Adhesin activity of Leptospira interrogans lipoprotein identified by in vivo and in vitro shotgun phage display

Abstract

Leptospira interrogans causes leptospirosis, one of the most common zoonotic diseases in the world. This pathogenic spirochete is able to bind to extracellular matrix, to express virulent factors and to cause host death. Until now, there is no effective human vaccine for the disease. Shotgun phage display genomic libraries of L. interrogans were constructed and used for in vivo biopanning in hamsters and screened for ligands able to bind to LLC-PK1 epithelial cells. In both panning procedures, clones coding for the putative lipoprotein LIC12976 were identified and, in order to confirm its adhesin activity, a recombinant protein was produced in Escherichia coli and showed to interact with A31 fibroblasts, LLC-PK1 and Vero epithelial cells in vitro. Moreover, rLIC12976 was shown to bind to laminin, indicating an adhesin function. This protein was also detected in extracts of L. interrogans from different serovars and it was found to be conserved among pathogenic leptospires. Further, the protein was tested as vaccine candidate and immunization of hamsters with LIC12976 did not confer protection against a lethal challenge with the homologous L. interrogans serovar Copenhageni. Nevertheless, LIC12976 seems to act as an adhesin, and may be important for the host-pathogen interaction, so that its study can contribute to the understanding of the virulence mechanisms in pathogenic leptospires.

Discovery of Leptospira spp. seroreactive peptides using ORFeome phage display

“…It has previously been used successfully to identify novel biomarkers from Salmonella typhimurium , Mycoplasma pneumoniae and Neisseria gonorrhea [14, 20, 38, 39]. Regarding Leptospira , it has been used successfully to identify (1) host proteins that interact with LipL32 [40], (2) LigB protein acting as heparin binding protein [41], (3) adhesin activity of Leptospira lipoprotein [42], and (4) mimotopes from monoclonal antibodies specific for Leptospira spp. [43].…”

Leptospira and Leptospirosis

“…This difficulty is compounded when the exposure of the protein of interest on the leptospiral cell is either not investigated (Lima et al 2013), is inconclusive, or cannot be verified , Table 3). It is therefore difficult to translate the meaning of these findings to natural infection.…”

Intermediate and C-terminal regions of leptospiral adhesin Lsa66 are responsible for binding with plasminogen and extracellular matrix components

“…It is possible that a number of these membrane proteins also mediate the initial adhesion to host cells (Barbosa et al , 2006; Choy et al , 2007; Merien et al , 2000). Indeed, several adhesins of Leptospira have been identified (Atzingen et al , 2008, 2009; Carvalho et al , 2009; Domingos et al , 2012; Fernandes et al , 2012; Hauk et al , 2008; Hoke et al , 2008; Lima et al , 2013; Lin et al , 2009a, b; Longhi et al , 2009; Mendes et al , 2011; Oliveira et al , 2010, 2011; Pinne et al , 2010, 2012; Souza et al , 2012; Stevenson et al , 2007; Vieira et al , 2010a). In addition, plasminogen (PLG) bound to some adhesins can be converted to plasmin, which could help the bacteria to overcome tissue barriers and dissemination (Domingos et al , 2012; Fernandes et al , 2012; Mendes et al , 2011; Oliveira et al , 2011; Souza et al , 2012; Verma et al , 2010; Vieira et al , 2010b).…”