Clostridial Glucosylating Toxins Enter Cells via Clathrin-Mediated Endocytosis

Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi α-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and α-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an acidified endosome for translocation of the catalytic domain into the cytosol. Here we studied the endocytic processes that mediate cell internalization of the CGTs. Intoxication of cells was monitored by analyzing cell morphology, status of Rac glucosylation in cell lysates and transepithelial resistance of cell monolayers. We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway. Additional evidence about the role of clathrin in the uptake of the prototypical CGT family member toxin B was achieved by expression of a dominant-negative inhibitor of the clathrin-mediated endocytosis (Eps15 DN) or by siRNA against the clathrin heavy chain. Accordingly, cells that expressed dominant-negative caveolin-1 were not protected from toxin B-induced cell rounding. In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs. Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin

Changes in photosynthetic capacity, carboxylation efficiency, and CO 2 compensation point associated with midday stomatal closure and midday depression of net CO 2 exchange of leaves of Quercus suber

The carbon-dioxide response of photosynthesis of leaves of Quercus suber , a sclerophyllous species of the European Mediterranean region, was studied as a function of time of day at the end of the summer dry season in the natural habitat. To examine the response experimentally, a “standard” time course for temperature and humidity, which resembled natural conditions, was imposed on the leaves, and the CO 2 pressure external to the leaves on subsequent days was varied. The particular temperature and humidity conditions chosen were those which elicited a strong stomatal closure at midday and the simultaneous depression of net CO 2 uptake. Midday depression of CO 2 uptake is the result of i) a decrease in CO 2 -saturated photosynthetic capacity after light saturation is reached in the early morning, ii) a decrease in the initial slope of the CO 2 response curve (carboxylation efficiency), and iii) a substantial increase in the CO 2 compensation point caused by an increase in leaf temperature and a decrease in humidity. As a consequence of the changes in photosynthesis, the internal leaf CO 2 pressure remained essentially constant despite stomatal closure. The effects on capacity, slope, and compensation point were reversed by lowering the temperature and increasing the humidity in the afternoon. Constant internal CO 2 may aid in minimizing photoinhibition during stomatal closure at midday. The results are discussed in terms of possible temperature, humidity, and hormonal effects on photosynthesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47468/1/425_2004_Article_BF00397440.pd