The Novel Leptospiral Surface Adhesin Lsa20 Binds Laminin and Human Plasminogen and Is Probably Expressed during Infection

Abstract

Leptospirosis is an emerging infectious disease caused by pathogenic species of Leptospira. In this work, we report the cloning, expression, purification, and characterization of two predicted leptospiral outer membrane proteins, LIC11469 and LIC11030. The LIC11469 protein is well conserved among leptospiral strains, while LIC11030 was identified only in Leptospira interrogans. We confirmed by surface proteolysis of intact leptospires with proteinase K that these proteins are most likely new surface leptospiral proteins. The recombinant proteins were evaluated for their capacity to attach to extracellular matrix (ECM) components and to plasminogen. The leptospiral protein encoded by LIC11469, named Lsa20 (leptospiral surface adhesin of 20 kDa), binds to laminin and to plasminogen. The binding with both components was not detected when Lsa20 was previously denatured or blocked with anti-Lsa20 antibodies. Moreover, Lsa20 binding to laminin was also confirmed by surface plasmon resonance (SPR). Laminin competes with plasminogen for binding to Lsa20, suggesting the same ligand-binding site. Lsa20-bound plasminogen could be converted to enzymatically active plasmin, capable of cleaving plasmin substrate D-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Lsa20 was recognized by antibodies in confirmed-leptospirosis serum samples, suggesting that this protein is expressed during infection. Taken together, our results indicate that Lsa20 is a novel leptospiral adhesin that in concert with the host-derived plasmin may help the bacteria to adhere and to spread through the hosts.

Characterization of Three Novel Adhesins of Leptospira interrogans

“…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.…”

“…Some of these proteins were identified as novel leptospiral adhesins that might be involved in the first steps of host-Leptospira interaction. [8][9][10][11][12][13][14][15][16][17][18] After attachment, leptospires have to overcome tissue barriers and reach target organs. Proteolytic activity by plasmin (PLA) generation at the surface of Leptospira spp.…”

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

“…Lsa27, Lsa20, Lsa25, Lsa33 e Lsa26 são proteínas de leptospiras identificadas quanto à sua capacidade de interação com laminina (DOMINGOS et al, 2012;LONGHI et al, 2009;MENDES et al, 2011;SIQUEIRA et al, 2013), enquanto outras proteínas, como LipL53 e Lsa21, foram descritas com base na sua ligação à laminina, colágeno IV, fibronectina celular e plasmática. Além disso, a expressão dessas proteínas parece ser restrita a linhagens virulenta e, ainda, Lsa21 parece ser regulada por condição osmótica e temperatura, sugerindo o seu potencial para patogênese da bactéria (ATZINGEN et al, 2008;OLIVEIRA 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).…”