Endoplasmic reticulum is a major target of cadmium toxicity in yeast.

Cadmium (Cd(2+)) is a very toxic metal that causes DNA damage, oxidative stress and apoptosis. Despite many studies, the cellular and molecular mechanisms underlying its high toxicity are not clearly understood. We show here that very low doses of Cd(2+) cause ER stress in Saccharomyces cerevisiae as evidenced by the induction of the unfolded protein response (UPR) and the splicing of HAC1 mRNA. Furthermore, mutant strains (Delta ire1 and Delta hac1) unable to induce the UPR are hypersensitive to Cd(2+), but not to arsenite and mercury. The full functionality of the pathways involved in ER stress response is required for Cd(2+) tolerance. The data also suggest that Cd(2+)-induced ER stress and Cd(2+) toxicity are a direct consequence of Cd(2+) accumulation in the ER. Cd(2+) does not inhibit disulfide bond formation but perturbs calcium metabolism. In particular, Cd(2+) activates the calcium channel Cch1/Mid1, which also contributes to Cd(2+) entry into the cell. The results reinforce the interest of using yeast as a cellular model to study toxicity mechanisms in eukaryotic cells.

Role of meprins to protect ileal mucosa of Crohn's disease patients from colonization by adherent-invasive E. coli.

Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to adhere to and invade intestinal epithelial cells (IEC), and to survive within macrophages. The interaction of AIEC with IEC depends on bacterial factors mainly type 1 pili, flagella, and outer membrane proteins. In humans, proteases can act as host defence mechanisms to counteract bacterial colonization. The protease meprin, composed of multimeric complexes of the two subunits alpha and beta, is abundantly expressed in IECs. Decreased levels of this protease correlate with the severity of the inflammation in patients with inflammatory bowel disease. The aim of the present study was to analyze the ability of meprin to modulate the interaction of AIEC with IECs. In patients with ileal CD we observed decreased levels of meprins, in particular that of meprin ß. Dose-dependent inhibition of the abilities of AIEC strain LF82 to adhere to and invade intestinal epithelial T84 cells was observed when bacteria were pre-treated with both exogenous meprin α and meprin ß. Dose-dependent proteolytic degradation of type 1 pili was observed in the presence of active meprins, but not with heat-inactivated meprins, and pretreatment of AIEC bacteria with meprins impaired their ability to bind mannosylated host receptors and led to decreased secretion of the pro-inflammatory cytokine IL-8 by infected T84 cells. Thus, decreased levels of protective meprins as observed in CD patients may contribute to increased AIEC colonization.

The cadmium transport sites of CadA, the Cd2+-ATPase from Listeria monocytogenes.

CadA, the Cd(2+)-ATPase from Listeria monocytogenes, belongs to the Zn(2+)/Cd(2+)/Pb(2+)-ATPase bacterial subfamily of P(1B)-ATPases that ensure detoxification of the bacteria. Whereas it is the major determinant of Listeria resistance to Cd(2+), CadA expressed in Saccharomyces cerevisiae severely decreases yeast tolerance to Cd(2+) (Wu, C. C., Bal, N., Pérard, J., Lowe, J., Boscheron, C., Mintz, E., and Catty, P. (2004) Biochem. Biophys. Res. Commun. 324, 1034-1040). This phenotype, which reflects in vivo Cd(2+)-transport activity, was used to select from 33 point mutations, shared out among the eight transmembrane (TM) segments of CadA, those that affect the activity of the protein. Six mutations affecting CadA were found: M149A in TM3; E164A in TM4; C354A, P355A, and C356A in TM6; and D692A in TM8. Functional studies of the six mutants produced in Sf9 cells revealed that Cys(354) and Cys(356) in TM6 as well as Asp(692) in TM8 and Met(149) in TM3 could participate at the Cd(2+)-binding site(s). In the canonical Cys-Pro-Cys motif of P(1B)-ATPases, the two cysteines act at distinct steps in the transport mechanism, Cys(354) being directly involved in Cd(2+) binding, while Cys(356) seems to be required for Cd(2+) occlusion. This confirms an earlier observation that the two equivalent Cys of Ccc2, the yeast Cu(+)-ATPase, also act at different steps. In TM4, Glu(164), which is conserved among P(1B)-ATPases, may be required for Cd(2+) release. Finally, analysis of the role of Cd(2+) in the phosphorylation from ATP and from P(i) of the mutants suggests that two Cd(2+) ions are involved in the reaction cycle of CadA.