Propionibacterium acnes GehA lipase, an enzyme involved in acne development, can be successfully inhibited by defined natural substances

Propionibacterium acnes, a usual inhabitant of human skin, plays an important role in acne development, related to the production of numerous enzymatic activities involved in the degradation of host molecules. Among these enzymes, P. acnes lipase (GehA, glycerol-ester hydrolase A) has been recognized as one of the major factors in the pathogenesis of acne, being responsible for the hydrolysis of sebum and the release of inflammatory compounds. Anti-acne treatments are based on the use of retinoids or benzoyl peroxide, frequently in combination with antibiotics. However, the low effectiveness of such treatments and the increasing antibiotic resistance has led to the development of alternative therapies such as Kampo formulations, containing traditional herbal drugs. Search for new anti-acne treatments led us to perform the cloning, characterization and inhibition of P. acnes GehA, considered an interesting pharmaceutical target for anti-acne therapies. The genetic, molecular and biochemical properties of the cloned lipase were analysed, and several inhibitor agents were tested, including natural substances like saponins, alkaloids or flavonoids. Among these, the flavonoids (±)-catechin and kaempferol were the most promising candidates for acne treatment, whereas saponins like glycyrrhicic acid and digitonin produced a lower inhibition of the enzyme. No inhibition by alkaloids was found. Therefore, the inhibition caused by (±)-catechin and kaempferol on GehA, together with their wide anti-acne properties and low toxicity, make them very suitable candidates for the treatment of acne and other P. acnes-related diseases.     

Growth engineering of Propionibacterium freudenreichii shermanii for organic acids and other value-added products formation

Propionic acid production from glucose was studied using Propionibacterium freudenreichii shermanii. Conditions were optimized for high yields of propionic acid and total organic acids by sequential optimization of parameters like pH, inoculum age, inoculum volume and substrate concentration. Near-theoretical yield (0.54?±?0.023?g/g) was achieved for propionic acid with fermentation of 1% glucose using 20% (v/v) of 48?hr old P. shermanii at 30°C, pH maintained at 5.5. Total organic acid yield under these conditions was 0.74?±?0.06?g/g. The study resulted in achieving 98% and 95% theoretical yields of propionic acid and total organic acids, respectively. Under optimized conditions, along with organic acids, P. shermanii also produced vitamin B12 and trehalose intracellularly, showing its potential to be used as a cell factory.     

Propionate acts as carboxylic group acceptor in aspartate fermentation by Propionibacterium freudenreichii

Cells of Propionibacterium freudenreichii ssp. shermanii and ssp. freudenreichii did not show significant growth or product formation in a mineral medium with 10 mM aspartate or 10 mM fumarate, vitamins, and a small amount (0.05% w/v) of yeast extract. In the presence of added propionate, growth with aspartate or fumarate was possible, and depended strictly on the amount of propionate provided, according to the equation: 3 aspartate + propionate 3 succinate + acetate + CO₂+3 NH₃. Cocultures of P. freudenreichii with the succinate-decarboxylating strain Ft2 converted 3 aspartate stoichiometrically to acetate and 2 propionate. High activity of methylmalonyl-CoA: pyruvate transcarboxylase, and lack of methylmalonyl-CoA decarboxylase and oxaloacetate decarboxylase activity in cell-free extracts of aspartate-grown cells indicated that failure to use aspartate as sole substrate was due to the inability of these strains to catalyze a net decarboxylation of C₄-dicarboxylic acids.Dedicated to Prof. Dr. Norbert Pfennig on occasion of his 65th birthday     

Efficient co‐production of propionic acid and succinic acid by Propionibacterium acidipropionici using membrane separation coupled technology

To improve the fermentation efficiency of Propionibacterium acidipropionici, a semi‐continuous coupled fermentation process was established to achieve co‐production of propionic acid (PA) and succinic acid (SA). First, the optimal proportion of glucose (Glc) and glycerol (Gl) as a mixed carbon source was determined, and the feeding procedure of Gl was optimized to make more energy flow in the direction of product synthesis. Then, ZGD630 anion exchange resin was used for efficient adsorption of PA, thereby eliminating the feedback inhibition effect of PA. Finally, an efficient, coupled fermentation process of P. acidipropionici characterized by membrane separation and chromatography technology was developed. The concentrations of PA and SA reached 62.22 ± 2.32 and 20.45 ± 1.34 g L⁻¹, with corresponding productivity of 0.43 and 0.14 g L⁻¹ h⁻¹, increased by 65.38% and 48.54%, respectively. Membrane separation coupled fermentation of PA and SA could significantly improve the process economics of P. acidipropionici, and has good prospects for industrial application.     

Caenorhabditis elegans mounts a p38 MAPK pathway‐mediated defence to Cutibacterium acnes infection

Cutibacterium acnes is capable of inducing inflammation in acne and can lead to a chronic prostatic infection. The diverse pathogenicity among different strains of C. acnes has been presented, but simple appropriate animal models for the evaluation of this bacterium are lacking. In this study, the nematode Caenorhabditis elegans was used as an invertebrate infection model. We revealed that C. acnes type strain ATCC 6919 caused lethal infections to C. elegans in solid and liquid culture media (p < .0001). Compared with the strain ATCC 6919, the antibiotic‐resistant strain HM‐513 was more virulent, resulting in reduced survival (p < .0001). Four different C. acnes strains killed worms with a p value of less than .0001 when provided to C. elegans at 4.8 × 10⁸ CFU/ml. The infection model was also employed to explore host defence responses. An increase in numerous immune effectors in response to C. acnes was detected. We focused on nine C‐type lectins, including: clec‐13, clec‐17, clec‐47, clec‐52, clec‐60, clec‐61, clec‐70, clec‐71 and clec‐227. The induced expression of these C‐type lectin genes was down‐regulated in mutant worms deficient in the p38 mitogen‐activated protein kinase (MAPK) pathway. Meanwhile, PMK‐1 (MAPK) was phosphorylated and activated at the onset of C. acnes infection. By monitoring the survival of mutant worms, we found that PMK‐1, SEK‐1 (MAPKK) and TIR‐1 (MAPKKK) were critical in responding to C. acnes infection. C. elegans pmk‐1 and tir‐1 mutants exhibited higher mortality to C. acnes infection (p < .0001). In conclusion, C. elegans serves as a simple and valuable model to study C. acnes virulence and facilitates improvements in understanding of host innate immune responses.     

Construction and characterization of ack knock-out mutants of Propionibacterium acidipropionici for enhanced propionic acid fermentation

Propionibacterium acidipropionici produces propionic acid from glucose with acetic acid, succinic acid, and CO2 as byproducts. In this work, inactivation of ack gene, encoding acetate kinase (AK), by gene disruption and integrational mutagenesis was studied as a method to reduce acetate formation in propionic acid fermentation. The partial ack gene of approximately 750 bp in P. acidipropionici was cloned using a PCR-based method with degenerate primers and sequenced. The deduced amino acid sequence had 88% similarity and 76% identity with the amino acid sequence of AK from Bacillus subtilis. The partial ack gene was used to construct a linear DNA fragment with an inserted tetracycline resistance cassette and a nonreplicative integrational plasmid containing a tetracycline resistance gene cassette. These DNA constructs were then introduced into P. acidipropionici by electroporation, resulting in two mutants, ACK-Tet and TAT-ACK-Tet, respectively. Southern hybridization confirmed that the ack gene in the mutant ACK-Tet was disrupted by the inserted tetracycline resistance gene. As compared to the wild-type, the activities of AK were reduced by 26% and 43% in ACK-Tet and TAT-ACK-Tet mutants, respectively. The specific growth rate of these mutants was reduced by approximately 25% to 0.10/h (0.13/h for the wild-type), probably because of reduced acetate and ATP production. Both mutants produced approximately 14% less acetate from glucose. Although ack disruption alone did not completely eliminate acetate production, the propionate yield was increased by approximately 13%.     

Enhanced resistance to Gram-positive bacterium and increased susceptibility to bacterial endotoxin in mice sensitized with Propionibacterium acnes: involvement of Toll-like receptor

Mice sensitized with Propionibacterium acnes showed an enhanced resistance against infection with Listeria monocytogenes in contrast to the increased susceptibility to LPS-induced endotoxin shock. The enhanced protection to L. monocytogenes was mediated by activated innate immunity but not by generation of Listeria-specific acquired immunity. After infection with L. monocytogenes, the elimination of bacteria was observed earlier in accordance with a higher level of endogenous cytokine production in P. acnes-sensitized mice than in control mice. Peritoneal cells from P. acnes-sensitized mice produced a larger amount of IL-12p70 and nitric oxide after stimulation with heat-killed L. monocytogenes or peptidoglycan purified from Staphylococcus aureus. RT-PCR analysis showed that the expression of TLR2 but not TLR1, TLR4 nor TLR6 was induced by injection of P. acnes in peritoneal cells. These results indicated that P. acnes-sensitization could induce the activation of innate immunity against L. monocytogenes through increased recognition of bacterial components by TLR2.     

Acetate oxidation through a modified citric acid cycle in Propionibacterium freudenreichii

Propionibacterium freudenreichii strain DSM 20271 was grown in a mineral medium containing 0.1% (w/v) yeast extract. Acetate was oxidized by growing cells with potassium hexacyanoferrate as electron acceptor, which was oxidized by a three-electrode poised-potential system at a redox potential of +510 mV. Growth with acetate under these conditions followed linear rather than expenential kinetics, whereas growth with other substrates such as lactate under the same conditions was exponential. Cell-free extracts of P. freudenreichii cells grown with acetate contained all enzymes of the classical citric acid cycle except 2-oxoglutarate-oxidizing activity. No activity of anaplerotic reactions such as isocitrate lyase or malate synthase was found. Instead, moderate activities of glutamate decarboxylase, 4-aminobutyrate:2-oxoglutarate aminotransferase, and succinate semialdehyde dehydrogenase were detected. In short-term radiolabeling experiments with U-¹⁴C-acetate, 4-aminobutyrate was identified as a major early intermediate in acetate oxidation by these cells. These findings allow the construction of a modified citric acid cycle that compensates the lack of 2-oxoglutarate dehydrogenase by a subcycle through glutamate, 4-aminobutyrate, and succinate semialdehyde. Lack of anaplerotic reactions explains subexponential growth kinetics during growth with acetate.     

Eradication of Propionibacterium acnes by its endogenic porphyrins after illumination with high intensity blue light

Propionibacterium acnes is a Gram-positive, microaerophilic bacterium that causes skin wounds. It is known to naturally produce high amounts of intracellular porphyrins. The results of the present study confirm that the investigated strain of P. acnes is capable of producing endogenic porphyrins with no need for any trigger molecules. Extracts from growing cultures have demonstrated emission peaks around 612 nm when excited at 405 nm, which are characteristic for porphyrins. Endogenic porphyrins were determined and quantified after their extraction from the bacterial cells by fluorescence intensity and by elution retention time on high-performance liquid chromatography (HPLC). The porphyrins produced by P. acnes are mostly coproporphyrin, as shown by the HPLC elution patterns. Addition of delta-aminolevulinic acid (ALA) enhanced intracellular porphyrin synthesis and higher amounts of coproporphyrin have been found. Eradication of P. acnes by its endogenic porphyrins was examined after illumination with intense blue light at 407-420 nm. The viability of 24 h cultures grown anaerobically in liquid medium was reduced by less than two orders of magnitude when illuminated once with a light dose of 75 J cm(-2). Better photodynamic effects were obtained when cultures were illuminated twice or three times consecutively with a light dose of 75 J cm(-2) and an interval of 24 h between illuminations. The viability of the culture under these conditions decreased by four orders of magnitude after two illuminations and by five orders of magnitude after three illuminations. When ALA-triggered cultures were illuminated with intense blue light at a light dose of 75 J cm(-2) the viability of the treated cultures decreased by seven orders of magnitude. This decrease in viability can occur even after a single exposure of illumination for the indicated light intensity. X-ray microanalysis and transmission electron microscopy revealed structural damages to membranes in the illuminated P. acnes. Illumination of the endogenous coproporphyrin with blue light (407-420 nm) apparently plays a major role in P. acnes photoinactivation. A treatment protocol with a series of several illuminations or illumination after application of ALA may be suitable for curing acne. Treatment by both pathways may overcome the resistance of P. acnes to antibiotic treatment.     

The effect of Propionibacterium acidipropionici, with or without Lactobacillus plantarum, on the fermentation and aerobic stability of wheat, sorghum and maize silages

AIMS: To determine the effect of Propionibacterium acidipropionici, alone or in combination with Lactobacillus plantarum, on the fermentation and aerobic stability of wheat, sorghum and maize silages. METHODS AND RESULTS: The inoculants were applied at 1.0 x 10(6) CFU g(-1). Silages with no additives served as control. Fresh forages were sampled prior to ensiling. Three jars per treatment were sampled on days 2, 4, 8, 16 and 60 after ensiling, for chemical and microbiological analysis. At the end of the ensiling period, the silages were subjected to an aerobic stability test. The P. acidipropionici-inoculated silages had significantly higher levels of acetic and propionic acid than the L. plantarum or P. acidipropionici + L. plantarum-inoculated silages (P < 0.05). Therefore, yeast activity was impaired in the P. acidipropionici-inoculated silages. As a result, P. acidipropionici decreased CO(2) production and improved aerobic stability of wheat, sorghum and maize silages. However, the combination of P. acidipropionici + L. plantarum did not improve aerobic stability of the silages. CONCLUSIONS: The P. acidipropionici was very effective in protecting the wheat, sorghum and maize silages exposed to air under laboratory conditions, probably because the acidic environment under ensiling conditions is favourable for this micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of P. acidipropionici, as a silage inoculant can improve the aerobic stability of silages by inhibition of yeast activity.