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Ribosomal proteins (r-proteins) from a type I strain (ATCC 11827) and a type II strain (ATCC 11828) of Propionibacterium acnes (P. acnes) and from a Propionibacterium granulosum (P. granulosum) strain (ATCC 11829-2) were analyzed using two-dimensional polyacrylamide gel electrophoresis (two-dimensional PAGE), for the purpose of genetical analysis of the P. acnes type I and type II strains and the P. granulosum strain. Among the fifty r-proteins in all, almost all of the proteins were electrophoretically different between both of the two types of P. acnes strains and the P. granulosum strain, whereas only five proteins were different between the type I and type II strains of P. acnes. Such differences of r-protein composition between the type I and type II strains of P. acnes indicate that the type I strain is genetically different from, but very closely related to, the type II strain. However, the P. granulosum strain is not so closely related to both the two types of P. acnes strains. This study thus suggests that the two-dimensional PAGE of r-proteins is an excellent tool for the taxonomy of P. acnes and the related bacteria.

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161.8 MHz 31P-NMR spectra were recorded from the light sensitive skin bacterium Propionibacterium acnes. The cells were grown anaerobically on synthetic phosphate-buffered Eagle's medium or on a complex yeast extract medium. The spectra showed a large accumulation of polyphosphates when grown on Eagles medium. A splitting of the inorganic phosphate peak indicated a difference between internal and external pH of the cells. Addition of glucose to the cell suspension gave rise to a change in the pH gradient across the cell membrane, as reported for other Gram-positive bacteria. A decrease in the polyphosphate peak was observed after addition of glucose. A lethal dose of broad-band near-ultraviolet light (corresponding to a 10% survival in a survival test), increased the amount of polyphosphates visible in the NMR-spectra. The addition of glucose to irradiated cells decreased the pH in the external solution, but no splitting of the inorganic phosphate peak could however be observed. 31P-NMR can, therefore, be used to study immediate near-ultraviolet-induced effects at the cellular level, at least in the case of P. acnes.

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The structure of polysaccharide prepared by lysozyme digestion from the cell wall of Propionibacterium acnes strain C7 was examined. The polysaccharide fraction was composed of glucose, galactose, mannose, galactosamine, and diaminomannuronic acid in a molar ratio of 1:1:0.3:1:2. By Smith degradation of the polysaccharide, diaminouronic acid-containing fractions were obtained, and the configuration of diaminouronic acid was identified as 2,3-diacetamido-2,3-dideoxymannuronic acid [Man(NAc)2A] by means of 1H-NMR and 13C-NMR spectroscopic analyses. The results of analyses involving methylation and partial acid hydrolysis led to the conclusion that the polysaccharide has the repeating unit----6)Gal(alpha 1----4)Man(NAc)2A(beta 1----6)Glc(alpha 1----4)Man(NAc)2A (beta 1----3)GalNAc(beta 1--. In addition, a portion of the galactose residues were substituted at C-4 by alpha 1----2 linked mannotriose.

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Splenic lymphocytes from mice treated with Propionibacterium acnes cells as well as with their cell walls were found to be variably active on the lymphoproliferative responsiveness. Furthermore, the effect of these bacterial agents on the ex vivo Con A response of the lymphocytes showed a certain stimulation that was higher with oral treatments. In the same conditions the influence of these agents on the LPS lymphocytes stimulation was almost without any statistical significance. In vitro blastogenesis experiments were undertaken in order to elucidate the influence of different amphiphilic molecules from peripheric bacterial structures on the lymphoproliferative response of murine splenocytes. Stimulation rates were also determined as a function of the (3H) thymidine incorporation. Combined effects of mitogens (Con A and LPS) with bacterial amphiphilic molecules were also evaluated as a function of the DNA synthesis variations. All cases resulted in a variable inhibition of the mitogenic response which appeared dose-dependent and more active for associations of Con A and amphiphilic molecules. The most effective intrinsic mitogenic activities were detected with teichoic acids and intracellular polysaccharides. These last molecules without purification, assayed as cytoplasmic fractions, appeared modified in the intensity of their action, depending on their carbohydrate/protein ratios.

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Evidence is presented which suggests that certain key markers of lepra bacilli reside collectively in Proprionibacterium acnes, Corynebacterium tuberculostearicum and Mycobacterium leprae. The unrestricted replication of Mycobacterium leprae depends most probably upon the presence of an immune-deficiency-inducing viral agent or possibly on the combined effects of the organisms considered.

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A previously undescribed component of the cell wall polysaccharide of Propionibacterium acnes, 2,3-diamino-2,3-dideoxyglucuronic acid, has been identified and synthesized. The component occurs to the extent of about 3 to 5% in the wall polysaccharides of P. acnes types I and II and in Propionibacterium avidum types I and II; it also appears to be present, but in much smaller amounts, in the cell wall of Propionibacterium granulosum.

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A semisynthetic medium, containing yeast extract as the only non defined component and glucose, glycerol, Tween 80 and mineral salts, has been developed to grow the strains of Propionibacterium acnes Beck 2037, Gerrath 2038 and Vogel 2039, at a rate comparable to that of complex media. An average of 10(12) cells per 1 (equivalent to approximately 1.5 g/l dry weight) was usually achieved. These yields are appropriated for biochemical and immunological studies, e.g. cell wall preparation, polysaccharides isolation, etc. In this work, some properties of the cell walls (sugar and amino acid composition) and the antigenic polysaccharides (neutral and amino sugar components) are described.

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The cell-wall skeletons of Listeria monocytogenes strain EGD and Propionibacterium acnes strain C7, which have the ability to induce macrophage activation, were analyzed, and the structures of the peptidoglycans were investigated. The analytical data indicate that both peptidoglycans have glucosamine residues with free amino groups, which are responsible for the resistance to lysozyme. Possible structures of these peptidoglycans were deduced from the composition and the results of determination of N- and C-terminal amino acids, together with the characterization of fragments obtained by enzymatic treatment and partial acid hydrolysis of both peptidoglycans. The results suggested that the peptidoglycan of L. monocytogenes contains a cross-linkage region of peptide chains with meso-diaminopimelic acid and D-alanine, which belongs to the A1 gamma type (Schleifer, K.H. & Kandler, O. (1972) Bacteriol. Rev. 36, 407-477), whereas the peptidoglycan of P. acnes contains a cross-linkage region of peptide chains with L,L-diaminopimelic acid and D-alanine, in which two glycine residues combine with amino and carboxyl groups of two L,L-diaminopimelic acid residues. The latter type should be classified as a new type. These cell-wall skeletons and peptidoglycans were shown to have immunoadjuvant activity on the induction of delayed-type hypersensitivity and suppressive activity on the growth of 3-methylcholanthrene-induced fibrosarcoma in BALB/c mice, and the peptidoglycans were shown to be an immunological-active principle of these cell-wall skeletons.

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A mannose specific lectin has been isolated by affinity chromatography from the cell wall of C. parvum. Polyacrylamide gel electrophoresis indicates that the lectin molecules lie in the molecular weight range 57,000--72,000. It appears that C. parvum like E. coli and salmonellae express lectins that bind to cells expressing mannose in their membranes. This may partly account for the interaction between C. parvum and the macrophage leading to the various immunological phenomena associated with C. parvum administration.

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Activation of the alternative pathway of complement is known to be initiated by bacterial structures. We have fractionated Propionibacterium acnes cells, purified various cell fractions, and tested their complement-activating ability in human serum chelated with ethyleneglycol bis-(beta-aminoethylether)-N,N1-tetraacetic acid. The majority of complement-activating activity was localized in the wall fraction. This activity was resistant to lipid extraction, protease, RNAse, DNAse and lysozyme treatment. NaIO4, formamide, and hot (but not cold) trichloroacetic acid (TCA) extraction ablated the complement-activating capacity of cell walls. Compounds removed by extraction failed to consume significant hemolytic activity against antibody-coated sheep erythrocytes (EA). Addition of TCA-extracted soluble material to cell wall suspensions resulted in an inhibition of hemolytic consumption by the cell wall. These results indicate that, in P. acnes, complement-activating molecules are located in the cell wall and are carbohydrate in nature. Peptidoglycan, lipid, protein, and nucleic acid do not appear to contribute to the cell wall's ability to activate complement.

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A gas liquid chromatography method for the separation of 10 volatile fatty acids (C1-C7 and isomers) has been improved by using oven temperature programmed conditions. In our conditions, the proprietary stationary phase SP 1220 introduced by Supelco Inc., gave sharp separation of volatile fatty acids in less than 8 min. This method was suitable for analyses with both thermal conductivity and flame ionization detectors.

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Mouse peritoneal macrophages, activated with Propionibacterium acnes (Corynebacterium parvum) in vivo, exhibited altered morphological characteristics, increased acid phosphatase activity, and release of [EH]thymidine-labeled DNA from tumor cells in vitro. Comparison with macrophages from mice injected with a control organism, P. jensenii, showed that the morphological changes, but not acid phosphatase, correlated with the development of tumoricidal activity. Investigation of the microbial component responsible for this activity indicated it was heat stable and refractory to extraction by methanol-chloroform, dilute acid, butanol, or a serial combination of extraction procedures. When the cells were disrupted by mechanical breakage, no activity could be found in the cell wall, soluble cytoplasm, or particulate cytoplasm. These results suggest that intact organisms are required for macrophage activation, and they may resolve conflicting reports on the nature of the immunostimulating activity of P. acnes.

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The prostaglandin-like substances (PLS) isolated from P. acnes were investigated by reversed phase chromatography and gas chromatography-mass spectrometry. These analyses demonstrated that PLS were not identical with PGE2, which supports a concept of PLS as a potential mediator of the inflammatory process in acne vulgaris.

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Three acidic polysaccharide (AP) fractions and the prostaglandin-like substances (PLS) isolated from P. acnes were investigated regarding their chemotactic activities on polymorphonuclear leukocytes. Both AP's and PLS induced a significant chemotatic response, which suggests their involvement in inflammatory acne vulgaris.

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A method is described in which washed whole cells of Corynebacterium parvum were chemically and enzymatically extracted to remove cytoplasm and cell-wall lipids. The resultant insoluble cell-wall residue possessed lympho-reticular stimulating properties as measured by their ability to increase spleen weight and protect against tumour-cell challenge. Analysis of the final product by chromatography and infrared spectroscopy has shown it to consist of carbohydrate and peptidoglycan, both of which appear to be necessary for the activities measured.

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