Propionibacterium (Cutibacterium) granulosum Extracellular DNase BmdE Targeting Propionibacterium (Cutibacterium) acnes Biofilm Matrix, a Novel Inter-Species Competition Mechanism.

Acne vulgaris is the most common dermatological disorder worldwide affecting more than 80% of adolescents and young adults with a global prevalence of 231 million cases in 2019. The involvement of the skin microbiome disbalance in the pathophysiology of acne is recognized, especially regarding the relative abundance and diversity of Propionibacterium acnes a well-known dominant human skin commensal. Biofilms, where bacteria are embedded into a protective polymeric extracellular matrix, are the most prevalent life style for microorganisms. P. acnes and its biofilm-forming ability is believed to be a contributing factor in the development of acne vulgaris, the persistence of the opportunistic pathogen and antibiotic therapy failures. Degradation of the extracellular matrix is one of the strategies used by bacteria to disperse the biofilm of competitors. In this study, we report the identification of an endogenous extracellular nuclease, BmdE, secreted by Propionibacterium granulosum able to degrade P. acnes biofilm both in vivo and in vitro. This, to our knowledge, may represent a novel competitive mechanism between two closely related species in the skin. Antibiotics targeting P. acnes have been the mainstay in acne treatment. Extensive and long-term use of antibiotics has led to the selection and spread of resistant bacteria. The extracellular DNase BmdE may represent a new bio-therapeutical strategy to combat P. acnes biofilm in acne vulgaris.

Transcriptomic analysis of Propionibacterium acnes biofilms in vitro.

Propionibacterium acnes is a well-known commensal of the human skin connected to acne vulgaris and joint infections. It is extensively studied in planktonic cultures in the laboratory settings but occurs naturally in biofilms. In this study we have developed an in vitro biofilm model of P. acnes and studied growth features, matrix composition, matrix penetration by fluorescent-labeled antibiotics as well as gene expression. Antibiotic susceptibility of biofilms was studied and could be enhanced by increased glucose concentrations. Biofilm cells were characterized by up-regulated stress-induced genes and up-regulation of genes coding for the potential virulence-associated CAMP factors. P. acnes can generate persister cells showing a reversible tolerance to 50 fold MIC of common antibiotics.

Microbial colonization of normal skin: Direct visualization of 194 skin biopsies.

Recent genetic studies have suggested the presence of numerous microbial species on and in the skin. We characterised microbial colonization of a large collection of skin biopsies from 194 healthy subjects by fluorescence assay. Forty per cent of all biopsies did not show any evidence for microbial colonization. Propionibacterium acnes was the sole predominant bacterial species in both sebaceous and non-sebaceous areas. Non- P. acnes species were present in approximately 30% of all colonized samples. . Only hair follicles and stratum corneum were colonized. Understanding of cutaneous microbiota requires validation from a variety of approaches and techniques.

Immobilization of active lipase B from Candida antarctica on the surface of polyhydroxyalkanoate inclusions.

Polyhydroxyalkanoate (PHA) beads, recombinantly produced in Escherichia coli, were functionalized to display lipase B from Candida antarctica as translational protein fusion. The respective beads were characterized in respect to protein content, functionality, long term storage capacity and re-usability. The direct fusion of the PHA synthase, PhaC, to lipase B yielded active PHA lipase beads capable of hydrolyzing glycerol tributyrate. Lipase B beads showed stable activity over several weeks and re-usability without loss of function.

Three dimensional distribution of Propionibacterium acnes biofilms in human skin.

Propionibacterium acnes is regarded as a common member of the human skin microbiota, often occurring in biofilms. Little is known about the size of bacterial biofilms in hair follicles as a few sections of biopsy tissue are routinely evaluated. Transversal sectioning provides a better opportunity for histological analyses of hair follicles which can be followed through the different morphological levels. Direct visualization of P. acnes biofilms in hundreds of consecutive sections allowed insight into the 3D distribution in human hair follicles as well as investigating the depth of biofilm distribution within hair follicles. Four distinct colonization patterns of P. acnes biofilms were revealed. Results have shown that an individual P. acnes biofilm can spread for 1900 µm in a terminal hair follicle. This information can be of help while designing potential antibiofilm treatment.

Microbiology of hidradenitis suppurativa (acne inversa): a histological study of 27 patients.

Hidradenitis suppurativa (acne inverse) (HS) is a chronic skin disease primarily affecting hair follicles. The aetiology of HS is unknown, but infection is believed to play some role. This retrospective study investigated the microbial colonization directly in skin appendices in HS skin samples. Archival samples from 27 patients with HS were screened by immunofluorescence labelling with monoclonal and polyclonal antibodies against Gram-positive bacteria, Propionibacterium acnes and Propionibacterium granulosum. Fluorescence in situ hybridization was used for further species identification of Staphylococcus spp. Overall, 17 patients (63%) were found positive for bacterial colonization. Of these, 15 showed colonization in hair follicles and/or sinus tracts. The most commonly identified bacteria were DAPI labelled coccoids that were seen in 71% of the positive patients in the form of biofilms and microcolonies. P. acnes was found as biofilms in hair follicles of two patients. Staphylococcus aureus and coagulase-negative staphylococci were not detected in any sample. The results of this study indicate a common bacterial presence in HS skin lesions. Bacterial biofilms are not uncommon and their pathogenic role needs further evaluation.

Microbiology of folliculitis: a histological study of 39 cases.

Folliculitis is a common inflammatory skin syndrome. Several microbial organisms have been put forward as causative agents, but few studies visualized microbes directly in inflamed hair follicles. This retrospective study investigated bacterial and fungal colonization of inflamed hair follicles in patients with clinically diagnosed non-infectious folliculitis. Skin biopsies from 39 folliculitis patients and 27 controls were screened by fluorescence in situ hybridization (FISH) using broad-range bacterial and fungal probes and by immunofluorescence microscopy using a monoclonal antibody towards Gram-positive bacteria. Specific monoclonal and polyclonal antibodies towards Staphylococcus spp. and Propionibacterium acnes were applied for further species identification. Inflamed follicles were associated with bacterial colonization in 10 samples (26%) and fungal colonization in three samples (8%). Staphylococcus spp. were observed in inflamed follicles in seven samples (18%). Two samples were positive for P. acnes, which were identified as either type II or type IB/type III. Both Staphylococcus spp. and P. acnes were seen in macrocolonies/biofilm structures. In conclusion, one-third of patients with clinically diagnosed, non-infectious folliculitis exhibited microbial colonization with predominance of Staphylococcus spp.

Simultaneous visualization of Propionibacterium acnes and Propionibacterium granulosum with immunofluorescence and fluorescence in situ hybridization.

Propionibacterium acnes (P. acnes) and Propionibacterium granulosum (P. granulosum) are common skin colonizers that are implicated as possible contributing factors in acne vulgaris development. We have established direct visualization tools for the simultaneous detection of these closely related species with immunofluorescence assay and fluorescence in situ hybridization (FISH). As proof of principle, we were able to distinguish P. acnes and P. granulosum bacteria in multi-species populations in vitro as well as in a mock skin infection model upon labelling with 16S rRNA probes in combinatorial FISH as well as with antibodies. Furthermore, we report the co-localization of P. acnes and P. granulosum in the stratum corneum and hair follicles from patients with acne vulgaris as well as in healthy individuals. Further studies on the spatial distribution of these bacteria in skin structures in various skin disorders are needed.

In vivo self-assembly of fluorescent protein microparticles displaying specific binding domains.

In this study, fluorescent proteins (FPs) were engineered to self-assemble into protein particles inside recombinant Escherichia coli while mediating the display of various protein functionalities such as maltose binding protein or IgG binding domains of Protein A or G, respectively. Escherichia coli produced functional FP particles of up to 30% of cellular dry weight. The use of respective FP particles displaying certain binding domains in diagnostics and as bioseparation resins was demonstrated by direct comparison to commercial offerings. It was demonstrated that variable extensions (AVTS, FHKP, LAVG, or TS) of the N-terminus of FPs (GFP, YFP, CFP, HcRed) in combination with large C-terminal extensions such as translational fusion of the polyester synthase from Ralstonia eutropha or an aldolase from Escherichia coli led to extensive intracellular self-assembly of strongly fluorescent fusion protein particles of oval shape (0.5×1 µm). The strong fluorescent label of these bioparticles in combination with covalent display of protein functions provides a molecular toolbox for the design of self-assembled microparticles suitable for antibody-capture or ligand binding based diagnostic assays as well as the high affinity purification of target compounds such as antibodies.

No link between rosacea and Propionibacterium acnes.

Rosacea is a common skin disease in adults affecting mainly the facial skin. Although inflammation appears to play a pathogenic role in rosacea, initiating factors are largely unknown. Microbial involvement in the development of rosacea has been suggested previously. We aimed to visualize Propionibacterium acnes in the skin compartments of rosacea patients. Facial skin biopsies from 82 rosacea patients and 25 controls were stained with a P. acnes-specific monoclonal antibody (QUBPa3). Seven of 82 patients (8.5%) tested positive for P. acnes which was present either as a biofilm (57% of positive) or a microcolony (43%) in colonized patients. Our results suggest that P. acnes does not play a major role in the pathogenesis of rosacea.

Relevant uses of surface proteins--display on self-organized biological structures.

Proteins are often found attached to surfaces of self-assembling biological units such as whole microbial cells or subcellular structures, e.g. intracellular inclusions. In the last two decades surface proteins were identified that could serve as anchors for the display of foreign protein functions. Extensive protein engineering based on structure-function data enabled efficient display of technically and/or medically relevant protein functions. Small size, diversity of the anchor protein as well as support structure, genetic manipulability and controlled cultivation of phages, bacterial cells and yeasts contributed to the establishment of designed and specifically functionalized tools for applications as sensors, catalysis, biomedicine, vaccine development and library-based screening technologies. Traditionally, phage display is employed for library screening but applications in biomedicine and vaccine development are also perceived. For some diagnostic purposes phages are even too small in size so other carrier materials where needed and gave way for cell and yeast display. Only recently, intracellular inclusions such as magnetosomes, polyhydroxyalkanoate granules and lipid bodies were conceived as stable subcellular structures enabling the display of foreign protein functions and showing potential as specific and tailor-made devices for medical and biotechnological applications.

Tolerance of the Ralstonia eutropha class I polyhydroxyalkanoate synthase for translational fusions to its C terminus reveals a new mode of functional display.

Here, the class I polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha was investigated regarding the functionality of its conserved C-terminal region and its ability to tolerate translational fusions to its C terminus. MalE, the maltose binding protein, and green fluorescent protein (GFP) were considered reporter proteins to be translationally fused to the C terminus. Interestingly, PhaC remained active only when a linker was inserted between PhaC and MalE, whereas MalE was not functional. However, the extension of the PhaC N terminus by 458 amino acid residues was required to achieve a functionality of MalE. These data suggested a positive interaction of the extended N terminus with the C terminus. To assess whether a linker and/or N-terminal extension is generally required for a functional C-terminal fusion, GFP was fused to the C terminus of PhaC. Both fusion partners were active without the requirement of a linker and/or N-terminal extension. A further reporter protein, the immunoglobulin G binding ZZ domain of protein A, was translationally fused to the N terminus of the fusion protein PhaC-GFP and resulted in a tripartite fusion protein mediating the production of polyester granules displaying two functional protein domains.

Bacterial polyhydroxyalkanoate granules: biogenesis, structure, and potential use as nano-/micro-beads in biotechnological and biomedical applications.

Polyhydroxyalkanoates (PHAs) are naturally occurring organic polyesters that are of interest for industrial and biomedical applications. These polymers are synthesized by most bacteria in times of unbalanced nutrient availability from a variety of substrates and they are deposited intracellularly as insoluble spherical inclusions or PHA granules. The granules consist of a polyester core, surrounded by a boundary layer with embedded or attached proteins that include the PHA synthase, phasins, depolymerizing enzymes, and regulatory proteins. Apart from ongoing industrial interest in the material PHA, more recently there has also been increasing interest in applications of the PHA granules as nano-/micro-beads after it was conceived that fusions to the granule associated proteins (GAPs) provide a way to immobilize target proteins at the granule surface. This review gives an overview of PHA granules in general, including biogenesis and GAPs, and focuses on their potential use as nano-/micro-beads in biotechnological and biomedical applications.

Multifunctional inorganic-binding beads self-assembled inside engineered bacteria.

Multifunctional shell-core nano/microbeads with a hydrophobic biopolymer core and a designed protein coat for selective binding of an inorganic substance and antibodies were self-assembled inside engineered bacteria. Hybrid genes were constructed to produce tailormade bead-coating proteins in the bacterium Escherichia coli. These fusion proteins contained a binding peptide for an inorganic material, the antibody binding ZZ domain, and a self-assembly promoting as well as biopolymer synthesizing enzyme. Production of these multidomain fusion proteins inside E. coli resulted in self-assembly of beads comprising a biopolyester core and displaying covalently bound binding sites for specific and selective binding of an inorganic substance and any antibody belonging to the immunoglobulin G class. Engineered beads were isolated and purified from the respective E. coli cells by standard cell disruption procedures. Bead morphology and the binding functionalities displayed at the bead surface were assessed by the enzyme-linked immunosorbent assay, transmission electron microscopy, elemental analysis, backscattering electron density, analytical density ultracentrifugation, and atomic force microscopy. These analyses showed that bacteria can be engineered to produce fusion proteins mediating self-assembly of spherical biopolymer beads with binding affinity to gold and/or silica and antibodies. Spherical structures of this type could conceivably serve as nano/microdevices for bioimaging in medical approaches where an antibody mediated targeted delivery of an inorganic contrast agent would be desired.