A domain of the Leptospira LigB contributes to high affinity binding of fibronectin

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Abstract

Adhesion of pathogenic Leptospira spp. to mammalian cells is mediated by their adhesins interacting with host cell receptors. In a previous study, we have identified two potential fibronectin (Fn) binding sites in central variable region (LigBCen) and C-terminal variable region (LigBCtv) of LigB, an adhesin of pathogenic Leptospira spp. In this study, we have further localized the Fn-binding site on LigBCen and found a domain of LigB (LigBCen2) (amino acids 1014–1165) strongly bound to Fn. LigBCen2 bound to a 70 kDa domain of Fn including N-terminal domain (NTD) and gelatin binding domain (GBD), but with a higher binding affinity to NTD (Kd = 272 nM) than to GBD (Kd = 1200 nM). Except Fn, LigBCen2 also bound laminin and fibrinogen. LigBCen2 could bind MDCK cells, and blocked the binding of Leptospira on MDCK cells by 45%. These results suggest that LigBCen2 contributed to high affinity binding on NTD or GBD of Fn, laminin, and fibrinogen and mediated Leptospira binding on host cells.

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Materials and methods

Bacterial strains and cell culture. Leptospira interrogans serovar Pomona (NVSL1427–35–093002) was used as previously described [11]. All experiments were performed with virulent, low-passage strains obtained by passage to golden syrian hamsters as previously described [16]. Leptospires were grown in EMJH medium at 30 °C for less than 5 passages and growth was monitored by dark-field microscopy. Madin–Darby canine kidney (MDCK) cells (ATCC CCL34™) were cultured in Dulbecco minimum essential

LigBCen2, contributed to high affinity of Fn binding

To localize the Fn-binding site on central variable region of LigBCen, three truncated LigBCen including LigBCen1, LigBCen2, and LigBCen3 (Fig. 1A) were expressed and purified to apply to isothermal titration calorimetry (ITC) titrated by Fn. (Table 1). As shown in Table 1, the dissociation constant (Kd) for Fn binding to LigBCen1 and LigBCen2 were 7200 nM and 170 nM, respectively. Binding of LigBCen3 was not detected by ITC (data not shown). The interaction appeared to be exothermic with a

Discussion

A pivotal factor for bacterial pathogenesis is the ability of the pathogenic organism to colonize host tissues. Leptospira spp. possess on their cell surface a number of MSCRAMMs [3], [4], [5], [12] that may promote the binding of the Leptospira to components of the host ECMs and play a pivotal role in leptospiral virulence. LigB, a Leptospira outer membrane protein proved to be an adhesin molecule, was reported to have significant Fn-binding activities [4], [5]. LigB also showed sequence

Acknowledgments

This work was supported in part by the Biotechnology Research and Development Corporation (BRDC), the Harry M. Zweig Memorial Fund for Equine Research, and the New York State Science and Technology Foundation (CAT). We thank Dr. Bhargavi Jayaraman and Ms. Charlene Mottler for help us in the ITC techniques and Dr. Marci Scidmore for the use of CLSM. Thanks are also to our laboratory members, especially to Dr. Tavan Janvilisri for his suggestions during the course of this study and Dr. John Leong

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