Biomedical subjects

C Frehel

Publications and source records attributed to C Frehel.

At least 19 recordsLinked to original sources

PubMed2001

The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan.

The emb genes are conserved among different mycobacteria. In Mycobacterium smegmatis and Mycobacterium tuberculosis, they belong to an operon comprising three genes, embC, embA, and embB. The EmbB protein has been proposed to be the target of ethambutol, a drug which is known to inhibit the synthesis of the arabinan portion of the mycobacterial cell wall arabinogalactan (AG). To further define the role of EmbB protein in arabinan biosynthesis, embA, -B, and -C genes were inactivated individually by homologous recombination in M. smegmatis. All three mutants were viable, and among the three, the slowest growing embB(-) mutant encountered profound morphological changes and exhibited a higher sensitivity to hydrophobic drugs and detergents, presumably due to an increase in cell wall permeability. Furthermore, chemical analyses showed that there was a diminution in the arabinose content of arabinogalactan from the embA(-) and embB(-) mutants. Specifically, in comparison with the wild-type strain, the crucial terminal hexaarabinofuranosyl motif, which is a template for mycolylation, was altered in both embA(-) and embB(-) mutants. Detailed nuclear magnetic resonance studies coupled with enzyme digestion, chromatography, and mass spectrometry analyses revealed that the disaccharide beta-d-Ara(f)-(1-->2)-alpha-d-Ara(f) extension from the 3-position of the 3,5-linked alpha-d-Ara(f) residue is markedly diminished. As a consequence, a linear terminal beta-d-Ara(f)-(1-->2)-alpha-d-Ara(f)-(1-->5)-alpha-d-Ara(f)-(1-->5)-alpha-d-Ara(f) is formed, a motif which is a recognized, nonreducing terminal feature of lipoarabinomannan but not of normal AG. Upon complementation with the embB and embA wild-type genes, the phenotype of the mutants reverted to wild-type, in that normal AG was resynthesized. Our results clearly show that both EmbA and EmbB proteins are involved in the formation of the proper terminal hexaarabinofuranoside motif in AG, thus paving the way for future studies to identify the complete array of arabinosyltransferases involved in the synthesis of mycobacterial cell wall arabinan.

Cell Wall↗

PubMed2001

Kinetics of the intracellular differentiation of Leishmania amazonensis and internalization of host MHC molecules by the intermediate parasite stages.

The establishment of Leishmania in mammals depends on the transformation of metacyclic promastigotes into amastigotes within macrophages. The kinetics of this process was examined using mouse macrophages infected with metacyclic promastigotes of L. amazonensis. The appearance of amastigote characteristics, including large lysosome-like organelles called megasomes, stage-specific antigens, high cysteine protease activity and sensitivity to L-leucine methyl ester, was followed over a 5-day period. Megasomes were observed at 48 h but probable precursors of these organelles were detected at 12h p.i. The promastigote-specific molecules examined were down-regulated within 5 to 12h after phagocytosis whereas the amastigote-specific antigens studied were detectable from 2 to 12-24 h. An increase in the cysteine protease activity and in sensitivity to L-leucine methyl ester of the parasites was detected from 24 h. The data indicate that at 48 h p.i., parasites exhibit several amastigote features but that complete differentiation requires at least 5 days. The appearance of megasomes or of megasome precursors and the rise in cysteine protease activity correlate quite well with the capacity of parasites to internalize and very likely degrade host MHC molecules. The fact that internalization by the parasites of host cell molecules occurs very early during the differentiation process argues for a role of this mechanism in parasite survival.

Animals↗

PubMed2001

Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence in Listeria monocytogenes.

The hly-encoded listeriolysin O (LLO) is a major virulence factor secreted by the intracellular pathogen Listeria monocytogenes, which plays a crucial role in the escape of bacteria from the phagosomal compartment. Here, we identify a putative PEST sequence close to the N-terminus of LLO and focus on the role of this motif in the biological activities of LLO. Two LLO variants were constructed: a deletion mutant protein, lacking the 19 residues comprising this sequence (residues 32-50), and a recombinant protein of wild-type size, in which all the P, E, S or T residues within this motif have been substituted. The two mutant proteins were fully haemolytic and were secreted in culture supernatants of L. monocytogenes in quantities comparable with that of the wild-type protein. Strikingly, both mutants failed to restore virulence to a hly-negative strain in vivo. In vitro assays showed that L. monocytogenes expressing the LLO deletion mutant was strongly impaired in its ability to escape from the phagosomal vacuole and, subsequently, to divide in the cytosol of infected cells. This work reveals for the first time that the N-terminal portion of LLO plays an important role in the development of the infectious process of L. monocytogenes.

Amino Acid Motifs↗

PubMed1999

ClpE, a novel member of the HSP100 family, is involved in cell division and virulence of Listeria monocytogenes.

We identified, in the facultative intracellular pathogen Listeria monocytogenes, a previously unknown Clp ATPase, unique among the HSP100 proteins because of the presence of a short N-terminal region with a potential zinc finger motif. This protein of 726 amino acids is highly homologous to ClpE of Bacillus subtilis, and is a member of a new subfamily of HSP100/Clp ATPases. The clpE gene is transcribed as a monocistronic mRNA from a typical consensus sigma A promoter. clpE is not stimulated by various stresses, but is upregulated in a clpC mutant. This is the first example of cross-regulation between Clp ATPases. By constructing a clpE mutant of L. monocytogenes, we found that ClpE is required for prolonged survival at 42 degrees C and is involved in the virulence of this pathogen. A double mutant deficient in both ClpE and ClpC was avirulent in a mouse model and completely eliminated in the liver. Electron microscopy studies did not show any morphological alterations in clpE or clpC mutants. In the clpE-clpC double mutant, however, cell division was affected, indicating that ClpE acts synergistically with ClpC in cell septation. These results show that the Clp chaperones play a crucial role in both cell division and virulence of L. monocytogenes.

Adenosine Triphosphatases↗

PubMed1996

Molecular characterization of a surface-exposed superoxide dismutase of Mycobacterium avium.

Mycobacterium avium is an intracellular pathogen capable of growing inside the phagosomal compartment of macrophages. In this work, we characterized the superoxide dismutase of M. avium, as a putative candidate to resist the oxidative stress. The gene sodA encoding superoxide dismutase (SOD:EC1.15.1.1) from Mycobacterium avium TMC724 was cloned and sequenced. It encodes a 23 kDa protein (207 aminoacids) showing identity with the Mycobacterium leprae SOD (91%) and the M. tuberculosis SOD (83%). This enzyme was functionally expressed in both Escherichia coli and Mycobacterium smegmatis, and identified as a manganese (Mn) SOD on the basis of sequence comparison with other MnSODs from different organisms, and by activity inhibition studies. By indirect immunogold labeling of M. avium with a mAb directed against M. leprae SOD, the enzyme was found to be exposed at the cell surface of M. avium. It was also shown that SOD was released in supernates of M. avium TMV724 during exponential growth, suggesting a role of this enzyme during interactions with the environment. When SOD was expressed in the non-pathogenic M. smegmatis, it was also exposed at the surface of bacteria and released in supernates, but this was not sufficient to protect this recombinant mycobacterium from the killing mechanisms of macrophages.

Amino Acid Sequence↗

PubMed1994

Distribution of MHC class I and of MHC class II molecules in macrophages infected with Leishmania amazonensis.

Macrophages, being apparently the only cells that in vivo allow the growth of the intracellular pathogen Leishmania, are likely candidates to present antigens to Leishmania-specific CD4+ and CD8+ T lymphocytes, known to be involved in the resolution or in the development of lesions induced by these parasites, and recognizing processed antigens bound to MHC class I and MHC class II molecules, respectively. In the present study, we analysed by confocal microscopy and by immunoelectron microscopy the subcellular distribution of both MHC class I and class II molecules in mouse (Balb/c and C57BL/6 strains) bone marrow-derived macrophages infected for 12 to 48 hours with Leishmania amazonensis amastigotes and activated with gamma interferon to determine the intracellular sites where Leishmania antigens and MHC molecules meet and can possibly interact. Double labelings with anti-MHC molecule antibodies and with either propidium iodide or an anti-amastigote antibody allowed localization of MHC molecules with regard to the endocytic compartments housing Leishmania amastigotes, organelles known as the parasitophorous vacuoles (PV) and which most likely contain the highest concentration of parasite antigens in the host cell. Both uninfected and infected macrophages from Balb/c mice expressed the MHC class I molecules H-2Kd and H-2Dd on their cell surface but no significant amount of these molecules could be detected in the PV, which indicates that, if infected macrophages play a role in the induction of Leishmania-specific CD8+ T lymphocytes, PV are probably not loading compartments for MHC class I molecules. In contrast, MHC class II molecules were found to be associated with the PV membranes as shown previously with microscopic techniques at lower resolution (Antoine et al. Infect. Immun. 59, 764-775, 1991). In addition, we show here that, 48 hours after infection of Balb/c macrophages, in about 90% of PV containing MHC class II molecules, the latter were mainly or solely localized at the attachment zone of amastigotes to PV membranes. This peculiar distribution, especially well demonstrated using confocal microscopy, was confirmed by subcellular fluorescence cytometry of infected macrophages stained for the MHC class II molecules. The following data agree with the idea that PV-associated MHC class II molecules establish specific interactions with plasma membrane components of amastigotes. First, the polarized localization of class II appeared specific to these molecules, since the distribution of the lysosomal glycoproteins Igp110 and Igp120, of the macrosialin (a macrophage-specific marker of endocytic compartments) and of the GTP-binding protein rab7p, shown here as being PV membrane components, was homogeneous.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Human platelet aggregation by Yersinia pseudotuberculosis is mediated by invasin.

Plasmid-free strains of Yersinia pseudotuberculosis induce aggregation of human platelets in vitro. It appears that this phenomenon is mediated by invasin (Inv), a 103-kDa outer membrane protein that permits bacteria to penetrate mammalian cells, since (i) an isogenic inv-deficient mutant failed to aggregate platelets compared with the parental strain; (ii) a monoclonal antibody directed against invasin inhibited platelet aggregation; (iii) Inv+ Escherichia coli HB101 promoted platelet aggregation. Platelet receptors for invasin were identified by using a panel of anti-platelet glycoprotein monoclonal antibodies in a bacterial adhesion assay. We found that bacteria bind to platelet membrane glycoproteins Ic and IIa. Electron microscopic study of bacterium-platelet interactions also revealed that bacteria expressing invasin attach to and are phagocytized by thrombocytes, in contrast to inv-deficient bacteria, indicating that these anucleated cells are able to internalize bacteria in vitro after specific interaction with invasin.

Adhesins, Bacterial↗

PubMed1991

Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci.

We report the identification of a previously unknown gene, inlA, which is necessary for the gram-positive intracellular pathogen Listeria monocytogenes to invade cultured epithelial cells. The inlA region was localized by transposon mutagenesis, cloned, and sequenced. inlA was introduced into Listeria innocua and shown to confer on this normally noninvasive species the ability to enter cells. Sequencing of inlA predicts an 80 kd protein, internalin. Two-thirds of internalin is made up of two regions of repeats, region A and region B, and the C-terminus of the molecule is similar to that of surface proteins from gram-positive cocci. Internalin has a high content of threonine and serine residues, and the repeat motif of region A has regularly spaced leucine residues. As evidenced by Southern blot analysis, inlA is part of a gene family. One of them is the gene situated directly downstream of inlA, called inlB, which also encodes a leucine-rich repeat protein.

Amino Acid Sequence↗

PubMed1991

Cellular oxidative responses and mycobacterial growth inhibition in aerosol and intradermal BCG-immunized guinea-pigs.

Although the dissemination of tuberculosis is aerogenic, less than 10% of infected subjects develop the active disease. Local immunity plays a major role in systemic cell-mediated immunity against this disease. BCG immunization may be more effective if administered via aerosol rather than intradermally. In this study, the immune responses seen in guinea-pigs vaccinated with a BCG aerosol were compared with those seen following intradermal vaccination. At regular intervals after each vaccination, the activation of alveolar macrophage was determined by their capacity to produce superoxides, phagosome-lysosome fusion and the inhibition of in vitro BCG growth. Concurrently, BCG multiplication or growth inhibition in the target organs was also determined. This study demonstrates that the alveolar route of BCG administration activated broncho-alveolar macrophage more effectively than the intradermal route. Superoxide production correlated with in vitro and in vivo inhibition of BCG growth. The spread, by the BCG inoculum, to the draining lymph nodes and spleen was similar for both test routes of administration. However, the lung BCG counts were significantly lower following intradermal vaccination. In contrast, the activation of broncho-alveolar macrophage was higher following aerogenic, rather than intradermal, BCG immunization.

Aerosols↗

PubMed1991

Localization of major histocompatibility complex class II molecules in phagolysosomes of murine macrophages infected with Leishmania amazonensis.

Leishmania-infected macrophages are potential antigen-presenting cells for CD4+ T lymphocytes, which recognize parasite antigens bound to major histocompatibility complex class II molecules (Ia). However, the intracellular sites where Ia and antigens may interact are far from clear, since parasites grow within the modified lysosomal compartment of the host cell, whereas Ia molecules seem to be targeted to endosomes. To address this question, the expression and fate of Ia molecules were studied by immunocytochemistry in Leishmania amazonensis-infected murine macrophages stimulated with gamma interferon. In uninfected macrophages, Ia molecules were localized on the plasma membrane and in perinuclear vesicles, but they underwent a dramatic redistribution after infection, since most of the intracellular staining was then associated with the periphery of the parasitophorous vacuoles (p.v.) and quite often polarized towards amastigote-binding sites. The Ii invariant chain, which is transiently associated with Ia during their intracellular transport, although well expressed in infected macrophages, apparently did not reach the p.v. Similar findings were observed with macrophages from mice either resistant or highly susceptible to Leishmania infection. In order to determine the origin of p.v.-associated Ia, the fate of plasma membrane, endosomal, and lysosomal markers, detected with specific antibodies, was determined after infection. At 48 h after infection, p.v. was found to exhibit a membrane composition typical of mature lysosomes. Overall, these data suggest that (i) Ia located in p.v. originate from secondary lysosomes involved in the biogenesis of this compartment or circulate in several endocytic organelles, including lysosomes and (ii) p.v. could play a role in antigen processing and presentation. Alternatively, the presence of high amounts of Ia in p.v. could be due to a Leishmania-induced mechanism by means of which this organism may evade the immune response.

Animals↗

PubMed1991

Intramacrophage growth of Mycobacterium avium during infection of mice.

Growth of the virulent Mycobacterium avium strain TMC 724 in host tissues during persistent infection of mice was studied. Following intravenous infection of C57BL/6 mice, the kinetics of bacterial growth was biphasic in the spleen and liver, with a significant reduction of the multiplication rate after day 21 to 28 of infection. An electron-microscopic study of the liver and spleen of infected mice showed that the bacteria were strictly intracellular. They were observed within inflammatory macrophages populating granulomas disseminated in host tissues. The bacteria were confined to the phagosome compartment, and they were encapsulated. Phagosome-lysosome fusions were encountered, but the bacteria showed no visible signs of degradation and continued to multiply. These results are the first in vivo evidence that virulent M. avium multiplies exclusively intracellularly and that encapsulated bacteria resist the microbicidal mechanisms of macrophages inside the phagosomal compartment.

Animals↗

PubMed1990

Characterization of adhesion zones in E. coli cells.

After plasmolysis of Escherichia coli cells, the adhesion zones were characterized using the cytochemical PTA and SP procedures which stain peptidoglycan and lipopolysaccharides (LPS) respectively. A PTA-stained layer was detected at the adhesion sites. This layer was visualized irrespective of the electron microscopy procedure used. Also, using SP staining an outer membrane in which LPS molecules were asymmetrically distributed, was observed.

Bacterial Adhesion↗

PubMed1990

Evidence that coating of Mycobacterium leprae surface antigens reduces its ability to hinder host microbicidal functions.

Mycobacterium leprae extracted and purified from experimentally infected armadillo was coated with rabbit sera raised against the total antigens of the following species of mycobacteria: M. leprae, M. avium, M. bovis BCG, and M. fallax. In addition, the bacteria were also coated either with serum from a lepromatous (LL), or a tuberculoid (TT) leprosy patient. The effectiveness of surface coating was verified by electron microscopy, with the aid of gold immunolabelling. The coated bacilli were phagocytized by mice bone marrow-derived macrophages, and the phagosome-lysosome fusions (PLF) were assessed during phagocytosis using acid-phosphatase (AcPase) cytochemistry. As compared to control preparations (like-wise treated with non-immune serum), significant but partial reversion of PLF inhibition was observed in all cases except when bacteria had been incubated with M. fallax antiserum (rapidly growing, non-pathogenic species). The results obtained suggest that some of the antimycobacterial antibodies may offer partial protection to the host during early events of infection by reverting the usual pattern of inhibition of PLF in infected macrophages.

Animals↗

PubMed1988

Nutritionally variant streptococci develop ultrastructural abnormalities during experimental endocarditis.

Nutritionally variant streptococci (NVS) are fastidious micro-organisms responsible for most of the so-called negative blood culture endocarditis. In patients, the relative resistance of these bacteria to antibiotic treatment may be relevant to bacterial alterations in infected tissues. We used here a rabbit experimental model of endocarditis in order to examine, under scanning and transmission electron microscope, the NVS ultrastructure inside cardiac vegetations and to follow alterations at the different stages of the disease. In the early phase (day 7) of endocarditis, NVS were found dispersed inside vegetations and exhibited a typical streptococcal morphology similar to that observed during an in vitro balanced growth. In contrast, on day 11 and day 18, bacteria were found gathered as large and numerous clusters, in which they exhibited abnormal ultrasturctural features similar to those previously described during in vitro unbalanced growth. At this stage, ruthenium red staining revealed a large amount of exopolysaccharide surrounding the bacteria. None of these bacterial alterations were observed inside the vegetations of rabbits treated from day 7 to day 11 with penicillin or vancomycin. These abnormalities might be mainly related to nutrient limitation inside the clusters and could contribute to the pathogenicity of these micro-organisms.

Animals↗

PubMed1987

Mycobacterium leprae surface components intervene in the early phagosome-lysosome fusion inhibition event.

Bone marrow-derived cultured macrophages were infected with Mycobacterium leprae. The bacteria were either used as freshly isolated organisms or incubated with M. leprae antiserum (1:5) for 30 min prior to phagocytosis. Immediately after inoculation (1 to 4 h) and at 1 to 8 days later, macrophages were stained for acid phosphatase activity to assess fusions between phagosomes and lysosomes. Inhibition of fusions was essentially apparent as an early event, which was partially reversed by antiserum treatment of the bacteria, suggesting a role for M. leprae immunogenic surface components in this early phenomenon. Later incubation times (1 to 8 days) did not show any considerable difference between antiserum-treated and nontreated bacteria. The formation of an electron-transparent zone around phagocytized bacteria and its role in phagosome-lysosome fusion was investigated, and a direct relationship could not be established.

Acid Phosphatase↗

PubMed1987

Cytochemical localization of lipopolysaccharides during peptidoglycan degradation of Escherichia coli cells.

The cytochemical reaction of Thiery [J. P. Thiery, J. Microsc. (Paris) 6:987-1018, 1976] was applied to several Escherichia coli strains having different lipopolysaccharide molecular structures. The granular deposit obtained strongly suggested that part of the R core exposed on the outer membrane was responsible for the staining. As this procedure specifically stains the outer membrane, it was possible to demonstrate that, in E. coli K-12, changes in lipopolysaccharide distribution occurred during autolysis and lysozyme treatment.

Autolysis↗

PubMed1986

Evidence for inhibition of fusion of lysosomal and prelysosomal compartments with phagosomes in macrophages infected with pathogenic Mycobacterium avium.

Bone marrow-derived cultured macrophages were infected with the pathogenic organism Mycobacterium avium. Immediately after infection and at 1 to 28 days later, cells either were stained for acid phosphatase activity or given horseradish peroxidase, which served as a pinocytotic marker. With the former, fusions between phagosomes and lysosomes exclusively were assessed; with the latter, those between phagosomes and both pinosomes and lysosomes were determined. As a control, similar experiments were undertaken by infecting macrophages with gamma ray-killed M. avium and the nonpathogenic live organisms Mycobacterium aurum and Bacillus subtilis. After infection with live M. avium, fusions between phagosomes and acid phosphatase-positive vesicles (lysosomes) were inhibited. The same inhibition was observed whether phagosomes contained damaged or structurally intact (presumed to be live) bacteria, except for the early time points. This inhibition was, however, partial, suggesting that some of the live bacteria are resistant to the hydrolytic enzymes of the phagolysosomal environment. Fusions between horseradish peroxidase-positive vesicles (pinosomes and lysosomes) and phagosomes depended upon the morphological state of the bacteria. Damaged bacteria did not inhibit fusions, whereas with intact bacteria, a partial inhibition which increased with time was observed. The two types of experiment suggest that viable M. avium can impair phagosome-pinosome fusions.

Acid Phosphatase↗

PubMed1985

Correlation between degradation and ultrastructure of peptidoglycan during autolysis of Escherichia coli.

The kinetics of peptidoglycan degradation were examined under different conditions of autolysis of Escherichia coli. With cephaloridine- or moenomycin-induced autolysis, degradation did not exceed 25 to 35%, whereas in EDTA-induced autolysis it rapidly reached 65 to 70%. When nonautolyzing cells were fixed overnight with glutaraldehyde, followed by an osmium fixation, and thin sections were stained by the phosphotungstic acid method, a dark, 15-nm-thick layer of uniform appearance and constant width occupied the whole area between the inner and outer membranes of the envelope. The stained material was tentatively identified with peptidoglycan. Ultrastructural changes in this phosphotungstic acid-stained periplasmic space were investigated at different time intervals after induction of autolysis. In all cases, breakdown proceeded over the whole cell surface. During antibiotic-induced autolysis a progressive thinning down limited to the inner side of the layer was observed. During EDTA-induced autolysis, the rapid decrease in thickness correlated well with the important loss of material labeled with [3H]diaminopimelic acid. Considering these changes and the insufficient amounts of peptidoglycan (1.3 U/nm2) necessary to account for a regularly structured polymer occupying the whole 15-nm layer, it was speculated that peptidoglycan might be unevenly distributed throughout the periplasmic space.

Anti-Bacterial Agents↗