Stage-specific expression of the proline-alanine transporter in the human pathogen Leishmania.

Leishmania are obligatory intracellular parasites that cycle between the sand fly midgut (extracellular promastigotes) and mammalian macrophage phagolysosomes (intracellular amastigotes). They have developed mechanisms of adaptation to the distinct environments of host and vector that favor utilization of both proline and alanine. LdAAP24 is the L. donovani proline-alanine transporter. It is a member of Leishmania system A that translocates neutral amino acids. Since system A is promastigote-specific, we aimed to assess whether LdAAP24 is also expressed exclusively in promastigotes. Herein, we established that upon exposing L. donovani promastigotes to amastigote differentiation signal (pH 5.5 and 37 °C), parasites rapidly and completely degrade LdAAP24 protein in both axenic and in spleen-derived amastigotes. In contrast, LdAAP24 mRNA remained unchanged throughout differentiation. Addition of either MG132 or Bafilomycin A1 partially inhibited LdAAP24 protein degradation, indicating a role for both lysosome- and proteasome-mediated degradation. This work provides the first evidence for post-translational regulation of stage-specific expression of LdAAP24.

Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression.

Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness.IMPORTANCE Aneuploidy is usually detrimental in multicellular organisms, but in several microorganisms, it can be tolerated and even beneficial. Leishmania-a protozoan parasite that kills more than 30,000 people each year-is emerging as a new model for aneuploidy studies, as unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription at initiation through promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo We show its adaptive impact on transcription and its interaction with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples without parasite isolation and that adequate methods should be developed for this.

In vivo efficiency of targeted norfloxacin against persistent, isoniazid-insensitive, Mycobacterium bovis BCG present in the physiologically hypoxic mouse liver.

Persistence of Mycobacterium tuberculosis is a hypoxia-inducible state in which the bacteria are phenotypically insensitive to currently available antituberculous drugs. In humans, persistent M. tuberculosis is found in granulomatous lesions, either inside macrophages or in necrotic tissue, where the partial oxygen pressure (pO(2)) is very low. Persistent bacteria can remain silent for decades before overt tuberculosis develops. Due to insensitivity to classical drugs, M. tuberculosis persistence prevents rapid and definitive clearance of bacteria. Consequently, therapeutic molecules are required that are both active against persistent bacilli and able to reach their intramacrophagic location. In contrast to its native form, norfloxacin is active in vivo against Mycobacterium bovis BCG present in the lungs when temporarily linked to a macromolecular carrier targeted to macrophages. To study the efficiency of this macromolecular prodrug targeted to persistent mycobacteria confined inside macrophages, we established a short-term in vivo model based on the physiological pO(2) differences between lungs, spleen and liver. Whereas lungs and spleen are well oxygenated, the liver has a low pO(2) due to its portal irrigation. Therefore, studying mycobacteria in the liver yields information about in vivo persistent bacilli exposed to low pO(2). To our knowledge, no similar short-term in vivo model has been published to date. Using this model, we demonstrated the insensitivity to isoniazid of M. bovis BCG present in hypoxic sites, and showed that norfloxacin given as a mannosylated macrophage-targeted prodrug was able to kill these isoniazid-insensitive mycobacteria. This demonstrates that intracellular persistent mycobacteria are amenable to antibiotic treatment.

[Preliminary study on isoniazid-epiroprim combination in a tuberculosis murine model]. / Etude préliminaire de l'association isoniazide-épiroprim dans un modèle murin de tuberculose.

The purpose of this study regarding isoniazid-epiroprim's association applied to antituberculosis chemotherapy, carried through murine model, initiated into Institut Pasteur of Côte d'Ivoire and worked out at Institut Pasteur of Paris was to evaluate the epiroprim's effect alone and associated with isoniazid on Mycobacterium tuberculosis. Sixteen mouses (lineage C57Bl/6) were inoculated by venous way with 10(5) viable bacillus (strain H37Rv) suspended in 500 microliters sterile physiological aqueous solution and were shared out into 4 sets. Fifteen days later the sets have been submitted or not to a daily treatment by gavage during three weeks (epiroprim, isoniazid, isoniazid plus epiroprim). The mouses were euthanasied, spleen and lung were removed from each animal. The titres of determined bacillus into those organs prove that isoniazid and epiroprim associated seem more efficacious than the isoniazid monotherapy for mouses pulmonary tuberculosis. Bacillus obtained are sensitive to isoniazid.

Mixed immune response induced in rodents by two naked DNA genes coding for mycobacterial glycosylated proteins.

Two genes of Mycobacterium tuberculosis, apa (Rv1860) and pro (Rv1796), coding for two glycosylated excreted proteins have been injected to mice and guinea pigs. They produce an extended immunological response of Th1 and Th2 types. Despite the fact that mycobacterial glycosylation is necessary for a high level of delayed-type hypersensitivity (DTH) reaction, plasmids bearing each of the two genes induced an elevated level of DTH sensitization. An inverse relation between the CpG-N hexamer cluster frequency and the protective effect of injected genes is described. A comparison of the strength of several eukaryotic promoters based on the diameter of the DTH reaction shows that CMVIE followed by the ubiquitin promoter are the most efficient among those tested. A significant protective effect (0.7 log unit CFU) in mice was found for the apa gene while the pro gene had no effect.

Role of Mycobacterium tuberculosis copper-zinc superoxide dismutase.

Superoxide dismutases (SODs) play an important role in protection against oxidative stress and have been shown to contribute to the pathogenicity of many bacterial species. To determine the function of the mycobacterial copper and zinc-cofactored SOD (CuZnSOD), we constructed and characterized Mycobacterium tuberculosis and Mycobacterium bovis BCG CuZnSOD null mutants. Both strains were more sensitive to superoxides and hydrogen peroxide than were their respective parental strains. The survival of M. bovis BCG in unstimulated as well as activated mouse bone marrow-derived macrophages was not affected by the loss of CuZnSOD. The survival of CuZnSOD deficient-M. tuberculosis in guinea pig tissues was comparable to that of its parental strain. These results indicate that the mycobacterial CuZnSOD is not essential for intracellular growth within macrophages and does not detectably contribute to the pathogenicity of M. tuberculosis.

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 vitro sensitized 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 in Escherichia coli was nearly unable to elicit DTH reactions in vivo or to stimulate T lymphocytes in vitro. Thus the observed biological effects were related to the extent of glycosylation of the antigen.

Deglycosylation of the 45/47-kilodalton antigen complex of Mycobacterium tuberculosis decreases its capacity to elicit in vivo or in vitro cellular immune responses.

A protection against a challenge with Mycobacterium tuberculosis is induced by previous immunization with living attenuated mycobacteria, usually bacillus Calmette-Guérin (BCG). The 45/47-kDa antigen complex (Apa) present in culture filtrates of BCG of M. tuberculosis has been identified and isolated based on its ability to interact mainly with T lymphocytes and/or antibodies induced by immunization with living bacteria. The protein is glycosylated. A large batch of Apa was purified from M. tuberculosis culture filtrate to determine the extent of glycosylation and its role on the expression of the immune responses. Mass spectrometry revealed a spectrum of glycosylated molecules, with the majority of species bearing six, seven, or eight mannose residues (22, 24, and 17%, respectively), while others three, four, or five mannoses (5, 9, and 14%, respectively). Molecules with one, two, or nine mannoses were rare (1.5, 3, and 3%, respectively), as were unglycosylated species (in the range of 1%). To eliminate the mannose residues linked to the protein, the glycosylated Apa molecules were chemically or enzymatically treated. The deglycosylated antigen was 10-fold less active than native molecules in eliciting delayed-type hypersensitivity reactions in guinea pigs immunized with BCG. It was 30-fold less active than native molecules when assayed in vitro for its capacity to stimulate T lymphocytes primed in vivo. The presence of the mannose residues on the Apa protein was essential for the antigenicity of the molecules in T-cell-dependent immune responses in vitro and in vivo.

[Comparative study of post-infarction myocardial viability after fibrinolysis by stress tomoscintigraphy and echography: can viability be detected without ischemia?]. / Etude comparée de la viabilité du myocarde en post-infarctus après fibrinolyse par tomoscintigraphie et échographie de stress: peut-on détecter la viabilité sans l'ischémie?

The objectives of this prospective study was to define the comparative ability of stress myocardial scintigraphy and dobutamine stress echocardiography to demonstrate post-MI myocardial viability, assessed on the functional recovery in terms of improvement of global and segmental kinetics by cardiac gamma-angiography after revascularization. 18 patients (11 anterior MI, 7 lateral or inferior MI) and 162 segments were analysed semiquantitatively. All patients with persistent significant stenosis underwent secondary revascularization of the artery responsible for myocardial infarction. The prevalence of viability was high, as only 34% of segments initially presented a segmental kinetic abnormality and contraction was improved at 6 months in 54% of cases. Stress scintigraphy and dobutamine echocardiography detected viability with a sensitivity of 96% and 70%, a specificity of 88% and 82%, a positive predictive value of 89% and 77% and a negative predictive value of 95% and 76%, respectively. Only the wall score index with low-dose dobutamine was correlated with the ejection fraction at 6 months. Stress echocardiography is a more reliable predictor of the degree of functional recovery after revascularization. Scintigraphy visualizes much more extensive abnormalities than echocardiography. This often corresponds to ischaemic territories with normal contraction under baseline conditions and low doses of dobutamine. It therefore seems preferable both examinations for optimal assessment of thrombolized patients following myocardial infarction.

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.

Isolation of a proline-rich mycobacterial protein eliciting delayed-type hypersensitivity reactions only in guinea pigs immunized with living mycobacteria.

Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced only by a previous immunization with living attenuated mycobacteria, usually bacillus Calmette-Guérin (BCG). Living and killed bacteria share a number of common antigens. To identify and to purify molecules that are dominant antigens during immunization with living bacteria, a two-step selection procedure was used. Quantitative delayed-type hypersensitivity (DTH) reactions elicited in guinea pigs immunized either with living or with killed BCG were used to select or counterselect antigens present in BCG culture filtrates. Each major fraction eluted from a series of HPLC columns (gel filtration, DEAE, reverse-phase chromatography) was assayed and titrated on guinea pigs of each group. A protein with an unusual amino acid composition (40% proline, 12% threonine) was purified and N-terminally sequenced. To our knowledge, the sequence Thr-Pro-Pro-Xaa-Glu-Xaa-Pro-Pro-Pro-Pro-Gln-Xaa-Val-Xaa-Leu has not been previously reported. The protein was 100-fold more potent on guinea pigs immunized with living bacteria than on guinea pigs immunized with dead bacteria to elicit a DTH reaction.

Identification of a Mycobacterium bovis BCG 45/47-kilodalton antigen complex, an immunodominant target for antibody response after immunization with living bacteria.

Increased protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by a previous immunization with living avirulent mycobacteria, usually Mycobacterium bovis BCG. Only a transient and marginal protection is obtained after immunization with bacterial extracts or dead bacteria. Both living and heat-killed bacteria share a number of common antigens. In order to identify mycobacterial molecules which are dominant antigens during immunization with living bacteria, a two-step selection method was used. Two groups of guinea pigs were immunized either with living or with heat-killed BCG. Sera were then collected and used to select and counterselect antigens present in BCG culture filtrates. Each major fraction eluted from a series of high-pressure liquid chromatography columns (gel filtration, DEAE, and reverse-phase chromatography) was run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on polyvinylidene difluoride sheets. The molecules present on twin immunoblots were stained with antibodies raised in guinea pigs immunized either with living or with heat-killed BCG. Cross-reactive antigens stained in twin immunoblots were eliminated. Major antigens interacting with antibodies raised after immunization only with living bacteria were further purified. A complex of 45- and 47-kDa major molecules (45/47-kDa complex) was thus identified and further purified. The complex was found to interact only with antibodies present in sera of guinea pigs immunized with living bacteria and not at all with antibodies raised after immunization with dead bacteria. The 45/47-kDa antigen complex molecules were resolved on two-dimensional electrophoresis in three major and seven minor proteins detected with silver staining. All the molecules interacted with the antibodies present in sera of guinea pigs immunized with living BCG. The three major proteins (two at 47 kDa and one at 45 kDa) were amino-terminal sequenced. The sequence A-P-E-P-A-P-P-V-P-P-A-A-A-A-P-P-A, which was not previously reported, was the same for these three molecules. By using a competitive enzyme-linked immunosorbent assay, the concentrations of the 45/47-kDa antigen complex were measured in BCG culture filtrates, freeze-dried BCG, and dried heat-killed BCG; they were, respectively, 2, 0.01, and 0.001% of the total mass. The low or very low values compared with the high antibody concentration emphasized the ability of the 45/47-kDa complex delivered through live BCG to trigger an antibody response.

Preparation of tuberculin antigen L.

The preparation of antigen L using culture filtrates of live BCG is described. The procedure consisted essentially of precipitating this filtrate with 10% trichloroacetic acid in the presence of N-butyl alcohol 4% final concentration. The precipitate was chromatographed on carboxy-methyl-trisacryl-M and then on DEAE-trisacryl-M; in both cases, maximal biological activity (delayed-type hypersensitivity) was found in the void volume. The product thus obtained was chromatographed on a molecular sieve so as to eliminate poorly active fractions. The final product was 30-fold more active on guinea-pigs sensitized with live BCG than on those sensitized with inactivated BCG.