Biology subjects

Braga, D.

Publications and source records attributed to Braga, D..

4 recordsLinked to original sources

bioRxiv2024

Lactobacillus paracasei CNCM I-5220-derived postbiotic counteracts skin inflammation and protects skin barrier integrity

IntroductionSkin inflammation and damage of skin barrier integrity contribute to the pathogenesis of inflammatory skin diseases such as psoriasis and atopic dermatitis. Nowadays, there is a growing interest in alternative and complementary strategies to counteract skin inflammation and treat dermatologic conditions. ObjectivesPostbiotics, metabolites released during bacterial fermentation, exert anti-inflammatory proprieties and contribute to the maintenance of epithelial barrier integrity. Therefore, we investigated the effect of LP-PBL, a novel Lactobacillus paracasei CNCM I-5220-derived postbiotic, in controlling skin inflammation and protecting the skin barrier. MethodsWe performed RNA-sequencing on Poly(I:C)-stimulated keratinocytes and investigated LP-PBL efficacy in regulating pro-inflammatory cytokine release. The production of most relevant cytokines was demonstrated by ELISA. Then, we tested postbiotic lenitive efficacy on healthy volunteers irritated skin. We compared the effect of a postbiotic-containing cream formulation versus placebo on sodium lauryl sulphate treated skin. ResultsWe demonstrate that LP-PBL has anti-inflammatory activity by modulating Poly(I:C)-dependent inflammatory pathways and pro-inflammatory cytokine release in keratinocytes. The postbiotic inhibited the upregulation of interleukin (IL)-23A, which is overexpressed in psoriatic skin, and of IL-33 and thymic stromal lymphopoietin (TSLP) that are upregulated in atopic dermatitis. Moreover, it increased filaggrin and zonula occludens (ZO)-1 expression in Poly(I:C)-stimulated keratinocytes, suggesting beneficial effects on inflamed and damaged skin. Consistently, a clinical test on healthy volunteers showed that topic LP-PBL treatment significantly reduced skin redness upon sodium lauryl sulphate challenge compared to placebo, leading to a rapid recovery of the irritated skin. ConclusionOverall, we demonstrated the protective role of LP-PBL on the skin, both in vitro and in the clinical test, suggesting that postbiotic topical application could represent an innovative and promising strategy to counteract skin inflammation and preserve skin barrier integrity.

microbiology↗

bioRxiv2021

Diversification by CofC and control by CofD govern biosynthesis and evolution of coenzyme F420 and its derivative 3PG-F420

Coenzyme F420 is a microbial redox cofactor that is increasingly used for biocatalytic applications. Recently, diversified biosynthetic routes to F420 and the discovery of a derivative, 3PG-F420, were reported. 3PG-F420 is formed via activation of 3-phospho-D-glycerate (3-PG) by CofC, but the structural basis of substrate binding, its evolution, as well as the role of CofD in substrate selection remained elusive. Here, we present a crystal structure of the 3-PG-activating CofC from Mycetohabitans sp. B3 and define amino acids governing substrate specificity. Site-directed mutagenesis enabled bidirectional switching of specificity and thereby revealed the short evolutionary trajectory to 3PG-F420 formation. Furthermore, CofC stabilized its product, thus confirming the structure of the unstable molecule, revealing its binding mode and suggesting a substrate channeling mechanism to CofD. The latter enzyme was shown to significantly contribute to the selection of related intermediates to control the specificity of the combined biosynthetic CofC/D step. Taken together, this work closes important knowledge gaps and opens up perspectives for the discovery, enhanced biotechnological production, and engineering of coenzyme F420 derivatives in the future. ImportanceThe microbial cofactor F420 is crucial for processes like methanogenesis, antibiotics biosynthesis, drug resistance, and biocatalysis. Recently, a novel derivative of F420 (3PG-F420) was discovered, enabling the production and use of F420 in heterologous hosts. By analyzing the crystal structure of a CofC homolog whose substrate choice leads to formation of 3PG-F420, we defined amino acid residues governing the special substrate selectivity. A diagnostic residue enabled reprogramming of the substrate specificity, thus mimicking the evolution of the novel cofactor derivative and successfully guiding the identification of further 3-PG-activating enzymes. Furthermore, a labile reaction product of CofC was revealed that has not been directly detected so far and CofD was shown to provide as another layer of specificity of the combined CofC/D reaction, thus controlling the initial substrate choice of CofC. The latter finding resolves a current debate in the literature about the starting point of F420 biosynthesis in various organisms.

microbiology↗

bioRxiv2019

Biosynthesis of the redox cofactor mycofactocin comprises oligoglycosylation by MftF in Mycolicibacterium smegmatis

Mycofactocin (MFT) is a redox cofactor involved in alcohol metabolism of mycobacteria including Mycobacterium tuberculosis. In recent years, a preliminary biosynthetic model of MFT has been established by in-vitro studies, while the final structure of MFT remained elusive. Here, we report the discovery of MFT by metabolomics and establish a model of its biosynthesis in Mycolicibacterium smegmatis. Structure elucidation revealed that MFT is decorated with up to nine {beta}-1,4-linked glucose residues. Dissection of biosynthetic genes demonstrated that the oligoglycosylation is catalyzed by the glycosyltransferase MftF. Furthermore, we confirm the cofactor function of MFT by activity-based metabolic profiling using the carveol dehydrogenase LimC and show that the MFT pool expands during cultivation on ethanol. Our results close an important gap of knowledge, will guide future studies into the physiological roles of MFT in bacteria and may inspire its utilization as a biomarker or potential drug target to combat mycobacterial diseases.

microbiology↗

bioRxiv2019

Metabolic pathway rerouting in Paraburkholderia rhizoxinica evolved long-overlooked derivatives of coenzyme F420

Coenzyme F420 is a specialized redox cofactor with a highly negative redox potential. It supports biochemical processes like methanogenesis, degradation of xenobiotics or the biosynthesis of antibiotics. Although well-studied in methanogenic archaea and actinobacteria, not much is known about F420 in Gram-negative bacteria. Genome sequencing revealed F420 biosynthetic genes in the Gram-negative, endofungal bacterium Paraburkholderia rhizoxinica, a symbiont of phytopathogenic fungi. Fluorescence microscopy, high-resolution LC-MS, and structure elucidation by NMR demonstrated that the encoded pathway is active and yields unexpected derivatives of F420 (3PG-F420). Further analyses of a biogas-producing microbial community showed that these derivatives are more widespread in nature. Genetic and biochemical studies of their biosynthesis established that a specificity switch in the guanylyltransferase CofC re-programmed the pathway to start from 3-phospho-D-glycerate, suggesting a rerouting event during the evolution of F420 biosynthesis. Furthermore, the cofactor activity of 3PG-F420 was validated, thus opening up perspectives for its use in biocatalysis. The 3PG-F420 biosynthetic gene cluster is fully functional in Escherichia coli, enabling convenient production of the cofactor by fermentation.

biochemistry↗