2003. complement control protein (CCP) domains had successively been deleted, we found that the aspartic acids at position 54 in the Dr fimbriae and in CCP domain 4 of hDAF played pivotal roles in the mobilization of the Src kinases and hDAF, respectively. INTRODUCTION Diffusely adhering (DAEC) belong to the sixth class of enterovirulent (29). Strains of DAEC expressing afimbrial (Afa) or fimbrial (Dr) adhesins have been subdivided into two classes, the typical Afa/Dr DAEC and the atypical Afa/Dr DAEC, each of which is further subdivided into two subclasses (52). The typical Afa/Dr DAEC class includes strains harboring AfaE-I, AfaE-II, AfaE-III, AfaE-V, Dr, Dr-II, F1845, and NFA-I adhesins, which (i) share the same genetic organization, (ii) are able to bind to human decay-accelerating factor (hDAF; CD55), and (iii) promote the mobilization of human hDAF around bacteria adhering to host cells. The typical subclass 1 strains, including strains harboring AfaE-III, Dr, and F1845 adhesins, (i) bind to members of the human carcinoembryonic antigen family (human carcinoembryonic antigen cell adhesion molecules [hCEACAMs]; Afa/DrhCEA) and (ii) mobilize hCEACAMs around bacteria adhering to host cells. In contrast, typical subclass 2 strains, including strains harboring afimbrial AfaE-I and Dr-II adhesins, do not bind to hCEACAM members. All these adhesins are able to bind to human receptors but do not recognize the counterpart mouse, rat, or pig receptors (26). Typical Afa/Dr adhesins are encoded by a Desoximetasone family of genes organized to form to expressing Dr fimbriae. We used the pyelonephritic Afa/Dr DAEC strain IH11128, which expresses Dr fimbriae. It belongs to the typical Afa/Dr DAEC subclass known to colonize both the DNM1 human urinary and intestinal tracts. This pathogenic group is frequently associated both with Desoximetasone recurrent urinary infections, such as cystitis or pyelonephritis, and with diarrhea in children. Some studies have reported that typical Afa/Dr DAEC strains, including IH11128, induce cellular modifications such as F-actin reorganization, brush border injuries, and the disruption of tight junctions and trigger proinflammatory responses (52). The pathogenesis of Afa/Dr DAEC is initiated by an initial adhesion step characterized by the recognition and mobilization of cellular receptors, including hDAF, around the bacteria. Here we show for the first time that activated Src kinases are necessary for the recruitment of hDAF around adhering Dr-positive bacteria, and we identify the c-Src kinases as being specifically involved in this process. We also show that the aspartic acids at position 54 in the DraE adhesin subunit and the CCP4 domain of hDAF are structural elements that regulate the Dr fimbria-induced hDAF mobilization and the activation of Src kinases. Finally, we demonstrate that the catalytic activity and the SH2 and SH3 protein-binding domains of Src kinases are crucial structural elements that contribute to the recruitment of hDAF around Dr-positive isolates. Taken together, these findings provide further insights into the molecular process underlying Afa/Dr DAEC adherence. Moreover, we demonstrated that Src kinases activated by Dr adhesin/hDAF interaction are involved in hCEACAM1-4L activation, suggesting that hDAF plays a central role in the organization of early events in response to infection Desoximetasone with Afa/Dr DAEC. MATERIALS AND METHODS Desoximetasone Reagents and antibodies. The antiprotease cocktail Triton X-100, methyl–cyclodextrin (MCD), and cytochalasin D were purchased from Sigma. The Src kinase inhibitor PP2 and its inactive analog, PP3, were purchased from Calbiochem. The antibodies used were goat anti-hDAF polyclonal antibody (pAb) (AF2009) from R&D systems; mouse monoclonal antibody (MAb) Desoximetasone 8D11, directed against the CCP4 domain of hDAF (Department of Pathology, Washington University School of Medicine, St. Louis, MO); rabbit anti-phospho-Src Tyr416 pAb, which recognizes phospho-Tyr416 (mammals) or phospho-Tyr418 (human), and rabbit anti-Src pAb, purchased from Cell Signaling (Denvers, MA); mouse anti-c-Src MAb (Millipore); mouse anti-Yes MAb (BD Transduction Laboratories); mouse anti-Fyn MAb and mouse anti-Lyn MAb (Exbio); rabbit antiactin pAb (Sigma);.