D. Gillet - Academia.edu (original) (raw)

Papers by D. Gillet

Antimicrobial agents and chemotherapy, Jan 18, 2015

The most deadly outbreak of E. coli O104:H4 occurred in Europe in 2011. Here, we evaluated the ef... more The most deadly outbreak of E. coli O104:H4 occurred in Europe in 2011. Here, we evaluated the effect of the retrograde trafficking inhibitor, Retro-2(cycl), in a murine model of O104:H4 E. coli infection. Systemic treatment by Retro-2(cycl) significantly reduced body-weight loss, and improved clinical score and survival in O104:H4-infected mice. The present data established that Retro-2(cycl) contributes to the protection of mice against O104:H4 infection and may represent a novel approach to limit STEC-induced toxicity.

Cancer research, Jan 15, 1992

Alteration of the p53 gene is the most frequent genetic feature of human cancer and leads to over... more Alteration of the p53 gene is the most frequent genetic feature of human cancer and leads to overexpression of the altered protein in the tumor cell nucleus. Two diagnostic procedures are currently available to assess p53 mutations: (a) molecular analysis of the gene sequence; and (b) immunohistochemical analysis of p53 protein accumulation. We now report a third approach, serological analysis. Fifteen % of primary breast cancer patients were found to have circulating antibodies to p53 protein by immunoprecipitation or immunoblotting. We have found a close correlation between the presence of such antibodies and bad prognosis such as high histological grade and the absence of hormone receptors. Furthermore, we found that the B-cell response to p53 protein is induced by two immunodominant regions located at the carboxy and amino termini of the protein, outside the central mutational hot spot region. These findings suggest that serological analysis, combined with molecular and histoche...

FEBS Letters, 1997

We have constructed two fusion proteins, DAB 389-raIL-3 and DAB 38 9-(Gly4Ser) 2-mIL-3, in which ... more We have constructed two fusion proteins, DAB 389-raIL-3 and DAB 38 9-(Gly4Ser) 2-mIL-3, in which the receptorbinding domain of diphtheria toxin is replaced by mouse interleukin-3 (IL-3). Cytotoxic activity of the fusion toxins was observed on three out of six cell lines assayed. This toxicity was mediated through binding to the IL-3 receptor as it was inhibited in a dose-dependent manner with murine IL-3 or anti-IL-3 neutralizing antibodies. DAB 3 89-(Gly4Ser) 2-mIL-3 was up to 5 times more toxic than DAB 3S g-mIL-3, depending on the cell line (0.8X10 10 M<IC 50 <3X10 10 M). These proteins can be used for the detection of IL-3 receptors on mouse cells and should allow for the selective elimination of IL-3 receptorpositive pluripotent hematopoietic stem cells prior to bone marrow transplantation.

Journal of Bacteriology, 1997

Epsilon-toxin is produced by Clostridium perfringens types B and D and is responsible for a rapid... more Epsilon-toxin is produced by Clostridium perfringens types B and D and is responsible for a rapidly fatal enterotoxemia in animals, which is characterized by edema in several organs due to an increase in blood vessel permeability. The Madin-Darby canine kidney (MDCK) cell line has been found to be susceptible to epsilon-toxin (D. W. Payne, E. D. Williamson, H. Havard, N. Modi, and J. Brown, FEMS Microbiol. Lett. 116:161-168, 1994). Here we present evidence that epsilon-toxin cytotoxic activity is correlated with the formation of a large membrane complex (about 155 kDa) and efflux of intracellular K+ without entry of the toxin into the cytosol. Epsilon-toxin induced swelling, blebbing, and lysis of MDCK cells. Iodolabeled epsilon-toxin bound specifically to MDCK cell membranes at 4 and 37 labeled C and was associated with a large complex (about 155 kDa). The binding of epsilon-toxin to the cell surface was corroborated by immunofluorescence staining. The complex formed at 37 degrees ...

Journal of Biological Chemistry, 2002

The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our i... more The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our insight into the interactions between proteins and membranes. During cell intoxication, this domain undergoes a change from a soluble and folded state at alkaline pH to a functional membrane-inserted state at acid pH. We found that hydrophobic and electrostatic interactions occur in a sequential manner between the domain and the membrane during the insertion. The first step involves hydrophobic interactions by the C-terminal region. This is because of the pH-induced formation of a molten globule specialized for binding to the membrane. Accumulation of this molten globule follows a precise molecular mechanism adapted to the toxin function. The second step, as the pH decreases, leads to the functional inserted state. It arises from the changes in the balance of electrostatic attractions and repulsions between the N-terminal part and the membrane. Our study shows how the structural changes and the interaction with membranes of the translocation domain are finely tuned by pH changes to take advantage of the cellular uptake system.

Clinical Diagnostic Systems: Technologies and Instrumentation, 2002

ABSTRACT

Journal of immunotherapy (Hagerstown, Md. : 1997)

The authors have investigated a new way of combining cytokines with tumor cells to prepare antica... more The authors have investigated a new way of combining cytokines with tumor cells to prepare anticancer vaccines. This method may offer an alternative to gene therapy approaches. It consists in anchoring recombinant cytokines to the cell membrane. Attachment is mediated by the transmembrane domain of diphtheria toxin (T) genetically fused to the cytokine and is triggered by an acid pH pulse. The authors found that the fusion protein T-hIL-2 anchored to the surface of tumor cells retained its IL-2 activity while remaining exposed for several days. Interestingly, vaccination of mice with these modified tumor cells induced a protective antitumor immunity mediated by tumor-specific cytotoxic T lymphocytes. This procedure presents several advantages as compared with the conventional approaches based on the transfection of tumor cells with cytokine genes. It does not require the culture of tumor cells from the patients and the selection of transfected clones, it eliminates the safety proble...

Cytokines have a strong potential for triggering anticancer immunity if released in the tumor mic... more Cytokines have a strong potential for triggering anticancer immunity if released in the tumor microenvironment. Successful vaccines have been engineered using tumor cells genetically modified to secrete the cytokines. Unfortunately, this approach remains difficult and hazardous to perform in the clinic. We describe a new way of combining cytokines with tumor cells to prepare anticancer vaccines. This consists in anchoring

Journal of Molecular Biology, 2012

Journal of Molecular Biology, 2011

The translocation domain of diphtheria toxin inserts in membrane and becomes functional when the ... more The translocation domain of diphtheria toxin inserts in membrane and becomes functional when the pH inside endosomes is acid. At that stage, the domain is in a partially folded state; this prevents the use of high-resolution methods for the characterization of its functional structure. On that purpose, we report here the use of hydrogen/deuterium exchange experiments coupled to mass spectrometry. The conformation changes during the different steps of insertion into lipid bilayer are monitored with a resolution of few residues. Three parts of the translocation domain can be distinguished. With a high protection against exchange, the C-terminal hydrophobic helical hairpin is embedded in the membrane. Despite a lower protection, a significant effect in the presence of lipid vesicles shows that the N-terminal part is in interaction with the membrane interface. The sensitivity to the ionic strength indicates that electrostatic interactions are important for the binding. The middle part of the domain has an intermediate protection; this suggests that this part of the domain can be embedded within the membrane but remains quite dynamic. These results provide unprecedented insight into the structure reorganization of the protein to go from a soluble state to a membrane-inserted one.

FEBS Letters, 2004

The botulinum neurotoxin A C-terminal fragment (Hc)

Journal of Biological Chemistry, 2008

The translocation of the catalytic domain through the membrane of the endosome to the cell cytopl... more The translocation of the catalytic domain through the membrane of the endosome to the cell cytoplasm is a key step of intoxication by botulinum neurotoxin (BoNT). This step is mediated by the translocation (T) domain upon endosome acidification, although the mechanism of interaction of the T domain with the membrane is still poorly understood. Using physicochemical approaches and spectroscopic methods, we studied the interaction of the BoNT/A T domain with the membrane as a function of pH. We found that the interaction with membranes does not involve major secondary or tertiary structural changes, as reported for other toxins like diphtheria toxin. The T domain becomes insoluble around its pI value and then penetrates into the membrane. At that stage, the T domain becomes able to permeabilize lipid vesicles. This occurs for pH values lower than 5.5, in agreement with the pH encountered by the toxin within endosomes. Electrostatic interactions are also important for the process. The role of the so-called belt region was investigated with four variant proteins presenting different lengths of the N-extremity of the T domain. We observed that this part of the T domain, which contains numerous negatively charged residues, limits the protein-membrane interaction. Indeed, interaction with the membrane of the protein deleted of this extremity takes place for higher pH values than for the entire T domain. Overall, the data suggest that acidification eliminates repulsive electrostatic interactions between the T domain and the membrane, allowing its penetration into the membrane without triggering detectable structural changes.