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 (III 2-10 , rIII 2-4 , rIII 5-7 , rIII 8 , rIII 9 , rIII 10 ) 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 III 2-10 and rIII 9 , and weakly with rIII 2-4 , rIII 10 and rIII 5-7 in Western blotting analysis. Binding of HB39 to Fn was inhibited in the presence of either rIII 9 or rIII 10 , but not in the presence of rIII 2-4 , rIII 5-7 , or rIII 8 . Binding of Fn to MW5 cells was strongly inhibited by both III 2-10 and rIII 9 , marginally inhibited by rIII 2-4 , but not affected by rIII 5-7 , rIII 8 , or rIII 10 . Significant binding of MW5 cells to immobilized rIII 9 and rIII 10 as well as immobilized III 2-10 was observed. The region of Fn recognized by C. perfringens was thus mapped to the region encompassed by III 9 and III 10 . [ABSTRACT FROM AUTHOR]