Biochemical and Biophysical Research Communications
A domain of the Leptospira LigB contributes to high affinity binding of fibronectin
Section snippets
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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