Engineering selectivity of Cutibacterium acnes phages by epigenetic imprinting.

Cutibacterium acnes (C. acnes) is a gram-positive bacterium and a member of the human skin microbiome. Despite being the most abundant skin commensal, certain members have been associated with common inflammatory disorders such as acne vulgaris. The availability of the complete genome sequences from various C. acnes clades have enabled the identification of putative methyltransferases, some of them potentially belonging to restriction-modification (R-M) systems which protect the host of invading DNA. However, little is known on whether these systems are functional in the different C. acnes strains. To investigate the activity of these putative R-M and their relevance in host protective mechanisms, we analyzed the methylome of six representative C. acnes strains by Oxford Nanopore Technologies (ONT) sequencing. We detected the presence of a 6-methyladenine modification at a defined DNA consensus sequence in strain KPA171202 and recombinant expression of this R-M system confirmed its methylation activity. Additionally, a R-M knockout mutant verified the loss of methylation properties of the strain. We studied the potential of one C. acnes bacteriophage (PAD20) in killing various C. acnes strains and linked an increase in its specificity to phage DNA methylation acquired upon infection of a methylation competent strain. We demonstrate a therapeutic application of this mechanism where phages propagated in R-M deficient strains selectively kill R-M deficient acne-prone clades while probiotic ones remain resistant to phage infection.

Solution Structure of the Cutibacterium acnes-Specific Protein RoxP and Insights Into Its Antioxidant Activity.

Cutibacterium acnes is a predominant bacterium on human skin and is generally regarded as commensal. Recently, the abundantly secreted protein produced by C. acnes, RoxP, was shown to alleviate radical-induced cell damage, presumably via antioxidant activity, which could potentially be harnessed to fortify skin barrier function. The aim of this study was to determine the structure of RoxP and elucidate the mechanisms behind its antioxidative effect. Here, we present the solution structure of RoxP revealing a compact immunoglobulin-like domain containing a long flexible loop which, in concert with the core domain, forms a positively charged groove that could function as a binding site for cofactors or substrates. Although RoxP shares structural features with cell-adhesion proteins, we show that it does not appear to be responsible for adhesion of C. acnes bacteria to human keratinocytes. We identify two tyrosine-containing stretches located in the flexible loop of RoxP, which appear to be responsible for the antioxidant activity of RoxP.

Engineering a genome-reduced bacterium to eliminate Staphylococcus aureus biofilms in vivo.

Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome-reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm-associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo. We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro, ex vivo, and in vivo. To our knowledge, this is the first engineered genome-reduced bacterium that can fight against clinically relevant biofilm-associated bacterial infections.

From Dysbiosis to Healthy Skin: Major Contributions of Cutibacterium acnes to Skin Homeostasis.

Cutibacterium acnes is the most abundant bacterium living in human, healthy and sebum-rich skin sites, such as the face and the back. This bacterium is adapted to this specific environment and therefore could have a major role in local skin homeostasis. To assess the role of this bacterium in healthy skin, this review focused on (i) the abundance of C. acnes in the skin microbiome of healthy skin and skin disorders, (ii) its major contributions to human skin health, and (iii) skin commensals used as probiotics to alleviate skin disorders. The loss of C. acnes relative abundance and/or clonal diversity is frequently associated with skin disorders such as acne, atopic dermatitis, rosacea, and psoriasis. C. acnes, and the diversity of its clonal population, contributes actively to the normal biophysiological skin functions through, for example, lipid modulation, niche competition and oxidative stress mitigation. Compared to gut probiotics, limited dermatological studies have investigated skin probiotics with skin commensal strains, highlighting their unexplored potential.

Gut-Skin Axis: Current Knowledge of the Interrelationship between Microbial Dysbiosis and Skin Conditions.

The microbiome plays an important role in a wide variety of skin disorders. Not only is the skin microbiome altered, but also surprisingly many skin diseases are accompanied by an altered gut microbiome. The microbiome is a key regulator for the immune system, as it aims to maintain homeostasis by communicating with tissues and organs in a bidirectional manner. Hence, dysbiosis in the skin and/or gut microbiome is associated with an altered immune response, promoting the development of skin diseases, such as atopic dermatitis, psoriasis, acne vulgaris, dandruff, and even skin cancer. Here, we focus on the associations between the microbiome, diet, metabolites, and immune responses in skin pathologies. This review describes an exhaustive list of common skin conditions with associated dysbiosis in the skin microbiome as well as the current body of evidence on gut microbiome dysbiosis, dietary links, and their interplay with skin conditions. An enhanced understanding of the local skin and gut microbiome including the underlying mechanisms is necessary to shed light on the microbial involvement in human skin diseases and to develop new therapeutic approaches.

Skin microbiome transplantation and manipulation: Current state of the art.

Many skin conditions are associated with an imbalance in the skin microbiome. In recent years, the skin microbiome has become a hot topic, for both therapeutic and cosmetic purposes. The possibility of manipulating the human skin microbiome to address skin conditions has opened exciting new paths for therapy. Here we review the skin microbiome manipulation strategies, ranging from skin microbiome transplantation, over skin bacteriotherapy to the use of prebiotics, probiotics and postbiotics. We summarize all efforts undertaken to exchange, manipulate, transplant or selectively apply the skin microbiome to date. Multiple microbial groups have been targeted, since they have been proven to be beneficial for skin health. We focus on the most common skin disorders and their associated skin microbiome dysbiosis and we review the existing scientific data and clinical trials undertaken to combat these skin conditions. The skin microbiome represents a novel platform for therapy. Transplantation of a complete microbiome or application of single strains has demonstrated beneficial therapeutic application.

MinION™ Nanopore Sequencing of Skin Microbiome 16S and 16S-23S rRNA Gene Amplicons.

Human skin microbiome dysbiosis can have clinical consequences. Characterizing taxonomic composition of bacterial communities associated with skin disorders is important for dermatological advancement in both diagnosis and novel treatments. This study aims to analyze and improve the accuracy of taxonomic classification of skin bacteria with MinION™ nanopore sequencing using a defined skin mock community and a skin microbiome sample. We compared the Oxford Nanopore Technologies recommended procedures and concluded that their protocols highly bias the relative abundance of certain skin microbiome genera, most notably a large overrepresentation of Staphylococcus and underrepresentation of Cutibacterium and Corynebacterium. We demonstrated that changes in the amplification protocols improved the accuracy of the taxonomic classification for these three main skin bacterial genera. This study shows that MinION™ nanopore could be an efficient technology for full-length 16S rRNA sequencing; however, the analytical advantage is strongly influenced by the methodologies. The suggested alternatives in the sample processing improved characterization of a complex skin microbiome community using MinION™ nanopore sequencing.

Characterization of different alginate lyases for dissolving Pseudomonas aeruginosa biofilms.

Aggregates of Pseudomonas aeruginosa form a protective barrier against antibiotics and the immune system. These barriers, known as biofilms, are associated with several infectious diseases. One of the main components of these biofilms is alginate, a homo- and hetero-polysaccharide that consists of ß-D-mannuronate (M) and α-L-guluronate (G) units. Alginate lyases degrade this sugar and have been proposed as biotherapeutic agents to dissolve P. aeruginosa biofilms. However, there are contradictory reports in the literature regarding the efficacy of alginate lyases against biofilms and their synergistic effect with antibiotics. We found that most positive reports used a commercial crude extract from Flavobacterium multivorum as the alginate lyase source. By using anion exchange chromatography coupled to nano LC MS/MS, we identified two distinct enzymes in this extract, one has both polyM and polyG (polyM/G) degradation activities and it is similar in sequence to a broad-spectrum alginate lyase from Flavobacterium sp. S20 (Alg2A). The other enzyme has only polyG activity and it is similar in sequence to AlyA1 from Zobellia galactanivorans. By characterizing both of these enzymes together with three recombinant alginate lyases (a polyM, a polyG and a polyM/G), we showed that only enzymes with polyM/G activity such as Alg2A and A1-II' (alginate lyase from Sphingomonas sp.) are effective in dissolving biofilms. Furthermore, both activities are required to have a synergistic effect with antibiotics.

Safety and Efficacy of Topically Applied Selected Cutibacterium acnes Strains over Five Weeks in Patients with Acne Vulgaris: An Open-label, Pilot Study.

Imbalance in skin microflora, particularly related to certain Cutibacterium acnes strains, may trigger acne. Application of non-acne-causing strains to the skin may modulate the skin microbiome and thereby lead to a reduction in acne. This pilot study evaluates the safety and efficacy of microbiome modulation on acne-prone skin. The study had 2 phases: active induction (5% benzoyl peroxide gel, 7 days) and interventional C. acnes strains treatment (5 weeks). Patients were randomized to either topical skin formulations PT1 (2 strains of C. acnes Single Locus Sequence Typing [SLST] type C3 and K8, 50% each) or PT2 (4 strains of C. acnes SLST type C3 [55%], K8 [5%], A5 [30%] and F4 [10%]). Safety and efficacy was evaluated in 14 patients (PT1=8/14, PT2=6/14). Skin microbiome composition shifted towards study formulations. No untoward adverse events, visible irritation, or significant flare-up were observed. Non-inflamed lesions and skin pH were reduced. Comedone counts improved clinically with no deterioration in inflammatory lesions.

Skin microbiome modulation induced by probiotic solutions.

BACKGROUND: The skin is colonized by a large number of microorganisms, most of which are beneficial or harmless. However, disease states of skin have specific microbiome compositions that are different from those of healthy skin. Gut microbiome modulation through fecal transplant has been proven as a valid therapeutic strategy in diseases such as Clostridium difficile infections. Therefore, techniques to modulate the skin microbiome composition may become an interesting therapeutic option in diseases affecting the skin such as psoriasis or acne vulgaris. METHODS: Here, we have used mixtures of different skin microbiome components to alter the composition of recipient skin microbiomes. RESULTS: We show that after sequential applications of a donor microbiome, the recipient microbiome becomes more similar to the donor. After intervention, an initial week-long phase is characterized by the dominance of donor strains. The level of engraftment depends on the composition of the recipient and donor microbiomes, and the applied bacterial load. We observed higher engraftment using a multi-strain donor solution with recipient skin rich in Cutibacterium acnes subtype H1 and Leifsonia. CONCLUSIONS: We have demonstrated the use of living bacteria to modulate skin microbiome composition.

Development of a replicating plasmid based on the native oriC in Mycoplasma pneumoniae.

Bacteria of the genus Mycoplasma have recently attracted considerable interest as model organisms in synthetic and systems biology. In particular, Mycoplasma pneumoniae is one of the most intensively studied organisms in the field of systems biology. However, the genetic manipulation of these bacteria is often difficult due to the lack of efficient genetic systems and some intrinsic peculiarities such as an aberrant genetic code. One major disadvantage in working with M. pneumoniae is the lack of replicating plasmids that can be used for the complementation of mutants and the expression of proteins. In this study, we have analysed the genomic region around the gene encoding the replication initiation protein, DnaA, and detected putative binding sites for DnaA (DnaA boxes) that are, however, less conserved than in other bacteria. The construction of several plasmids encompassing this region allowed the selection of plasmid pGP2756 that is stably inherited and that can be used for genetic experiments, as shown by the complementation assays with the glpQ gene encoding the glycerophosphoryl diester phosphodiesterase. Plasmid-borne complementation of the glpQ mutant restored the formation of hydrogen peroxide when bacteria were cultivated in the presence of glycerol phosphocholine. Interestingly, the replicating plasmid can also be used in the close relative, Mycoplasma genitalium but not in more distantly related members of the genus Mycoplasma. Thus, plasmid pGP2756 is a valuable tool for the genetic analysis of M. pneumoniae and M. genitalium.

In situ overlap and sequence synthesis during DNA assembly.

Modern cloning methods are independent from restriction enzyme recognition sites. However, nearly all current cloning methods still require the introduction of overlaps by PCR, which can introduce undesired mutations. Here, we investigated whether overlaps needed for DNA assembly can be synthesized in situ and we tested if the de novo synthesis of sequences can be simultaneously combined with the assembly of larger double-stranded DNA fragments. We showed in a set of 44 cloning experiments that overlaps of 20 bp needed for DNA assembly can be synthesized in situ from single-stranded oligonucleotides. Short sequences of 30-255 bp can be synthesized from single-stranded oligonucleotides concurrently with DNA assembly, and both techniques can be combined. The assembly of similar constructs by state-of-the-art techniques would have required multiple rounds of cloning or tedious sample preparations, whereas our approach is a one-step reaction.

Large-scale metabolome analysis and quantitative integration with genomics and proteomics data in Mycoplasma pneumoniae.

Systems metabolomics, the identification and quantification of cellular metabolites and their integration with genomics and proteomics data, promises valuable functional insights into cellular biology. However, technical constraints, sample complexity issues and the lack of suitable complementary quantitative data sets prevented accomplishing such studies in the past. Here, we present an integrative metabolomics study of the genome-reduced bacterium Mycoplasma pneumoniae. We experimentally analysed its metabolome using a cross-platform approach. We explain intracellular metabolite homeostasis by quantitatively integrating our results with the cellular inventory of proteins, DNA and other macromolecules, as well as with available building blocks from the growth medium. We calculated in vivo catalytic parameters of glycolytic enzymes, making use of measured reaction velocities, as well as enzyme and metabolite pool sizes. A quantitative, inter-species comparison of absolute and relative metabolite abundances indicated that metabolic pathways are regulated as functional units, thereby simplifying adaptive responses. Our analysis demonstrates the potential for new scientific insight by integrating different types of large-scale experimental data from a single biological source.

Intrinsic dynamics in ECFP and Cerulean control fluorescence quantum yield.

Enhanced cyan fluorescent protein (ECFP) and its variant Cerulean are genetically encoded fluorophores widely used as donors in FRET-based cell imaging experiments. First, we have confirmed through denaturation experiments that the double-peak spectroscopic signature of these fluorescent proteins originates from the indole ring of the chromophore. Then, to explain the improvement in the fluorescence properties of Cerulean compared to those of ECFP, we have determined the high-resolution crystal structures of these two proteins at physiological pH and performed molecular dynamics simulations. In both proteins, the N-terminal half of the seventh strand exhibits two conformations. These conformations both have a complex set of van der Waals interactions with the chromophore and, as our simulations suggest, they interconvert on a nanosecond time scale. The Y145A and H148D mutations in Cerulean stabilize these interactions and allow the chromophore to be more planar, better packed, and less prone to collisional quenching, albeit only intermittently. As a consequence, the probability of nonradiative decay is significantly decreased. Our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins and provide insights that should allow the design of mutants with enhanced optical properties.

The reovirus sigma1 aspartic acid sandwich: a trimerization motif poised for conformational change.

Reovirus attachment protein sigma1 mediates engagement of receptors on the surface of target cells and undergoes dramatic conformational rearrangements during viral disassembly in the endocytic pathway. The sigma1 protein is a filamentous, trimeric molecule with a globular beta-barrel head domain. An unusual cluster of aspartic acid residues sandwiched between hydrophobic tyrosines is located at the sigma1 subunit interface. A 1.75-A structure of the sigma1 head domain now reveals two water molecules at the subunit interface that are held strictly in position and interact with neighboring residues. Structural and biochemical analyses of mutants affecting the aspartic acid sandwich indicate that these residues and the corresponding chelated water molecules act as a plug to block the free flow of solvent and stabilize the trimer. This arrangement of residues at the sigma1 head trimer interface illustrates a new protein design motif that may confer conformational mobility during cell entry.