Role of Supramolecule ErpY-Like Lipoprotein of Leptospira in Thrombin-Catalyzed Fibrin Clot Inhibition and Binding to Complement Factors H and I, and Its Diagnostic Potential

Abstract

Leptospirosis is one of the most widespread zoonoses caused by pathogenic Leptospira spp. In this study, we report that the LIC11966/ErpY-like lipoprotein is a surface-exposed outer membrane protein exclusively present in pathogenic species of Leptospira. The recombinant ErpY (rErpY)-like protein is recognized by the immunoglobulins of confirmed leptospirosis sera of diverse hosts (human, bovine, and canine), suggesting the expression of the native leptospiral surface protein during infection. Circular dichroism of pure rErpY-like protein showed the secondary structural integrity to be uncompromised during the purification process. Analysis of the rErpY-like protein by native polyacrylamide gel electrophoresis, chemical cross-linking, dynamic light scattering, and field emission transmission electron microscopy demonstrated it undergoes supramolecular assembly. The rErpY-like protein can bind to diverse host extracellular matrices, and it presented a saturable and strong binding affinity (dissociation constant [KD] of 70.45 ± 4.13 nM) to fibrinogen, a central host plasma component involved in blood clotting. In the presence of the rErpY-like supramolecule, thrombin-catalyzed fibrin clot formation is inhibited up to 7%, implying its role in inhibiting blood coagulation during Leptospira infection. In addition, binding of the rErpY-like supramolecule to complement factors H and I suggests the protein also contributes to Leptospira evading innate host defense during infection by inactivating alternative complement pathways. This study reveals that rErpY-like protein is functionally active in the supramolecular state and performs moonlighting activity under the given in vitro conditions.

ErpY-like protein, a promising antigen to leptospirosis control: characterization of antigenic and immunogenic potential

“…Since the rErpY-like protein was recognized by the antibodies present in the sera from naturally infected human, canine, and swine, this protein may have a role in the Leptospira infection process. This result corresponds with previous reports, in which RlpA (Gamberini et al, 2005), LemA, LIC10009, LIC11567, LIC13305, LIC13059, and LIC20172 (Hartwig et al, 2011), OmpL36 (Oliveira et al, 2015, LigB (Conrad et al, 2017) and ErpY-like (Ghosh et al, 2019) were recognized by the antibodies present in the sera from naturally and experimentally infected animals.…”

“…Additionally, in an ELISA, from the diagnosis of swine leptospirosis, the rErpY-like protein presented 96.8% of sensitivity, 100% of specificity and 99% of accuracy (Padilha et al, 2019). The identification and characterization of the novel leptospiral antigens represent an important advance in the search for new methods to control leptospirosis (Conrad et al, 2017;Ghosh et al, 2019;Hartleben et al, 2013;Hartwig, Seixas, Cerqueira, McBride, & Dellagostin, 2011;Nagalingam et al, 2015).…”

“…The ErpY-like (LIC11966) protein is a L. interrogans antigen, with a sequence similarity to virulence factors from other pathogens (such as the outer membrane protein ErpY from Borrelia burgdorferi) (Eshghi, Cullen, Cowen, Zuerner, & Cameron, 2009). Furthermore, the protein is expressed during in vivo infection and interacts with the extracellular matrix (ECM) (Ghosh et al, 2019;Oliveira et al, 2018), therefore suggesting its importance in infectious processes and its potential to be used in diagnosis and to develop a vaccine. Recently, the rErpY-like protein was used as a subunit vaccine in protection assays against a virulent strain of L. interrogans, with survival rates of 62.5% in a hamster animal model (Oliveira et al, 2018).…”

A Review on Host-Leptospira Interactions: What We Know and Future Expectations

“…with fibrinogen is mediated by several outer membrane proteins. To date, the fibrinogen-binding proteins identified include: LigA and LigB ( Choy et al., 2011 ; Lin et al., 2011 ), OmpL37 ( Pinne et al., 2010 ), Lsa33, Lsa25, Lsa30 and OmpL1 ( Oliveira et al., 2013 ), Lsa23, Lsa36 ( Siqueira et al., 2013 ), Lsa37 ( Silva et al., 2016 ), rLIC10508 ( Siqueira et al., 2015 ), Lsa25.6 and Lsa16 ( Pereira et al., 2017 ), ErpY ( Ghosh et al., 2019 ), rLIC10774 ( Passalia et al., 2020a ), and rLIC13086 ( Passalia et al., 2021 ). The interactions with most of these proteins were found to be dose-dependent and specific.…”

“…Several leptospiral proteins have been identified as FH and C4BP receptors. Endostatin-like (Len) proteins A (LenA) and B (LenB) ( Stevenson et al., 2007 ), EF-Tu protein ( Wolff et al., 2013 ) and Erp-Y-like lipoprotein ( Ghosh et al., 2019 ) were identified as ligands of FH. Among these proteins, only LenA and EF-Tu showed the ability to inactivate C3b.…”