Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Abstract

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. . Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17-27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that

Fibronectin Binds to and Induces Conformational Change in a Disordered Region of Leptospiral Immunoglobulin-like Protein B

“…LigB binds to Fn, fibrinogen, laminin, elastin, proelastin, and collagen and hence probably plays a substantial role in adhesion for pathogenic Leptospira (13,14,44,45). We have shown that LigBCen2 strongly binds to the NTD and weakly binds to the GBD of Fn (14) (NTD, K D ϭ 272 nM; GBD, K D ϭ 1200 nM).…”

“…Also, a non-immunoglobulin-like repeat region found on the C-terminal tail of LigB is not found in LigA (10 -12). Lig proteins are categorized as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) 2 due to their ability to bind to eukaryotic cells (13) through their interactions with extracellular matrix components, including fibronectin (Fn), laminin, collagens, elastin, and tropoelastin (13,14,45). Previously, a high affinity Fn-binding region was localized to LigBCen2, which includes the partial eleventh and complete twelfth immunoglobulin-like repeat region and the first 47 amino acids of the non-repeat regions of LigB (15).…”

Novel Mycobacteria Antigen 85 Complex Binding Motif on Fibronectin

“…The cell-free extract was loaded onto a 10-ml nickel-nitrilotriacetic acid or GST column that was equilibrated with PBS. The protein purification procedures were the same as described previously (27,28).…”

“…Protein Binding Assays by ELISA-To measure the binding affinity of MAP Ag85 (Ag85A, Ag85B, and Ag85C) to Fn, human plasma Fn (0.5 g) was coated on microtiter plate wells using 0.1 M NaHCO 3 (pH 9.3) coating buffer at 4°C for overnight and blocked by PBS plus 3% BSA at 37°C for 1 h. Subsequently, various concentrations (0.1, 0.2, 0.4, 0.8, and 1.6 M) of His-tagged MAP Ag85 were added to each well and incubated at 37°C for 1 h. After three washes with PBST (PBS plus 0.05% Tween 20), bound Ag85 was detected by mouse anti-histidine (1:1000) and HRP-conjugated goat anti-mouse IgG (1:1000), serving as primary and secondary antibodies, respectively (27,28). To investigate the MAP Ag85B-binding domains of Fn, 0.5 g of full-length Fn, four Fn proteolytic domains (including NTD, GBD, CBD, and 40-kDa domain) or BSA (negative control) was coated on microtiter plate wells.…”

Bacterial adhesion to animal tissues: protein determinants for recognition of extracellular matrix components

“…In FnBPA, the FBD1-2 region was shown to display adhesion properties against elastin in addition to fibronectin (Keane et al, 2007). In LigB, the Ig-like repeats are involved in chargecharge interactions with elastin (Lin et al, 2009). While structure-function analysis is awaited, OmpL37 demonstrates a pronounced specificity for elastin (Pinne et al, 2010).…”