Determination of the Clostridium perfringens-binding site on fibronectin.

The extracellular matrix protein fibronectin (Fn) is known to bind to the surface of Clostridium perfringens cells. Fn is a disulfide-linked homodimer protein, with each Fn polypeptide consisting of three types of repeating modules: 12 type I, 2 type II, and 15-17 type III modules. To determine the epitope on Fn recognized by C. perfringens cells, anti-Fn monoclonal antibodies (mAbs) and various Fn fragments (III2-10, rIII2-4, rIII5-7, rIII8, rIII9, rIII10) were employed. Although two C. perfringens-derived Fn-binding proteins, FbpA and FbpB, have been reported, they appear not to be the bacterium's surface Fn receptor. Moreover, both FbpA and FbpB were found to bind to C. perfringens cells. To avoid confusion, a mutant C. perfringens lacking both the fbpA and fbpB genes (MW5) was prepared using an in-frame deletion system. MW5 cells bound Fn on their surface, suggesting the presence of a putative Fn receptor(s) on C. perfringens cells. Of several anti-Fn mAbs, both HB39 and MO inhibited the binding of Fn to MW5 cells. HB39 reacted strongly with III2-10 and rIII9, and weakly with rIII2-4, rIII10 and rIII5-7 in Western blotting analysis. Binding of HB39 to Fn was inhibited in the presence of either rIII9 or rIII10, but not in the presence of rIII2-4, rIII5-7, or rIII8. Binding of Fn to MW5 cells was strongly inhibited by both III2-10 and rIII9, marginally inhibited by rIII2-4, but not affected by rIII5-7, rIII8, or rIII10. Significant binding of MW5 cells to immobilized rIII9 and rIII10 as well as immobilized III2-10 was observed. The region of Fn recognized by C. perfringens was thus mapped to the region encompassed by III9 and III10.