Characterization of Novel OmpA-Like Protein of Leptospira interrogans That Binds Extracellular Matrix Molecules and Plasminogen

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.

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

“…Circular dichroism (CD) spectroscopy. Purified recombinant proteins were dialysed against sodium phosphate buffer (pH 7.4) and CD spectroscopy measurements were performed as described previously (Oliveira et al, 2011). The residual molar ellipticity was expressed in degree cm 2 dmol 21 .…”

“…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.…”

Avaliação e caracterização de candidatos vacinais voltados para o controle da leptospirose.

“…A proteína Lsa66, outra proteína com domínio OmpA-like, quando teve seu papel avaliado quanto à interação com ECM, demonstrou propriedades de interação com laminina. Além disso, essa proteína foi reconhecida pelo soro de indivíduos contaminados, sugerindo sua expressão durante a infecção (OLIVEIRA et al, 2011). Outra proteína identificada com domínio OmpA-like foi denominada de OmpA70, porém seu papel na adesão a ECM não foi investigado (FRAGA et al, 2010).…”

“…Além disso, foi demonstrado que a atividade de plasmina pode conferir alguma vantagem relacionada à evasão imune, visto que plasmina gerada na superfície das leptospiras foi capaz de interferir na deposição de IgG e C3b na superfície da bactéria, reduzindo o processo de opsonização (VIEIRA et al, 2011). Diante da importância desse sistema, diversas proteínas de membrana de leptospiras têm sido avaliadas quanto à interação com PLG, sendo que uma quantidade bastante expressiva de proteínas foi identificada como receptores de PLG (DOMINGOS et al, 2012;FERNANDES et al, 2014;FERNANDES et al, 2012;MENDES et al, 2011;OLIVEIRA et al, 2011;SIQUEIRA et al, 2013;SIQUEIRA et al, 2016;SOUZA et al, 2012;VIEIRA et al, 2010a;.…”

“…Para isso, laminina, fibronectina celular, elastina, colágeno tipo I, colágeno tipo IV e as proteínas controles BSA e fetuína foram imobilizados em placas de microdiluição e a ligação das proteínas recombinantes foi avaliada por ELISA, após o uso dos anticorpos policlonais obtidos contra cada proteína recombinante. Várias proteínas de leptospiras com propriedades de ligação específica a laminina e a outros componentes da ECM foram identificadas pelo nosso grupo (ATZINGEN et al, 2008;ATZINGEN et al, 2009;BARBOSA et al, 2006;DOMINGOS et al, 2015;DOMINGOS et al, 2012;FERNANDES et al, 2014;FERNANDES et al, 2012;LONGHI et al, 2009;MENDES et al, 2011;OLIVEIRA et al, 2011;OLIVEIRA et al, 2010; __________________________________________________________Resultados e Discussão 113 2013; SOUZA et al, 2012;VIEIRA et al, 2010b). Outros grupos também identificaram proteínas com capacidade de adesão (CARVALHO et al, 2009;CHOY et al, 2007;HAUK et al, 2008;HOKE et al, 2008;LIN et al, 2009;STEVENSON et al, 2007).…”