ompetition between TcdA, TcdA1874 and TcdA1875710 for cell binding by FACS analyses. Interestingly, neither full length TcdA nor the isolated CROPs compete with the truncated toxin for binding sites at HT29 cells indicating that TcdA1874 and TcdA1875710 bind to different surface structures March 2011 | Volume 6 | Issue 3 | e17623 CROP-Mediated Endocytosis of TcdA 12 March 2011 | Volume 6 | Issue 3 | e17623 CROP-Mediated Endocytosis of TcdA . Rather, pre-incubation with TcdA1875710 resulted in enhanced fluorescence intensity emitted from truncated TcdA which might be based either on binding of the N-terminal domain of TcdA1874 to the immobilized CROPs or to a potentially activated receptor. The first assumption refers to the study of Pruitt and co-workers who described an interaction of the glucosyltransferase domain of TcdA to its repetitive sequences at neutral pH. Since CROP-truncated TcdA lacks its autoligand, the N-terminus might interact 16632257 with CROPs immobilized at the cell surface leading to the observed dramatic increase of fluorescence intensity. The observation of TcdA1874 sequestering the CROPs in solution additionally supports this hypothesis. Another reason for increased fluorescence intensity of truncated TcdA following pre-incubation with TcdA1875710 could be the nature of the receptor: Binding of the CROPs to the cell surface might induce conformational changes and activates the specific receptor. This might be a prerequisite for binding of TcdA1874 through binding sites located in the intermediate part of the toxin. Hence, purchase Brivanib uptake of full length TcdA might occur in a two-step process explaining the potent endocytosis and increased toxin potency observed towards many cells compared to the truncated toxin. Even if the hypothesis has to be examined in more detail, we conclude that TcdA and TcdA1874 predominantly bind to different but not independent receptor structures. We further investigated the hypothesis that internalization of TcdA and/or TcdB additionally occur via alternative routes. This hypothesis was evaluated and substantiated by comparative analyses of the toxin-induced reduction of transepithelial electrical resistance following apical or basolateral toxin uptake into CaCo-2 cells. Basolaterally applied TcdB possesses considerably higher potency in destroying epithelial integrity of monolayer than apically applied TcdB. This observation implies that expression of the TcdB-specific receptor is more or less restricted to the basolateral membrane, as also suggested by Stubbe and coworkers. 18290633 This discrepancy in sensitivity of apical and basolateral membrane surfaces was also observed towards CROP-deleted TcdB, although TcdB1852 
was less potent than full length TcdB. Interestingly, this was not the case regarding TcdA. While potency of full length TcdA is almost independent of the site of application, basolateral membranes show considerable increased endocytotic capacity towards CROP- truncated TcdA, resembling those effects observed for TcdB. This finding emphasizes the assumption of an additional alternative uptake process, at least for TcdA, which still might be based either on the recognition of different receptor structures or on the use of other associated endocytotic pathways. Thus, different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of C. difficile. The current study proved that the C-terminal repeats of TcdA a
