TLR9-dependent and independent pathways drive activation of the immune system by Propionibacterium acnes

Propionibacterium acnes is usually a relatively harmless commensal. However, under certain, poorly understood conditions it is implicated in the etiology of specific inflammatory diseases. In mice, P. acnes exhibits strong immunomodulatory activity leading to splenomegaly, intrahepatic granuloma formation, hypersensitivity to TLR ligands and endogenous cytokines, and enhanced resistance to infection. All these activities reach a maximum one week after P. acnes priming and require IFN-γ and TLR9. We report here the existence of a markedly delayed (1-2 weeks), but phenotypically similar TLR9-independent immunomodulatory response to P. acnes. This alternative immunomodulation is also IFN-γ dependent and requires functional MyD88. From our experiments, a role for MyD88 in the IFN-γ-mediated P. acnes effects seems unlikely and the participation of the known MyD88-dependent receptors, including TLR5, Unc93B-dependent TLRs, IL-1R and IL-18R in the development of the alternative response has been excluded. However, the crucial role of MyD88 can partly be attributed to TLR2 and TLR4 involvement. Either of these two TLRs, activated by bacteria and/or endogenously generated ligands, can fulfill the required function. Our findings hint at an innate immune sensitizing mechanism, which is potentially operative in both infectious and sterile inflammatory disorders.     

Studies of the extracellular proteolytic activity produced by Propionibacterium acnes

Of several commercial media tested, trypticase soya both containing 0.4% (w/v) D-sorbitol was superior as a growth medium for the production of extracellular proteinase by Propionibacterium acnes (strain P-37). Extracellular proteinase, production of which was shown to be growth-associated by both batch and continuous culture studies, was partially purified by 70% (NH4)2SO4 saturation. Sephadex G-75 chromatography and ion exchange on DEAE-Sephadex A-50. It was shown to be a heterogeneous mixture of at least three molecular species of enzyme. Proteinase I was inhibited by EDTA (10(-3) mol/l) and PMSF (5 millimol/l) and stimulated by CaCl2 (190% at 10(-3) mol/l). It had a molecular weight of 20 to 30000 and a broad pH optimum from 6.5 to 7.5. Proteinase II was an alkaline proteinase with a molecular weight of 30 to 40000 which was not significantly inhibited by EDTA (10(-2) mol/l) nor stimulated by CaCl2. Proteinase III represented a minor proportion of the recovered proteolytic activity, had a molecular weight of 20 to 30000 and was most active in the alkaline pH range. This enzyme was inhibited by EDTA (10(-4) mol/l) and PMSF (5 millimol/l), and stimulated by CaCl2 (250% at 10(-2) mol/l).     

Studies of the extracellular proteolytic activity produced by Propionibacterium acnes

Of several commercial media tested, trypticase soya broth containing 0.4% (w/v) D-sorbitol was superior as a growth medium for the production of extracellular proteinase by Propionibacterium acnes (strain P-37). Extracellular proteinase, production of which was shown to be growth-associated by both batch and continuous culture studies, was partially purified by 70% (NH₄)₂SO₄ saturation, Sephadex G-75 chromatography and ion exchange on DEAE-Sephadex A-50. It was shown to be a heterogeneous mixture of at least three molecular species of enzyme. Proteinase I was inhibited by EDTA (10^-3 mol/l) and PMSF (5 millimol/l) and stimulated by CaCl₂ (190% at 10^-3 mol/l). It had a molecular weight of 20 to 30000 and a broad pH optimum from 6.5 to 7.5. Proteinase II was an alkaline proteinase with a molecular weight of 30 to 40000 which was not significantly inhibited by EDTA (10^-2 mol/l) nor stimulated by CaCl₂. Proteinase III represented a minor proportion of the recovered proteolytic activity, had a molecular weight of 20 to 30000 and was most active in the alkaline pH range. This enzyme was inhibited by EDTA (10^-4 mol/l) and PMSF (5 millimol/l), and stimulated by CaCl₂ (250% at 10^-2 mol/l).     

Optimization of electrotransformation conditions for Propionibacterium acnes

Propionibacterium acnes has been known to be involved in the pathology of acne. However, the definite mechanism in the development of acne and the inflammation are unknown. For P. acnes, a transformation method has not been established, although it is believed to be a basic tool for gene manipulation. This study attempted to develop a P. acnes transformation method by using electroporation. Various parameters were used to develop and optimize the transformation of P. acnes. Among them two factors were crucial in the transformation for P. acnes: one was the E. coli strain from which the plasmid DNA had been isolated and the other the growth temperature of P. acnes-competent cells. It was essential to prepare plasmid DNA from a dam(-) E. coli strain, ET12567. When plasmid DNAs isolated from the other E. coli strains such as JM109 and HB101 were tested, transformation efficiency was extremely low. When P. acnes cells were cultivated at 24 degrees C for competent cell preparation, transformation efficiency increased considerably. When plasmid DNA isolated from a dam(-) mutant strain of E. coli was used for transformation of P. acnes which had been grown at 24 degrees C, maximum transformation efficiency of 1.5 x 10(4) transformants per mug of plasmid DNA was obtained at a field strength of 15 kV/cm with a pulse time of 3.2 ms. This is believed to be the first report on the transformation of P. acnes which can be employed for gene manipulations including knock-out of specific genes.     

A simple method for differentiation of Propionibacterium acnes and Propionibacterium propionicum

Abstract TLC glycolipid profiles of several culture collection and clinical strains of Propionibacterium acnes and Propionibacterium propionicum were examined. The former were characterized by weak orcinol-positive minor glycolipids of type g, while the others had mainly strong orcinol-positive major glycolipids of type G. The simple and rapid small scale procedure seemed to be useful for differentiation of these phenotypically similar and genotypically closely related species irrespective of their serotypes.     

Propionibacterium acnes in the etiology of endocarditis]

Propionibacterium acnes is the gram positive anaerobic bacteria belongs to the normal skin and oral microbial flora. The participation of this microorganism in the infective endocarditis is still controversial. The aim of the study was to perform the diagnostic and therapeutic difficulties in 5 patients with infective endocarditis caused by Propionibacterium acnes. In 3 out of 5 patients the infective endocarditis developed after prosthesis valve replacement, in 2 others on the native valves. The inserted prostheses were mechanical ones, propionibacterium acnes was identified as causative organisms in all of the causes (two positive blood and/or valve culture). The bacterial strains were sensitive to the antibiotics as: penicillins, cephalosporins, clindamycin, and vancomycin, however cephalosporins used at the beginning of the treatment in 3 patients and clindamycin in 1 patient had limited clinical efficacy. Later treatment with timentin, augmentin and tienamycin was successful in 3 patients; one patient was cured with vancomycin. One patient died because of septic, embolic complication in early stage of illness. We conclude the effectiveness of penicillins in combination with clavulanic acid and tienamycin in therapy of infective endocarditis due to Propionibacterium acnes. The treatment should be lasted during 4-6 weeks.     

Comparative studies of porphyrin production in Propionibacterium acnes and Propionibacterium granulosum.

Porphyrin production by Propionibacterium acnes and that by Propionibacterium granulosum were compared. Porphyrin synthesized by both organisms was identified as coproporphyrin III on the bases of absorption and fluorescence spectra and behavior on paper chromatography and thin-layer chromatography. Quantitative, rather than qualitative, differences in production were found between these organisms. In general, P. granulosum produced significantly greater amounts (P less than 0.001) of porphyrin than did P. acnes. delta-Aminolevulinic acid synthetase appeared to be the rate-limiting enzyme of the heme biosynthetic pathway in both organisms. The increased porphyrin production in P. granulosum is apparently associated with increased delta-aminolevulinic acid synthetase activity.     

Role of respiratory-burst products from polymorphonuclear leukocytes in the antitumor activity of Propionibacterium acnes vaccine

Summary Tumor cells injected into Balb/c mice together with heat-killed 48-h P. acnes cells were rendered nontumorigenic as early as 12 h after injection, as determined by the inability of the tumor cells to give rise to tumors when transferred to a new host. Determination of tumor cell antigen levels by ELISA indicated that the tumor antigens had virtually disappeared by 24 h after injection of tumor cells and P. acnes. In contrast, in control animals injected with tumor cells only, there was an initial drop in tumor antigen levels at 12 h, after which the level rose steadily and tumors developed in 7–10 days. Since the cellular exudate at 12 h was almost entirely composed of polymorphonuclear leukocytes (PMN), we tested the ability of PMN, stimulated by phagocytosis of 48-h P. acnes cells, to produce substances toxic to tumor cells. Results indicated that the supernatant fluid from a phagocytosis mixture of PMN and P. acnes contained material toxic to tumor cells and also to Chinese hamster ovary cells. Tests with scavengers and inhibitors of oxygen-derived radicals suggested that the toxic material is either hydrogen peroxide (H₂O₂) or hydroxyl radicals (OH). Suspensions of 12-h P. acnes, P. acnes cells walls, P. freudenrichii, or latex beads were ineffective in preventing tumor growth, and induced little toxicity when phagocytosed. We conclude that in this test system 48-h P. acnes prevents tumor growth by stimulating the production of toxic oxygen metabolites during phagocytosis by PMN.     

The microaerophily and photosensitivity of Propionibacterium acnes

E. MARTIN GRIBBON, J.G. SHOESMITH, W.J. CUNLIFFE AND K.T. HOLLAND. 1994. The effect of oxygen on the in vitro propagation of Propionibacterium acnes was investigated under defined culture conditions. This micro-organism is the predominant bacterial resident within the pilosebaceous follicles of sebum-rich areas of human skin. The organism was grown in continuous culture in defined synthetic medium with glucose as the main carbon-energy source at various air saturation concentrations and in the presence and absence of light. Steady state continuous cultures were achieved at very low oxygen tensions in the presence of light, and at higher levels of oxygen when non-illuminated. Culture biomass yields were higher than those of anaerobic cultures. Bacterial cells were inactivated in the presence of light at high oxygen concentrations because of photosensitization reactions involving excess oxygen and microbial porphyrin species.     

In situ assessment of porphyrin photosensitizers in Propionibacterium acnes

Porphyrins are known to be efficient photosensitizer molecules and the combined action of light and porphyrins in Propionibacterium acnes have a lethal action on the cells. Identification and quantification of in situ porphyrins in P. acnes have been done using an integrating sphere connected to an ordinary absorption spectrophotometer, and the amounts of porphyrins in the cells were quantified by measuring scattering free absorption spectra of the cell suspensions. The concentration of porphyrins in P. acnes cells were increased in either of two ways; by the addition of delta-aminolevulinic acid (ALA), which lead to the formation of coproporphyrin III under the incubation conditions used in these experiments, or by the addition of protoporphyrin IX (PPIX) to the cell suspension. In the latter case, PPIX molecules are taken up by the cells in a membrane-mediated uptake mechanism, and accumulate in the cells either on a monomeric or a particular aggregate form. The fraction of porphyrins on aggregate form increased with increasing PPIX additions. In the case of ALA induced porphyrin production, only monomeric porphyrins were stored in the cells. In both cases, the cells have a limited binding capacity of monomeric porphyrins, which is estimated to be 3 x 10(5) molecules/cell, or one porphyrin molecule to every 100st lipid molecule in the cell membrane.     

Proliferative response of murine lymphocytes caused by the activity of amphiphilic molecules from Propionibacterium acnes

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.     

Purification and characterisation of lipoglycan macroamphiphiles from Propionibacterium acnes

Lipidated macroamphiphiles such as the lipoteichoic acids and mycobacterial lipoarabinomannans are cell envelope components of Gram-positive bacteria that have been extensively associated with the pathogenesis of disease. In order to study such associations, purification of these macroamphiphiles is essential for resolving their structures and diverse biological effects. We describe herein a method for purification of lipoglycan components from Propionibacterium acnes. This method uses the existing phenol-water extraction, followed by hydrophobic interaction chromatography and an additional purification step that utilises preparative electrophoresis for the separation of two lipoglycan components. Analysis of these lipoglycans revealed evidence for a lipid anchor based on fatty acids whilst the polysaccharide moiety contained significant amounts of mannose, glucose and galactose, together with an amino sugar suspected of being a diaminohexuronic acid. These latter components have been previously identified as components of the P. acnes cell wall polysaccharide. Consequently, it is proposed that there may be a relationship between the structures of these distinctive cell envelope polymers.     

Isolation of Propionibacterium acnes from central nervous system infections

Propionibacterium acnes is a common skin colonizer and its involvement in central nervous system (CNS) infections may be related with previous neurosurgical procedures. P. acnes was isolated in pure or mixed cultures from ten patients with CNS infections during a 5-year period. The clinical presentation, treatment and outcome were retrospectively reviewed. Nine out of 11 patients had CNS infections after a neurosurgical procedure. The clinical presentation was: brain abscess (five patients), subdural or epidural empyema (four patients) and shunt meningitis (one patient). Three patients had also secondary meningitis. All patients received antibiotic therapy and all abscesses and empyemas were drained. The patient with shunt meningitis cured without catheter removal. Only one patient with a brain abscess by P. acnes died, but several months thereafter and as a consequence of a Gram-negative superinfection. P. acnes is a pathogen for the CNS and infections must be surgically managed under adequate antibiotic treatment.     

Genome sequence and analysis of a Propionibacterium acnes bacteriophage

Cutaneous propionibacteria are important commensals of human skin and are implicated in a wide range of opportunistic infections. Propionibacterium acnes is also associated with inflammatory acne vulgaris. Bacteriophage PA6 is the first phage of P. acnes to be sequenced and demonstrates a high degree of similarity to many mycobacteriophages both morphologically and genetically. PA6 possesses an icosahedreal head and long noncontractile tail characteristic of the Siphoviridae. The overall genome organization of PA6 resembled that of the temperate mycobacteriophages, although the genome was much smaller, 29,739 bp (48 predicted genes), compared to, for example, 50,550 bp (86 predicted genes) for the Bxb1 genome. PA6 infected only P. acnes and produced clear plaques with turbid centers, but it lacked any obvious genes for lysogeny. The host range of PA6 was restricted to P. acnes, but the phage was able to infect and lyse all P. acnes isolates tested. Sequencing of the PA6 genome makes an important contribution to the study of phage evolution and propionibacterial genetics.     

Susceptibility of Propionibacterium acnes to the essential oil of Melaleuca alternifolia

The susceptibility of 32 strains of Propionibacterium acnes to the essential oil of Melaleuca alternifolia , tea tree oil, was examined using a broth dilution method. The minimum bactericidal concentration of tea tree oil for five strains was 0.25% or less while, for the remainder, it was 0.50%.     

Analysis of clinical isolates of Propionibacterium acnes by optimised RAPD

Random amplification of polymorphic DNA (RAPD) was evaluated as a genotypic method for typing clinical strains of Propionibacterium acnes. RAPD can suffer from problems of reproducibility if parameters are not standardised. In this study the reaction conditions were optimised by adjusting template DNA concentration and buffer constituents. All isolates were typeable using the optimised RAPD protocol which was found to be highly discriminatory (Simpson's diversity index, 0.98) and reproducible. Typing of P. acnes by optimised RAPD is an invaluable tool for the epidemiological investigation of P. acnes for which no other widely accepted method currently exists.     

Sampling and detection of skin Propionibacterium acnes: Current status

A connection between acne vulgaris and Propionibacterium acnes has long been suggested. Over the years, several human skin microbiota sampling methods have been evolved and applied, e.g. swab, scrape, extraction techniques including cyanoacrylate gel sampling as well as punch biopsy. Collected samples have been processed following various methodologies ranging from culture studies to probe labelling and molecular analysis. Direct visualization techniques have recently shown the existence of anatomically distinct skin P. acnes populations: epidermal and follicular. P. acnes biofilms appear to be a common phenomenon. Current sampling approaches target different skin populations of P. acnes and the presence of microbial biofilms can influence the retrieval of P. acnes. The anatomical considerations must be taken into account while interpreting microbiological data.