Felix Romain

ESAT-6 from Mycobacterium tuberculosis Dissociates from Its Putative Chaperone CFP-10 under Acidic Conditions and Exhibits Membrane-Lysing Activity

The 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10 of Mycobacterium tuberculosis are secreted by the ESX-1 system into the host cell and thereby contribute to pathogenicity. Although different studies performed at the organismal and cellular levels have helped to explain ESX-1-associated phenomena, not much is known about how ESAT-6 and CFP-10 contribute to pathogenesis at the molecular level. In this study we describe the interaction of both proteins with lipid bilayers, using biologically relevant liposomal preparations containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol, and cholesterol. Using flotation gradient centrifugation, we demonstrate that ESAT-6 showed strong association with liposomes, and in particular with preparations containing DMPC and cholesterol, whereas the interaction of CFP-10 with membranes appeared to be weaker and less specific. Most importantly, binding to the biomembranes no longer occurred when the proteins were present as a 1:1 ESAT-6.CFP-10 complex. However, lowering of the pH resulted in dissociation of the protein complex and subsequent protein-liposome interaction. Finally, cryoelectron microscopy revealed that ESAT-6 destabilized and lysed liposomes, whereas CFP-10 did not. In conclusion, we propose that one of the main features of ESAT-6 in the infection process of M. tuberculosis is the interaction with biomembranes that occurs after dissociation from its putative chaperone CFP-10 under acidic conditions typically encountered in the phagosome.

Decreased Capacity of Recombinant 45/47-kDa Molecules (Apa) of Mycobacterium tuberculosis to Stimulate T Lymphocyte Responses Related to Changes in Their Mannosylation Pattern*

The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bovis, or BCG have been identified as major immunodominant antigens. Mass spectrometry analysis indicated similar mannosylation, a complete pattern from 1 up to 9 hexose residues/mole of protein, of the native species from the 3 reference strains. The recombinant antigen expressed in M. smegmatis revealed a different mannosylation pattern: species containing 7 to 9 sugar residues/mole of protein were in the highest proportion, whereas species bearing a low number of sugar residues were almost absent. The 45/47-kDa recombinant antigen expressed in E. coli was devoid of sugar residues. The proteins purified from M. tuberculosis, M. bovis, or BCG have a high capacity to elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and to stimulate in vitrosensitized T lymphocytes of guinea pigs immunized with living BCG. The recombinant Apa expressed in Mycobacterium smegmatis was 4-fold less potent in vivo in the DTH assay and 10-fold less active in vitro to stimulate sensitized T lymphocytes than the native proteins. The recombinant protein expressed inEscherichia coli was nearly unable to elicit DTH reactionsin vivo or to stimulate T lymphocytes in vitro. Thus the observed biological effects were related to the extent of glycosylation of the antigen.

Conferring a template-dependent polymerase activity to terminal deoxynucleotidyltransferase by mutations in the Loop1 region

Terminal deoxynucleotidyltransferase (Tdt) and DNA polymerase μ (pol μ) are two eukaryotic highly similar proteins involved in DNA processing and repair. Despite their high sequence identity, they differ widely in their activity: pol μ has a templated polymerase activity, whereas Tdt has a non-templated one. Loop1, first described when the Tdt structure was solved, has been invoked as the major structural determinant of this difference. Here we describe attempts to transform Tdt into pol μ with the minimal number of mutations in and around Loop1. First we describe the effect of mutations on six different positions chosen to destabilize Tdt Loop1 structure, either by alanine substitution or by deletion; they result at most in a reduction of Tdt activity, but adding Co++ restores most of this Tdt activity. However, a deletion of the entire Loop1 as in pol λ does confer a limited template-dependent polymerase behavior to Tdt while a chimera bearing an extended pol μ Loop1 reproduces pol μ behavior. Finally, 16 additional substitutions are reported, targeted at the two so-called ‘sequence determinant’ regions located just after Loop1 or underneath. Among them, the single-point mutant F401A displays a sequence-specific replicative polymerase phenotype that is stable upon Co++ addition. These results are discussed in light of the available crystal structures.

Comparative electrophoresis, isoelectric focusing and numerical taxonomy of some isolates of Fonsecaea pedrosoi and allied fungi

The proteins in broken-cell extracts from eight isolates of Fonsecaea pedrosoi, the principal agent of chromomycosis, were studied and compared by electrophoresis and isoelectric focusing. A type pattern was established with 16 fractions ranging in molecular weight between 7600 and 78500 daltons and 16 fractions varying in isoelectric point between 4.95 and 7.90. A genetic distance of 0.1 found by the numerical study applied to both analyses reveals a considerable similarity among the isolates studied. This resemblance was moreover observed between F. pedrosoi and other related dematiaceous fungi.

Structural basis for a novel mechanism of DNA bridging and alignment in eukaryotic DSB DNA repair.

Eukaryotic DNA polymerase mu of the PolX family can promote the association of the two 3′‐protruding ends of a DNA double‐strand break (DSB) being repaired (DNA synapsis) even in the absence of the core non‐homologous end‐joining (NHEJ) machinery. Here, we show that terminal deoxynucleotidyltransferase (TdT), a closely related PolX involved in V(D)J recombination, has the same property. We solved its crystal structure with an annealed DNA synapsis containing one micro‐homology (MH) base pair and one nascent base pair. This structure reveals how the N‐terminal domain and Loop 1 of Tdt cooperate for bridging the two DNA ends, providing a templating base in trans and limiting the MH search region to only two base pairs. A network of ordered water molecules is proposed to assist the incorporation of any nucleotide independently of the in trans templating base. These data are consistent with a recent model that explains the statistics of sequences synthesized in vivo by Tdt based solely on this dinucleotide step. Site‐directed mutagenesis and functional tests suggest that this structural model is also valid for Pol mu during NHEJ.

Characterization of murine monoclonal antibodies specific for the 45/47 kDa antigen complex (APA) of Mycobacterium tuberculosis, M. bovis and BCG

The alanine-proline antigen (APA), representing less than 2% of the released or excreted material during Mycobacterium tuberculosis or bacillus Calmette-Guérin (BCG) growth, is a dominant antigen present during M. tuberculosis infection or BCG immunization. To obtain new tools to dissect the major epitopes of the APA molecules, seven monoclonal antibodies (mAbs) against the purified molecules were developed. Epitope maps of the mAbs were obtained on APA molecules absorbed on plastic surfaces or in solution (BIAcore technology). The mAbs were found to be independent or to be different despite binding to adjacent or overlapping epitopes. In Western blot assays some proteins secreted in culture fluid by M. avium, M. kansasii, M. smegmatis or M. xenopi were also labelled by six of the seven antibodies. Conversely one antibody was specific for the proteins from the M. tuberculosis complex (I10-0,3) demonstrating that the APA molecules have some properties or general conformations that are specific for M. tuberculosis and M. bovis.