Peptides were eluted from the analytical column at a flow rate of 250 nL/min using a 30-min gradient from 5% to 35% of solution B (0.1% formic acid, 100% acetonitrile). Trap and analytical column were packed with ReproSil-Pur C18-AQ 3 μm resin (Dr. The peptides were eluted and separated on a 15 cm analytical column (75 μm i.d.), pulled in-house (P2000 laser puller Sutter Instrument, Novato, CA, USA). The trypsin-digested samples were suspended in 0.1% formic acid, injected, trapped and desalted on a precolumn. Nano-electrospray ionization MS/MS (nanoESI-MS/MS) analyses were performed on an EASY-nLC II system (Thermo-Fisher Scientific, Waltham, MA, USA) connected to a TripleTOF 5600+ mass spectrometer (AB SCIEX, Framingham, MA, USA) operated under Analyst TF 1.6.1 control. Protein bands in the MW range 80–90 kDa were excised for in-gel tryptic digestion. acnes Proteins and Detection of HYL by Mass Spectrometry In addition, the Δ hyl mutant strain used in this study was sequenced to confirm that the ermE resistance cassette was inserted in the right location. Only few clones were obtained and tested by PCR to validate the correct knock-out of PPA0380. After electroporation and one-day recovery, cells were plated out on agar plates containing 10 µg/mL erythromycin. A protocol for preparing competent cells and electroporation settings were described previously. The KPA171202 wild-type strain is susceptible to erythromycin. As selection marker, the erythromycin-resistance cassette ermE of Saccharopolyspora erythraea (DSM no. To amplify the 500 bp regions up- and downstream of the gene PPA0380 ( hyl) the primer combinations PPA0380_1/PPA0380_2 and PPA0380_3/PPA0380_4, respectively, were used ( Table S2). acnes strain KPA171202 was used as the wild-type strain. Other glycosaminoglycans (GAGs) like chondroitin 4-sulfate (CSA), dermatan sulfate (CSB) and chondroitin 6-sulfate (CSC) are also degraded by both vertebrate and bacterial HYLs, but at a considerable slower rates. Both keratinocytes in the epidermis and fibroblasts in the dermis produce HA. The molecule is an abundant extracellular polysaccharide in the epidermis and the dermis up to 56% of the total body HA resides in the skin. HA is a non-sulfated glycosaminoglycan (GAG) of high molecular weight it is composed of repeated units of β-1,4-glucunoric acid and 1,3- N-acetylglucosamine disaccharides, connected through a β-linkage. HYLs are ubiquitous enzymes that function primarily in the degradation of hyaluronic acid (HA). One genomic difference between type IA and type IB/II strains concerned the gene hyl that encodes a hyaluronic acid lyase/hyaluronidase (HYL). acnes phylotypes were mapped and catalogued. In a recent study, genomic differences between the different P. It was recently proposed to reclassify type I strains as P. acnes consists of several phylogenetic subtypes commonly designated IA1, IA2, IB, IC, II and III. Based on several phylotyping schemes and complete genome sequencing, the population of P. The bacterium has also been associated with opportunistic infections such as prosthetic joint infection, sarcoidosis, endocarditis, and several other diseases. Despite its low virulence, it is commonly known to play a role in the pathogenesis of the skin disorder acne vulgaris. The bacterium is a major commensal of the human skin, accounting for a large proportion of the total human skin microbiota. Propionibacterium acnes is a Gram-positive anaerobic but aerotolerant bacterium present in sebaceous follicle-rich areas of human skin, such as the face, upper chest, and back. Whereas type IA strains are primarily found on the skin surface and associated with acne vulgaris, type IB/II strains are more often associated with soft and deep tissue infections, which would require elaborate tissue invasion strategies, possibly accomplished by a highly active HYL-IB/II. Our findings could explain some of the observed differences between P. The other variant, HYL-IA, has low activity, resulting in incomplete HA degradation it is present in type IA strains. One variant, HYL-IB/II, is highly active, resulting in complete HA degradation it is present in strains of the phylotypes IB and II. acnes employs two distinct variants of HYL. acnes hyl knockout mutant and HYL activity assays to determine the substrate range and formed products. Investigations include the generation of a P. acnes and investigated the genotypic and phenotypic characteristics. In this study, we identified the HYL of P. One of the most prevalent skin microorganisms, Propionibacterium acnes, possesses HA-degrading activity, possibly conferred by the enzyme hyaluronate lyase (HYL). Hyaluronic acid (HA) and other glycosaminoglycans are extracellular matrix components in the human epidermis and dermis.
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