we questioned whether the plant genes that are employed in response to Salmonella infection are different from those of animals Results Proliferation in plants does not alter the virulence for mammalian cells We examined the virulence potential of Salmonella enterica subsp

April 5, 2017

of full length and CROP-deleted toxins A and B towards polarized cells depend on the site of application To further investigate an alternative internalization mechanism for CROP- truncated C. difficile toxins, toxin uptake into polarized CaCo-2 cells was analyzed. CaCo-2 cells serve as model for different receptor structures and/or protein composition of the apical and basolateral membrane. Toxin-induced disturbance of the intestinal barrier function, monitored by reduction of transepithelial electrical resistance, was measured as marker for toxin internalization. In coherence with previous data, TcdA possesses almost identical cytotoxicity when applied apically or basolaterally whereas cytotoxic potency of TcdB strongly depends on the site of application. Interestingly, basolaterally applied TcdA1874 exhibits considerably higher cytotoxic potency than apically applied or even full length TcdA. This observation was confirmed by intracellular 16632257 Rac1-glucosylation. In contrast to TcdA, TcdB was more potent than its truncated form TcdB1852, independent on the site of application. Thus, the CaCo-2 model is a cell line where different endocytotic routes are present and can be studied separately. These findings strongly speak in favor for different Discussion The current study investigates the role of the C-terminal repeats of C. difficile TcdA regarding toxin functionality. The report by Amimoto showed that the novel identified TpeL from C. perfringens, which is homologous to large clostridial glucosylating toxins, possesses cytotoxic activity. This toxin, however, lacks the C-terminal repeats that are typical for clostridial glucosyltransferases and serve as receptor binding domain. Based on this finding we dispute the necessity of the CROP domain concerning functional properties of the C. difficile toxins A and B. By recombinant expression and purification of full length TcdA and truncated TcdA1874 we were able to compare the cytopathic impact of both TcdA forms. Indeed, CROP-truncated TcdA1874 induced time- and concentration dependent rounding of host cells. Since the N-terminal- and transmembrane domain-covering mutant TcdA1101 was completely inactive in cell rounding and March 2011 | Volume 6 | Issue 3 | e17623 CROP-Mediated Endocytosis of TcdA 10 March 2011 | Volume 6 | Issue 3 | e17623 CROP-Mediated Endocytosis of TcdA Rac1 glucosylation assays and MedChemExpress AEB 071 inhibition of endosomal acidification prevented TcdA1874-induced effects, we excluded nonspecific cellular uptake. This was in line with previously reported lack of cytotoxic potency of TcdA1065. Recently, Demarest and co-workers described 21609844 potent toxin neutralization by a combination of monoclonal antibodies directed against multiple sites of TcdA localized within the proposed receptor binding domain. This is in absolute accordance with our findings and the question arises why antibody solely directed against the C-terminal repeats is able to neutralize the effect of TcdA. This phenomenon might be explained by simultaneous steric inhibition of the vicinal intermediate domain or by hindering conformational changes of the domains as a prerequisite for binding to the functional receptor. In this study we clearly show that TcdA can display its cytopathic action in the absence of the C- terminal repeats. This finding is new and puts light on the intermediate and transmembrane domain as target for further receptor interaction. It can be assumed that the same is true for TcdB. In coherence with our