. 8,15,16,20,40 We have also described leptospiral proteins that in addition to binding PLG can also interact with the complement regulator C4BP. 17,18 We evaluated whether the Lsa23, Lsa26, and Lsa36 proteins could can also interact with plasma components in vitro.…”

“…19 Furthermore, our group has identified several leptospiral proteins as PLG-binding receptors. 8,[15][16][17][18]20 Recently, we have reported two leptospiral proteins capable of binding the complement regulator C4BP, an interaction that might help the bacteria evade the immune system and increase survival. 17,18 In the present study, we report the characterization of three novel leptospiral proteins.…”

Functional and immunological evaluation of two novel proteins of Leptospira spp.

“…In our previous work, we showed that leptospires bind PLG on their surface and we also described several proteins that are probably PLG receptors (Vieira et al, 2010b). Thus, the recombinant proteins were assayed for their ability to adhere to human PLG in vitro.…”

“…P la s m a f ib r o n e c t in C e ll u la r f ib r o n e c t in E la s t in G e la t in B S A F e t u in serum serum recombinant proteins Fernandes et al, 2012;Mendes et al, 2011;Oliveira et al, 2011;Souza et al, 2012;Verma et al, 2010;Vieira et al, 2010b), a microplate was coated with Lsa44 or Lsa45, incubated with PLG, a urokinase (uPA)-type PLG activator and a PLAspecific chromogenic substrate (see Methods). The PLG bound to the proteins could be converted into PLA, as demonstrated indirectly by the specific proteolytic activity (Fig.…”

“…The detection of bound protein was performed as described above. For accessing the PLA generation from PLG bound to the recombinant proteins, we performed 96-well ELISAs as described previously (Vieira et al, 2010b).Effect of antibodies against Lsa44 and Lsa45 on leptospiral binding to laminin and PLG. L. interrogans serovar Copenhageni strain FIOCRUZ L1-130 was harvested from serum-depleted medium culture at 6000 g for 15 min, washed twice and resuspended in lowsalt PBS (lsPBS).…”