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C Frehel

Publications and source records attributed to C Frehel.

At least 37 records · Page 2Linked to original sources
PubMed1981

Multiple drug resistance in Mycobacterium avium: is the wall architecture responsible for exclusion of antimicrobial agents?

Whole cells of Mycobacterium avium, characterized by their negative response in the nine biochemical tests used for mycobacterial identification in our laboratory, turned positive for nitrate reductase, Tween-80 hydrolysis, beta-glucosidase, acid phosphatase, alkaline phosphatase, penicillinase, and trehalase after their wall portion was removed to yield spheroplasts. This suggested that the negative results in most of the biochemical procedures were caused by the exclusion mechanism at the wall level. Preliminary transmission and scanning electron microscopic studies showed differences at wall level between laboratory-maintained opaque, dome-shaped (SmD) and host-recycled smooth, transparent (SmT) colony type variants of M. avium and suggested the presence of an outer regularly structured layer in SmT variants. Comparative ultrastructural studies utilizing different polysaccharide coloration methods confirmed the presence of an outer polysaccharide layer in SmT variants which was probably related to their enhanced pathogenicity for experimental animals and drug resistance as compared to that of SmD variants. These findings are discussed with respect to multiple drug resistance, virulence, and gene expression of M. avium.

Anti-Bacterial Agents↗
PubMed1980

Bacillus megaterium sporal peptidoglycan synthesis studied by high-resolution autoradiography.

Cells of a Dap- Lys- mutant strain of Bacillus megaterium were pulse labeled with [3H]diaminopimelic acid at different times of growth and sporulation. They were processed for radioactivity measurements and high-resolution autoradiography either just after the pulse or after a chase in a nonradioactive medium until refractile forespores started to appear at time (t)4,5. In the pulse-labeled cells, autoradiographs and radioactivity measurements showed that the radioactivity incorporated during a pulse decreased abruptly after t0 and stayed at a low level until t5, although the forespore wall and cortex were formed between t4 and t5. In the pulse-chased bacteria, the acid-insoluble radioactivity, as well as the number of silver grains on autoradiographs, increased during the chase in cells labeled at t1 to t2, whereas it decreased in those labeled before t0. Furthermore, analysis of silver grain distribution showed that, in stage IV bacteria, grains were distributed at the outside of the forespore, mostly on the sporangium cell wall, when pulse-labeling occurred before or at t0; they were located along the cortex and in the forespore cytoplasm when labeling was made at t1 or t2. These facts show that [3H]diaminopimelic acid necessary for spore envelope synthesis was incorporated before their morphological appearance. Free or small diaminopimelic acid precursors entered the sporangium between t1 and t2. The appearance of silver grains in the forespore cytoplasm suggests that the forespore is implicated in sporal peptidoglycan synthesis.

Autoradiography↗
PubMed1979

Peptidoglycan turnover during growth of a Bacillus megaterium Dap- Lys- mutant.

The aim of this study was to ascertain whether or not the absence of cell wall growth zones, deduced from the analysis of autoradiographs of DL-[3H]mesodiaminopimelic acid pulse-labeled cells of a Dap- Lys- mutant of Bacillus megaterium, was due to a high peptidoglycan turnover. Turnover was determined in very precise experimental conditions because two kinds of turnover occurred: a low, acid-soluble turnover and a high, acid-insoluble one. The latter was detected during a chase in the culture medium when bacteria were centrifuged before treatment with trichloroacetic acid. Otherwise the acid-insoluble released material precipitated with the bacteria. In the electron microscope this material presented a globular structure and contained both peptidoglycan and teichoic acid. The acid-insoluble turnover was mainly produced by a lytic acitivity that was released into the culture medium. This thermolabile activity was not due to cell lysis. It was implicated in septum cleavage and in the detachment of wall fragments from the cell surface, but did not seem indispensable for cell elongation. The acid-soluble turnover was much weaker and seemed to be indispensable for cell elongation.

Bacillus megaterium↗
PubMed1979

Electron microscopic study of Bacillus subtilis protoplast fusion.

When protoplasts derived from sporulating cells of Bacillus subtilis were fused by exposure to polyethylene glycol (PEG) and fixed immediately thereafter, protoplasts with two enclosed prespores could be seen by electron microscope. The number of fusion events was greatly increased, and multiply fused protoplasts appeared, when the PEG-treated suspension was diluted in hypertonic broth and reincubated before fixation. This post-PEG incubation effect is taken to indicate a fusion mechanism of two steps: a short, PEG-dependent step of membrane activation, followed by a slow, metabolism-requiring step completing fusion. When prespore-bearing protoplasts from two genetically different strains were mixed and fused, the extent of fusion could also be followed by counting clones of recombinant bacteria. Maximal from the start, their number (1% of each parent type protoplast present) was unaffected by post-PEG incubation. Fusion in this case is apparently completed after plating on the wall-regeneration medium. After optimal post-PEG incubation, the majority of the protoplasts were seen to participate in fusion, and the cytological fusion observed, corrected for wall-regeneration frequency, accounted quantitatively for the prototrophic bacteria eventually recovered. These results are in good agreement with those obtained independently by Sanchez-Rivas and Garro (J. Bacteriol. 137:1340--1345, 1979).

Bacillus subtilis↗
PubMed1975

Cell wall growth of Bacillus megaterium: cytoplasmic radioactivity after pulse-labeling with tritiated diaminopimelic acid.

Study of the cell wall growth in Bacillus megaterium by pulse-labeling a DAP- Lys- mutant with tritiated diaminopimelic acid (DAP) had revealed the presence of intracytoplasmic radioactivity. The nature of this radioactivity was studied on one hand by autoradiographic analysis of bacteria treated in different ways and on the other hand by chromatography of the radioactive compounds extracted with boiling water. It is shown that cytoplasmic radioactivity corresponds neither to free DAP nor to DAP metabolized into lysine, but to murein precursors. Autoradiographic analysis of bacteria in which all murein precursors were removed gives exactly the same cell wall growth pattern as the one previously obtained for untreated bacteria. It can be concluded that, in B. megaterium, cell wall elongation occurs by diffuse intercalation of newly synthesized murein along the cylindrical part of the cell wall and that only cross wall formation occurs in a precise growth zone.

Autoradiography↗
PubMedDate not supplied

The electron-transparent zone in phagocytized Mycobacterium avium and other mycobacteria: formation, persistence and role in bacterial survival.

After phagocytosis by bone-marrow macrophages, Mycobacterium avium was surrounded by a thick electron-transparent zone (ETZ). The use of various fixation and embedding procedures showed that ETZ did not seem to be an artifactual structure. A quantitative assessment of ETZ frequency was performed at different times after infection of macrophages with SmD and SmT colony variants of M. avium. For SmT-variant-infected macrophages, a higher percentage of ETZ+ bacilli paralleled a higher percentage of intact bacilli than was the case for SmD-infected macrophages. Macrophages were also infected with bacteria killed with UV or gamma rays, H2O2, heat or glutaraldehyde. About 50% of bacilli killed with any of these treatments were found ETZ+ instead of 80-85% with live bacteria. Unlike live bacilli, for which the percentage of ETZ frequency remained stable throughout incubation time, ETZ frequency for killed bacilli decreased with time. ETZ assessment performed on M. tuberculosis H37 Rv for comparison showed that, despite a very low ETZ frequency (8-15%), the percentage of intact bacteria was identical to that observed with M. avium. In contrast, three rapidly growing non-pathogenic species (M. smegmatis, M. phlei and M. fallax) presented a low ETZ frequency after phagocytosis and were rapidly degraded. The process of ETZ formation and its role in bacterial survival are discussed.

Animals↗
PubMedDate not supplied

Macrophage interaction with mycobacteria including M. leprae.

Resistance properties of pathogenic mycobacteria to macrophage bactericidal activity seems to be due mostly to the composition and constitution of their cell walls. In the case of Mycobacterium tuberculosis, sulfatides and polyglutamic acid could be implicated in the phenomenon of fusion inhibition between phagosomes and lysosomes. M. leprae and M. lepraemurium, which do not seem to inhibit fusions are protected by a thick electron transparent zone (ETZ) that seems to be composed of mycosides. This layer would inhibit lysosomal enzyme diffusion inside phagosomes. As ETZ does not exist in mycobacteria before their phagocytosis, we have tried to see when and how it is formed inside macrophages. We have compared ETZ formation in M. leprae and M. avium which both contain mycosides. These two species were allowed to be phagocytized by mouse bone-marrow derived macrophage and samples were taken for electron microscopy during the first hours of phagocytosis and also during several weeks of incubation. In M. avium ETZ appeared within 1 to 2 hours after phagocytosis. It seems to be formed by a sort of swelling of the thin electron transparent layer of the bacterial cell wall. This swelling occurs only in regions where the external polysaccharide layer of M. avium starts to disappear. After 1 to 3 hours, this layer was completely absent and all bacteria were enveloped in a thick ETZ. In M. leprae, the ETZ is also formed within one hour after ingestion. However, the presence in some bacteria of a very thin dense layer located at the original place of the outer dense layer of the cell wall does not fit well with the idea of ETL swelling. In addition, the appearance of a thick dense layer located between the ETZ and the phagosome membrane is not yet understood. The ETZ formed also rapidly in macrophages infected with heat killed cells of M. avium or M. leprae. This shows that its formation does not require the active participation of the bacterium. As already proposed ETZ seems to lessen considerably the diffusion of lysosomal enzymes towards the bacterium in both species. In M. leprae it seems especially efficient because despite acid phosphatase activity found in many phagosomes, neither the number of bacteria per macrophage nor their state of degradation changed during 3 and a half months of macrophage culture.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

Cytochemical characterization of mycobacterial outer surfaces.

A cytochemical study of mycobacterial outer surfaces was carried out on both pathogenic (M. leprae, M. avium) and non pathogenic (M. aurum) strains. Different cytochemical markers were used: Ruthenium Red, Concanavalin A, Wheat Germ Agglutinin, Colloidal Iron and Cationized Ferritin. The cytochemical staining pattern varied according to the species studied. The relationship between outer surface properties of mycobacteria and their capacity of adhesion to or ingestion by bone marrow macrophages was also considered.

Cell Wall↗