Glycerol fermentation by skin bacteria generates lactic acid and upregulates the expression levels of genes associated with the skin barrier function.

Commensal bacteria play a major role in multiple skin functions by providing the first layer of defense against pathogens and maintaining the skin barrier. Staphylococcus epidermidis is one of the most common skin commensals. In this study, we showed that S. epidermidis ferments glycerol and uses it as a nutrient, while producing short-chain and organic fatty acids, with the most notable being lactic acid. Lactic acid is an alpha-hydroxy acid that inhibits the growth of pathogenic bacteria, without any negative effect on the commensal bacteria itself. Using in vivo experiments, we validated our in vitro results, showing that the skin microbiome is also capable of doing this. Finally, using 2D and 3D skin culture models, we showed that the fermentation of glycerol, mainly lactic acid, as determined by analytical methods, upregulates the expression levels of several key genes that are associated with the barrier properties of the skin. While the hydration effect of glycerol on the skin is well known, our study shows the overall benefits of glycerol on the skin microbiome, while revealing an alternate mode of action of glycerol for multiple skin benefits.

Dysbiotic Lesional Microbiome With Filaggrin Missense Variants Associate With Atopic Dermatitis in India.

Background: Atopic Dermatitis (AD) has been associated with the loss of function (LoF) mutations in Filaggrin (FLG) gene and increase in relative abundance of specific microbes in the lesional skin, predominantly in Caucasians. Our study aims to determine, in Indian AD patients, (a) the prevalence of FLG LoF and missense mutations, and (b) the nature and extent of dysbiosis and altered microbial pathways with and without mutations in FLG. AD patients (n = 34) and healthy controls (n = 54) were recruited from India in this study and shotgun sequencing was carried out in a subset of samples with adequate microbiome DNA concentration. Host DNA from the same subset of samples was subjected to FLG coding region sequencing and host-microbiome association was estimated. Results: The prevalence of FLG LoFs that are associated with AD globally were significantly lesser in our cases and controls (8.6%, 0%) than those reported in Europeans (27%, 2.6%). Staphylococcus aureus was present only on AD skin [abundance in Pediatric AD: 32.86%; Adult AD: 22.17%], but not on healthy skin on which Staphylococcus hominis (Adult controls: 16.43%, Adult AD: 0.20%; p = 0.002), Cutibacterium acnes (Adult controls:10.84%, Adult AD: 0.90%; p = 0.02), and Malassezia globosa (Adult controls: 8.89%, Adult AD: 0.005%; p = 0.001) were significantly more abundant. Microbial pathways mostly associated with skin barrier permeability, ammonia production and inflammation (Arginine and Proline metabolism, Histidine Metabolism and Staphylococcus aureus infection) were significantly enriched on AD skin metagenome. These pathways are also reported to impair antimicrobial peptide activity. Among AD patients with missense single nucleotide polymorphisms harboring "potentially damaging" alleles in FLG gene, damaging allele dosage was significantly (p < 0.02) positively correlated with relative abundance of phylum_Proteobacteria up to order_Pseudomonadales and negatively correlated with phylum_Firmicutes up to species_Staphylococcus aureus. Conclusion: Our study has provided evidence that host DNA profile is significantly associated with microbiome composition in the development of AD. Species and strain level analysis showed that the microbial pathways enriched in AD cases were mostly found in MRSA strains. These evidences can be harnessed to control AD by modulating the microbiome using a personalized strategy. Our findings on the association of FLG genotypes with the microbiome dysbiosis may pave the way for a personalized strategy to provide a more effective control of AD.

Sebum and Hydration Levels in Specific Regions of Human Face Significantly Predict the Nature and Diversity of Facial Skin Microbiome.

The skin microbiome varies across individuals. The causes of these variations are inadequately understood. We tested the hypothesis that inter-individual variation in facial skin microbiome can be significantly explained by variation in sebum and hydration levels in specific facial regions of humans. We measured sebum and hydration from forehead and cheek regions of healthy female volunteers (n = 30). Metagenomic DNA from skin swabs were sequenced for V3-V5 regions of 16S rRNA gene. Altogether, 34 phyla were identified; predominantly Actinobacteria (66.3%), Firmicutes (17.7%), Proteobacteria (13.1%) and Bacteroidetes (1.4%). About 1000 genera were identified; predominantly Propionibacterium (58.6%), Staphylococcus (8.6%), Streptococcus (4.0%), Corynebacterium (3.6%) and Paracoccus (3.3%). A subset (n = 24) of individuals were sampled two months later. Stepwise multiple regression analysis showed that cheek sebum level was the most significant predictor of microbiome composition and diversity followed by forehead hydration level; forehead sebum and cheek hydration levels were not. With increase in cheek sebum, the prevalence of Actinobacteria (p = 0.001)/Propionibacterium (p = 0.002) increased, whereas microbiome diversity decreased (Shannon Index, p = 0.032); this was opposite for other phyla/genera. These trends were reversed for forehead hydration levels. Therefore, the nature and diversity of facial skin microbiome is jointly determined by site-specific lipid and water levels in the stratum corneum.

Functional characterization of piggyBat from the bat Myotis lucifugus unveils an active mammalian DNA transposon.

A revelation of the genomic age has been the contributions of the mobile DNA segments called transposable elements to chromosome structure, function, and evolution in virtually all organisms. Substantial fractions of vertebrate genomes derive from transposable elements, being dominated by retroelements that move via RNA intermediates. Although many of these elements have been inactivated by mutation, several active retroelements remain. Vertebrate genomes also contain substantial quantities and a high diversity of cut-and-paste DNA transposons, but no active representative of this class has been identified in mammals. Here we show that a cut-and-paste element called piggyBat, which has recently invaded the genome of the little brown bat (Myotis lucifugus) and is a member of the piggyBac superfamily, is active in its native form in transposition assays in bat and human cultured cells, as well as in the yeast Saccharomyces cerevisiae. Our study suggests that some DNA transposons are still actively shaping some mammalian genomes and reveals an unprecedented opportunity to study the mechanism, regulation, and genomic impact of cut-and-paste transposition in a natural mammalian host.

Characterization of the TnsD-attTn7 complex that promotes site-specific insertion of Tn7.

The bacterial transposon Tn7 is distinguished by its ability to recognize a specific site called attTn7, and insert just downstream of the highly conserved chromosomal glmS gene. TnsD is one of four transposon-encoded polypeptides (TnsABC+D) required for site-specific insertion of Tn7 into attTn7, and is the target site-selector that binds to a highly conserved sequence in the end of the glmS protein coding region. In this study, we identified important nucleotides within this region that are crucial for TnsD-attTn7 interaction. We also probed the regions of TnsD that interact with attTn7 and found that there are important DNA-binding determinants throughout the entire length of the protein, including an amino-terminal CCCH zinc-finger motif. A key role of TnsD is to recruit the non-sequence specific DNA-binding protein TnsC to attTn7; TnsC also interacts with and controls both the TnsA and TnsB subunits of the Tn7 transposase. TnsC stimulates the binding of TnsD to attTn7 in vivo, and TnsCD and TnsD can also interact in the absence of DNA and localize their interaction domains to the N-terminal region of each protein.

piggyBac can bypass DNA synthesis during cut and paste transposition.

DNA synthesis is considered a defining feature in the movement of transposable elements. In determining the mechanism of piggyBac transposition, an insect transposon that is being increasingly used for genome manipulation in a variety of systems including mammalian cells, we have found that DNA synthesis can be avoided during piggyBac transposition, both at the donor site following transposon excision and at the insertion site following transposon integration. We demonstrate that piggyBac transposon excision occurs through the formation of transient hairpins on the transposon ends and that piggyBac target joining occurs by the direct attack of the 3'OH transposon ends on to the target DNA. This is the same strategy for target joining used by the members of DDE superfamily of transposases and retroviral integrases. Analysis of mutant piggyBac transposases in vitro and in vivo using a piggyBac transposition system we have established in Saccharomyces cerevisiae suggests that piggyBac transposase is a member of the DDE superfamily of recombinases, an unanticipated result because of the lack of sequence similarity between piggyBac and DDE family of recombinases.

Site-specific Tn7 transposition into the human genome.

The bacterial transposon, Tn7, inserts into a single site in the Escherichia coli chromosome termed attTn7 via the sequence-specific DNA binding of the target selector protein, TnsD. The target DNA sequence required for Tn7 transposition is located within the C-terminus of the glucosamine synthetase (glmS) gene, which is an essential, highly conserved gene found ubiquitously from bacteria to humans. Here, we show that Tn7 can transpose in vitro adjacent to two potential targets in the human genome: the gfpt-1 and gfpt-2 sequences, the human analogs of glmS. The frequency of transposition adjacent to the human gfpt-1 target is comparable with the E.coli glmS target; the human gfpt-2 target shows reduced transposition. The binding of TnsD to these sequences mirrors the transposition activity. In contrast to the human gfpt sequences, Tn7 does not transpose adjacent to the gfa-1 sequence, the glmS analog in Saccharomyces cerevisiae. We also report that a nucleosome core particle assembled on the human gfpt-1 sequence reduces Tn7 transposition by likely impairing the accessibility of target DNA to the Tns proteins. We discuss the implications of these findings for the potential use of Tn7 as a site-specific DNA delivery agent for gene therapy.

Transposition of hAT elements links transposable elements and V(D)J recombination.

Transposons are DNA sequences that encode functions that promote their movement to new locations in the genome. If unregulated, such movement could potentially insert additional DNA into genes, thereby disrupting gene expression and compromising an organism's viability. Transposable elements are classified by their transposition mechanisms and by the transposases that mediate their movement. The mechanism of movement of the eukaryotic hAT superfamily elements was previously unknown, but the divergent sequence of hAT transposases from other elements suggested that these elements might use a distinct mechanism. Here we have analysed transposition of the insect hAT element Hermes in vitro. Like other transposons, Hermes excises from DNA via double-strand breaks between the donor-site DNA and the transposon ends, and the newly exposed transposon ends join to the target DNA. Interestingly, the ends of the donor double-strand breaks form hairpin intermediates, as observed during V(D)J recombination, the process which underlies the combinatorial formation of antigen receptor genes. Significant similarities exist in the catalytic amino acids of Hermes transposase, the V(D)J recombinase RAG, and retroviral integrase superfamily transposases, thereby linking the movement of transposable elements and V(D)J recombination.

Molecular characterisation of rough variants of Vibrio cholerae isolated from hospitalised patients with diarrhoea.

Seven rough isolates of Vibrio cholerae isolated as the sole infecting agent from patients with cholera-like diarrhoea were examined for the presence of the regulatory element toxR and certain virulence-associated genes of the CTX genetic element and V. cholerae pathogenicity island (VPI). Multiplex PCR analysis with wb-specific genes of either O1 or O139 origin showed that six of the seven isolates produced an O1 wb-specific amplicon and the remaining isolate produced an O139-specific amplicon. Analysis of lipopolysaccharide profiles of smooth variants of V. cholerae revealed the presence of long repeated units of 'O' polysaccharide side chains but all the rough variants appeared to be devoid of the latter and possessed only core oligosaccharide. PCR amplification with primers specific to the ctxA, ctxB, tcpA, tagA, int, aldA, toxT, LJ, RJ and toxR genes revealed that six of the seven rough isolates were positive for these genes. One isolate was found to be negative for tagA and RJ, indicating the presence of an altered VPI. Each of these isolates showed media-dependent expression of cholera toxin (CT) and produced more toxin than the reference V. cholerae O1 El Tor strain VC20 or O139 strain SG24 under comparable conditions. Studies on the clonality of these isolates by the analysis of rRNA genes indicated their relatedness to strains of V. cholerae O1 El Tor or O139, isolated during the same time period.

Response of wild-type mutants of Vibrio cholerae O1 possessing different combinations of virulence genes in the ligated rabbit ileal loop and in Ussing chambers: evidence for the presence of additional secretogen.

Five wild-type mutant strains of Vibrio cholerae serogroup O1 that lacked the CTX virulence cassette, or contained a natural deletion of a virulence gene within the CTX virulence cassette, or possessed an additional virulence gene, along with a prototype toxigenic strain representing the El Tor classical biotypes were examined by in-vivo and in-vitro methods to determine their enterotoxic potential. The ability of whole cells and culture supernates of the strains to cause fluid accumulation in the rabbit ileal loop model revealed a pattern consistent with the presence of the various virulence gene(s), with those possessing the intact CTX virulence cassette being the most secretogenic. Culture supernates of strains without the CTX virulence cassette or the strain with an incomplete cassette were also able to evoke mild to moderate fluid accumulation in the rabbit ileal loop. Of the various media used, AKI and brain heart infusion broth appeared to support the production of a hitherto unknown secretogenic factor, because culture supernates of the non-toxigenic V. cholerae O1 strains showed higher fluid accumulation ratios when grown in these media than in the others. To confirm that the fluid accumulation elicited by these strains in the ileal loop was due to enterotoxin activity, the effect of supernate of the strains was examined in rabbit small intestine mounted on Ussing chambers. Increases in short circuit current and tissue conductance, as compared with the medium control, were observed even with the strains that did not possess the CTX virulence cassette, confirming their ability to disrupt the function of intestinal tissue. From these studies, it was concluded that strains of V. cholerae O1 devoid of the CTX virulence cassette were still able to elicit a secretory response in the ileal loop and displayed enterotoxic activity in an in-vitro experimental model.