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

Abstract

This work shows the production and characterization of two novel putative lipoproteins encoded by the genes LIC10645 and LIC10731 identified in the genome sequences of Leptospira interrogans. In silico conservation analysis indicated that the proteins are well conserved among pathogenic leptospiral serovars and species. Recombinant proteins were obtained in Escherichia coli BL21(DE3) Star pLysS strain, purified by metal-affinity chromatography, and used for characterization and immunological evaluations. Recombinant proteins were capable of eliciting a combination of humoral and cellular immune responses in animal models, and could be recognized by antibodies present in human serum samples. The recombinant proteins Lsa44 and Lsa45 were able to bind laminin, and were named Lsa44 and Lsa45 for leptospiral surface adhesins of 44 and 45 kDa, respectively. The attachment to laminin was dose-responsive with K D values of 108.21 and 250.38 nM for Lsa44 and Lsa45, respectively. Moreover, these proteins interact with plasminogen (PLG) with K D values of 53.56 and 36.80 nM, respectively. PLG bound to the recombinant proteins could be converted to plasmin (PLA) in the presence of an activator. Cellular localization assays suggested that the Lsa44 and Lsa45 were surface-exposed. These are versatile proteins capable of interacting with laminin and PLG/PLA, and hence could mediate bacterial adhesion and contribute to tissue penetration. INTRODUCTIONLeptospirosis is a zoonosis of global importance caused by pathogenic bacterial species of the genus Leptospira (Bharti et al., 2003;Faine et al., 1999). In urban environments, rodents are the main host reservoirs of leptospires, shedding live bacteria through their urine (Ko et al., 1999;Vinetz et al., 1996). Humans are infected via contact with urine of wild or domestic animal carriers, either directly or indirectly through contaminated water or soil (Adler & de la Peña Moctezuma, 2010). The disease presents a broad spectrum of symptoms, including fever, vomiting, headache, diarrhoea, and abdominal and generalized muscle pain. Due to these flu-like signs, leptospirosis remains under-diagnosed. Progression to multiorgan system complications, known as Weil's syndrome, occurs in 5-15 % of cases, with mortality rates of 5-40 % (Faine et al., 1999;Ko et al., 1999;Levett, 2001; Plank & Dean, 2000).Currently available commercial vaccines are based on inactivated whole-cell or membrane preparations of pathogenic leptospires, named bacterins. They confer protective responses mostly through the induction of antibodies against LPS antigens (Adler & de la Peña Moctezuma, 2010; de la Peña-Moctezuma et al., 1999). Nevertheless, these vaccines do not induce long-term protection against infection and do not provide cross-protective immunity against leptospiral serovars not included in the vaccine preparation (Adler & de la Peña Moctezuma, 2010). Due to the large number of serovars (Bharti et al., 2003), the search for conserved and protective antigens is being pursued (Koizumi & Watanabe, 2005...

Induction of Boosted Immune Response in Mice by Leptospiral Surface Proteins Expressed in Fusion with DnaK

“…This improved effect is particularly evident with the recombinant Lsa21, a protein that has been previously shown to be present only in low passage, virulent strain of Leptospira , with a possible role in pathogenesis [18]. Similar to the data we have previously obtained with the adhesin Lsa44 of Leptospira [41], these fusion proteins did not induce either TNF-alfa or IL-4. IL-10 was described to have a special role in restraining and preventing an excessive immune response and collateral damage [42].…”

“…We have previously shown that OmpL1 of L. interrogans stimulated both Th1 and Th2 immune response in BALB/c mice [40]. More recently, we have reported that two leptospiral adhesins, Lsa44 and Lsa45, were capable of inducing a combination of humoral and cellular immune response in mice, characterized by high concentration of antibodies, induction of cellular lymphoproliferation and increased level of cytokines [41]. …”

Interação de proteínas de membrana de Leptospira com os reguladores Fator H e C4BP do sistema complemento humano.

“…Há vários anos foi relatado que L. interrogans interage com PLG e que, na presença dos ativadores exógenos presentes no hospedeiro como uPA, é ativado em PLA (Vieira et al, 2009). Até a presente data não foi descrito em leptospira nenhum ativador endógeno de PLG, mas já foram identificadas várias proteínas de membrana como ligantes desta molécula: LipL32, rLIC12730, rLIC10494, Lp29, Lp49, Lp40, MPL36, rLIC12238 (Vieira et al, 2010), LenA 120 (Verma et al, 2010), OmpA-like (Oliveira et al, 2011), Lsa20 (Mendes et al, 2011), OmpL-1 (Fernandes et al, 2012), Lsa23, Lsa26, e Lsa36 (Siqueira et al,2013, Lsa 33 ), Lsa30 (Spuza et al, 2012) EF-Tu , Lsa44 e Lsa45 (Fernandes et al, 2014) e enolase (Nogueira et al, 2013).…”

“…Também foi demonstrado que o plasminogênio ligado à leptospira pode ser ativado em plasmina na presença de uPA. As proteínas de membrana de leptospira que até o momento mostraram interação com plasminogênio foram LipL32, rLIC12730, rLIC10494, Lp29, Lp49, Lp40, MPL36, rLIC12238 (Vieira et al, 2010) e LenA (Verma et al, 2010), OmpA-like (Oliveira et al, 2011), Lsa20 (Mendes et al, 2011), OmpL-1 (Fernandes et al, 2012), Lsa23, Lsa26, e Lsa36 (Siqueira et al, 2013), EF-Tu , enolase (LA1951) (Nogueira et al, 2013) Lsa44 e Lsa45 (Fernandes et al, 2014).…”

Avaliação do papel de duas proteínas de <i>Leptospira interrogans</i> na patogênese da leptospirose.

“…Como descrito anteriormente, a Leptospira é capaz de capturar o PLG presente no soro do hospedeiro, utilizando diversas proteínas descritas e transforma-lo em sua forma ativa plasmina, utilizando-se do ativador uroquinase do hospedeiro (FERNANDES et al, 2014;SOUZA et al, 2012;SIQUEIRA et al 2016;TEIXEIRA et al, 2015;VIEIRA et al, 2010VIEIRA et al, , 2011…”