Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) andPseudomonasexotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1–DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derivedDPHgene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus,DPHgene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which theDPHgene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.

Identification of a Novel Virulence Factor in Clostridium Difficile That Modulates Toxin Sensitivity of Cultured Epithelial Cells

ABSTRACTTwo glucosylating toxins named toxins A and B play a role in the pathogenesis ofClostridium Difficileinfection. The interaction of the toxins with host cell factors proceeds to downstream stages of cytotoxic effects in cells, in which involvement of otherC. difficilefactors remains unknown. We utilized culture filtrate ofC. difficilewith a low dilution to characterize the influence of putative minor proteins on the organization of the actin cytoskeleton in cultured epithelial cells and found a previously uncharacterized F-actin aggregated structure, termed “actin aggregate,” at the juxtanuclear region. We reasoned that formation of actin aggregate was due to an additional factor(s) in the culture filtrate rather than the glucosylating toxins, because treatment of purified toxins rarely caused actin aggregate in cells. We focused on a previously uncharacterized hypothetical protein harboring a KDEL-like sequence as a candidate. The product of the candidate gene was detected in culture filtrate ofC. difficileATCC 9689 and was renamed Srl. Purified glutathioneS-transferase-tagged Srl triggered formation of actin aggregate in the cells in the presence of either toxin A or B and enhanced cytotoxicity of each of the two toxins, including decreases in both cell viability and transepithelial resistance of cultured epithelial monolayer, although the recombinant Srl alone did not show detectable cytotoxicity. Srl-neutralized culture filtrate partially inhibited morphological changes of the cells in parallel with decreased actin aggregate formation in the cells. Thus, Srl might contribute to the modulation of toxin sensitivity of intestinal epithelial cells by enhancing cytotoxicity ofC. difficiletoxins.