Rapid Identification of Malaria Vaccine Candidates Based on α-Helical Coiled Coil Protein Motif

To identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally “native” epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens

In Silico Identification of Specialized Secretory-Organelle Proteins in Apicomplexan Parasites and In Vivo Validation in Toxoplasma gondii

Apicomplexan parasites, including the human pathogens Toxoplasma gondii and Plasmodium falciparum, employ specialized secretory organelles (micronemes, rhoptries, dense granules) to invade and survive within host cells. Because molecules secreted from these organelles function at the host/parasite interface, their identification is important for understanding invasion mechanisms, and central to the development of therapeutic strategies. Using a computational approach based on predicted functional domains, we have identified more than 600 candidate secretory organelle proteins in twelve apicomplexan parasites. Expression in transgenic T. gondii of eight proteins identified in silico confirms that all enter into the secretory pathway, and seven target to apical organelles associated with invasion. An in silico approach intended to identify possible host interacting proteins yields a dataset enriched in secretory/transmembrane proteins, including most of the antigens known to be engaged by apicomplexan parasites during infection. These domain pattern and projected interactome approaches significantly expand the repertoire of proteins that may be involved in host parasite interactions

« L'Europe à reculons ? Les partis politiques et l'euro-scepticisme dans les pays candidats à l'Union Européenne », Timm Beichelt

traduction de l'anglais , parue dans Terrains & Travaux, n°8, avril 2005, pp.109-133

Rapid Diagnosis of Extrapulmonary Tuberculosis by PCR: Impact of Sample Preparation and DNA Extraction

In cases of suspected extrapulmonary tuberculosis, rapid and accurate laboratory diagnosis is of prime importance, since traditional techniques of detecting acid-fast bacilli have limitations. The major difficulty with mycobacteria is achieving optimal cell lysis. Buffers used in commercial kits do not allow this complete lysis in a number of clinical specimens. A comparison of two sample preparation methods, pretreatment with proteinase K (PK-Roche) and complete DNA purification (cetyltrimethylammonium bromide [CTAB]-Roche), was conducted on 144 extrapulmonary specimens collected from 120 patients to evaluate the impact on the Cobas-Amplicor method. Thirty patients were diagnosed with tuberculosis, with 15 patients culture positive for Mycobacterium tuberculosis. Amplification and detection of the amplicons were impaired by a high number of inhibitory specimens (39 to 52%). CTAB-Roche allowed the detection of more culture-positive specimens by PCR than PK-Roche. Comparison with the final diagnoses of tuberculosis confirmed that CTAB-Roche produced the best sensitivity (53.8%) compared to culture (43.3%), PK-Roche (16%), and smear (13%). However, the specificity of the PCR assay with CTAB-Roche-extracted material was always lower (78.8%) than those with culture (100%) and PK-Roche (96.5%). False-positive specimens were lung biopsy material, lymph node biopsy material and aspirate, or bone marrow aspirate, mainly from immunocompromised patients. Despite the efficiency of complete DNA extraction for the rapid diagnosis by PCR of extrapulmonary tuberculosis, the false-positive results challenge our understanding of PCR results

Sub-localisation of novel proteins with unique dynamics during assembly and maintenance of the eukaryotic flagellum

International audienceCilia and flagella are complex organelles composed of up to 500 proteins. We have purified intact flagella from the model organism Trypanosoma brucei using mechanical shearing. Scanning and transmission electron microscopy confirmed the quality and the purity of flagella and biochemical analysis demonstrated a 15-fold enrichment of flagellar markers. Mass spectrometry investigation carried out on 5 separate experiments led to the identification of 387 proteins, 55 of which had never reported to be associated to the flagellum. 10 out of the 12 proteins investigated experimentally were indeed associated to the flagellum but turned out to localise to several sub-localisations with unique profiles: flagellar membrane, axoneme, paraflagellar rod (an extra-axonemal structure) and the adhesion zone. Two of them, termed FLAMM6 and FLAMM8 showed restricted distribution to the proximal part and to the far distal end of the axoneme, respectively. Dynamics analysis revealed that membrane proteins were incorporated by the proximal end and showed a rapid turnover whereas axonemal and PFR proteins were added to the distal end of elongating flagella but showed stable association to their structure. FLAMM6 was found only in the first half of the flagellum no matter its length, a process dependent on IFT. Finally, FLAMM8 was progressively incorporated to the elongating axoneme accumulating at the distal tip where it showed very slow turnover after flagellum formation was complete. These data highlight the existence of specific micro-domains within the eukaryotic flagellum, each with its own dynamics for assembly and turnover

Light microscopy in trypanosomes : use of fluorescent proteins and tags

Fluorescence microscopy enables the localization of proteins to specific structures within a cell which have either been fused to a fluorescence protein or detected by immunofluorescence. Here, we describe the various procedures that can be used to prepare both the procyclic form and bloodstream form of the human pathogen Trypanosoma brucei for fluorescence microscopy. The choice of procedure to be used is determined by various parameters, including protein characteristics and the scientific question being investigated